Fibrosis of Two: Epithelial Cell-Fibroblast Interactions in Pulmonary Fibrosis

PubMed Central

Sakai, Norihiko; Tager, Andrew M.

2013-01-01

Idiopathic pulmonary fibrosis (IPF) is characterized by the progressive and ultimately fatal accumulation of fibroblasts and extracellular matrix in the lung that distorts its architecture and compromises its function. IPF is now thought to result from wound-healing processes that, although initiated to protect the host from injurious environmental stimuli, lead to pathological fibrosis due to these processes becoming aberrant or over-exuberant. Although the environmental stimuli that trigger IPF remain to be identified, recent evidence suggests that they initially injure the alveolar epithelium. Repetitive cycles of epithelial injury and resultant alveolar epithelial cell death provoke the migration, proliferation, activation and myofibroblast differentiation of fibroblasts, causing the accumulation of these cells and the extracellular matrix that they synthesize. In turn, these activated fibroblasts induce further alveolar epithelial cell injury and death, thereby creating a vicious cycle of pro-fibrotic epithelial cell-fibroblast interactions. Though other cell types certainly make important contributions, we focus here on the “pas de deux” (steps of two), or perhaps more appropriate to IPF pathogenesis, the “folie à deux” (madness of two) of epithelial cells and fibroblasts that drives the progression of pulmonary fibrosis. We describe the signaling molecules that mediate the interactions of these cell types in their “fibrosis of two”, including transforming growth factor-β, connective tissue growth factor, sonic hedgehog, prostaglandin E2, angiotensin II and reactive oxygen species. PMID:23499992

Autonomous assembly of epithelial structures by subrenal implantation of dissociated embryonic inner-ear cells.

PubMed

Wang, Li; Zhang, Kaiqing; Zhu, Helen He; Gao, Wei-Qiang

2015-05-27

Microenvironment and cell-cell interactions play an important role during embryogenesis and are required for the stemness and differentiation of stem cells. The inner-ear sensory epithelium, containing hair cells and supporting cells, is derived from the stem cells within the otic vesicle at early embryonic stages. However, whether or not such microenvironment or cell-cell interactions within the embryonic otic tissue have the capacity to regulate the proliferation and differentiation of stem cells and to autonomously reassemble the cells into epithelial structures is unknown. Here, we report that on enzymatic digestion and dissociation to harvest all the single cells from 13.5-day-old rat embryonic (E13.5) inner-ear tissue as well as on implantation of these cells under renal capsules; the dissociated cells are able to reassemble themselves to form epithelial structures as early as 7 days after implantation. By 25 days after implantation, more mature epithelial structures are formed. Immunostaining with cell-type-specific markers reveals that hair cells and supporting cells are not only formed, but are also well aligned with the hair cells located in the apical layer surrounded by the supporting cells. These findings suggest that microenvironment and cell-cell interactions within the embryonic inner-ear tissue have the autonomous signals to induce the formation of sensory epithelial structures. This method may also provide a useful system to study the potential of stem cells to differentiate into hair cells in vivo.

Infection of human intestinal epithelial cells with invasive bacteria upregulates apical intercellular adhesion molecule-1 (ICAM)-1) expression and neutrophil adhesion.

PubMed Central

Huang, G T; Eckmann, L; Savidge, T C; Kagnoff, M F

1996-01-01

The acute host response to gastrointestinal infection with invasive bacteria is characterized by an accumulation of neutrophils in the lamina propria, and neutrophil transmigration to the luminal side of the crypts. Intestinal epithelial cells play an important role in the recruitment of inflammatory cells to the site of infection through the secretion of chemokines. However, little is known regarding the expression, by epithelial cells, of molecules that are involved in interactions between the epithelium and neutrophils following bacterial invasion. We report herein that expression of ICAM-1 on human colon epithelial cell lines, and on human enterocytes in an in vivo model system, is upregulated following infection with invasive bacteria. Increased ICAM-1 expression in the early period (4-9 h) after infection appeared to result mainly from a direct interaction between invaded bacteria and host epithelial cells since it co-localized to cells invaded by bacteria, and the release of soluble factors by epithelial cells played only a minor role in mediating increased ICAM-1 expression. Furthermore, ICAM-1 was expressed on the apical side of polarized intestinal epithelial cells, and increased expression was accompanied by increased neutrophil adhesion to these cells. ICAM-1 expression by intestinal epithelial cells following infection with invasive bacteria may function to maintain neutrophils that have transmigrated through the epithelium in close contact with the intestinal epithelium, thereby reducing further invasion of the mucosa by invading pathogens. PMID:8755670

Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

PubMed

Silva, Carlos A M; Danelishvili, Lia; McNamara, Michael; Berredo-Pinho, Márcia; Bildfell, Robert; Biet, Franck; Rodrigues, Luciana S; Oliveira, Albanita V; Bermudez, Luiz E; Pessolani, Maria C V

2013-07-01

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

Rab5-regulated endocytosis plays a crucial role in apical extrusion of transformed cells.

PubMed

Saitoh, Sayaka; Maruyama, Takeshi; Yako, Yuta; Kajita, Mihoko; Fujioka, Yoichiro; Ohba, Yusuke; Kasai, Nobuhiro; Sugama, Natsu; Kon, Shunsuke; Ishikawa, Susumu; Hayashi, Takashi; Yamazaki, Tomohiro; Tada, Masazumi; Fujita, Yasuyuki

2017-03-21

Newly emerging transformed cells are often eliminated from epithelial tissues. Recent studies have revealed that this cancer-preventive process involves the interaction with the surrounding normal epithelial cells; however, the molecular mechanisms underlying this phenomenon remain largely unknown. In this study, using mammalian cell culture and zebrafish embryo systems, we have elucidated the functional involvement of endocytosis in the elimination of RasV12-transformed cells. First, we show that Rab5, a crucial regulator of endocytosis, is accumulated in RasV12-transformed cells that are surrounded by normal epithelial cells, which is accompanied by up-regulation of clathrin-dependent endocytosis. Addition of chlorpromazine or coexpression of a dominant-negative mutant of Rab5 suppresses apical extrusion of RasV12 cells from the epithelium. We also show in zebrafish embryos that Rab5 plays an important role in the elimination of transformed cells from the enveloping layer epithelium. In addition, Rab5-mediated endocytosis of E-cadherin is enhanced at the boundary between normal and RasV12 cells. Rab5 functions upstream of epithelial protein lost in neoplasm (EPLIN), which plays a positive role in apical extrusion of RasV12 cells by regulating protein kinase A. Furthermore, we have revealed that epithelial defense against cancer (EDAC) from normal epithelial cells substantially impacts on Rab5 accumulation in the neighboring transformed cells. This report demonstrates that Rab5-mediated endocytosis is a crucial regulator for the competitive interaction between normal and transformed epithelial cells in mammals.

Columnar cell lesions and pseudoangiomatous hyperplasia like stroma: is there an epithelial-stromal interaction?

PubMed

Recavarren, Rosemary A; Chivukula, Mamatha; Carter, Gloria; Dabbs, David J

2009-10-10

The significance of association between cancer and its microenvironment has been increasingly recognized. It has been shown in animal models that interaction between neoplastic epithelial cells and adjacent stroma can modulate tumor behavior. Carcinoma associated stromal cells can transform normal epithelial cells into neoplastic cells. In breast, columnar cell lesions are non-obligate precursors of low grade ductal carcinoma in situ. Columnar cell lesions can be seen intimately associated with PASH-like-stroma, a lesion we termed as CCPLS. Our aim is to investigate epithelial-stromal interactions in CCPLS and compare them to PASH without columnar cell lesions in breast core needle biopsies. Normal terminal duct lobular unit (TDLU) epithelium was seen in association with columnar cell lesions as well as PASH. Eight (8) cases of each category were examined by a panel of immunostains: CD117 (C-kit), CD34, CD105, bFGF, AR, ER-beta, MIB-1. We observed a markedly decreased expression of c-kit in columnar cell lesions compared to TDLU-epithelium. CD105 showed a quantitative increase in activated vessels in CCPLS compared to PASH. A subset of CCPLS and PASH were androgen receptor positive. A strong nuclear positivity for ER-beta is observed in the epithelium and stroma of all CCPLS cases. We conclude that (1) activated blood vessels predominate in CCPLS; (2) A molecular alteration is signified by c-kit loss in columnar cell lesions; (3) ER-beta and androgen receptor positivity indicate CCPLS are hormonally responsive lesions. Our study suggests an intimate vascular and hormone dependent epithelial-stromal interaction exists in CCPLS lesions.

Feedback amplification loop drives malignant growth in epithelial tissues.

PubMed

Muzzopappa, Mariana; Murcia, Lada; Milán, Marco

2017-08-29

Interactions between cells bearing oncogenic mutations and the surrounding microenvironment, and cooperation between clonally distinct cell populations, can contribute to the growth and malignancy of epithelial tumors. The genetic techniques available in Drosophila have contributed to identify important roles of the TNF-α ligand Eiger and mitogenic molecules in mediating these interactions during the early steps of tumor formation. Here we unravel the existence of a tumor-intrinsic-and microenvironment-independent-self-reinforcement mechanism that drives tumor initiation and growth in an Eiger-independent manner. This mechanism relies on cell interactions between two functionally distinct cell populations, and we present evidence that these cell populations are not necessarily genetically different. Tumor-specific and cell-autonomous activation of the tumorigenic JNK stress-activated pathway drives the expression of secreted signaling molecules and growth factors to delaminating cells, which nonautonomously promote proliferative growth of the partially transformed epithelial tissue. We present evidence that cross-feeding interactions between delaminating and nondelaminating cells increase each other's sizes and that these interactions can explain the unlimited growth potential of these tumors. Our results will open avenues toward our molecular understanding of those social cell interactions with a relevant function in tumor initiation in humans.

Activated fluid transport regulates bacterial-epithelial interactions and significantly shifts the murine colonic microbiome

PubMed Central

Keely, Simon; Kelly, Caleb J.; Weissmueller, Thomas; Burgess, Adrianne; Wagner, Brandie D.; Robertson, Charles E.; Harris, J. Kirk; Colgan, Sean P.

2012-01-01

Within the intestinal mucosa, epithelial cells serve multiple functions to partition the lumen from the lamina propria. As part of their natural function, intestinal epithelial cells actively transport electrolytes with passive water movement as a mechanism for mucosal hydration. Here, we hypothesized that electrogenic Cl- secretion, and associated mucosal hydration, influences bacterial-epithelial interactions and significantly influences the composition of the intestinal microbiota. An initial screen of different epithelial secretagogues identified lubiprostone as the most potent agonist for which to define these principles. In in vitro studies using cultured T84 cells, lubiprostone decreased E. coli translocation in a concentration-dependent manner (p < 0.001) and decreased S. typhimurium internalization and translocation by as much as 71 ± 6% (p < 0.01). Such decreases in bacterial translocation were abolished by inhibition of electrogenic Cl- secretion and water transport using the Na-K-Cl- antagonist bumetanide (p < 0.01). Extensions of these findings to microbiome analysis in vivo revealed that lubiprostone delivered orally to mice fundamentally shifted the intestinal microbiota, with notable changes within the Firmicutes and Bacteroidetes phyla of resident colonic bacteria. Such findings document a previously unappreciated role for epithelial Cl- secretion and water transport in influencing bacterial-epithelial interactions and suggest that active mucosal hydration functions as a primitive innate epithelial defense mechanism. PMID:22614705

Engineering epithelial-stromal interactions in vitro for toxicology assessment.

PubMed

Belair, David G; Abbott, Barbara D

2017-05-01

Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues. Published by Elsevier B.V.

Engineering epithelial-stromal interactions in vitro for toxicology assessment

PubMed Central

Belair, David G.; Abbott, Barbara D.

2018-01-01

Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues. PMID:28285100

Lung epithelial stem cells and their niches: Fgf10 takes center stage.

PubMed

Volckaert, Thomas; De Langhe, Stijn

2014-01-01

Throughout life adult animals crucially depend on stem cell populations to maintain and repair their tissues to ensure life-long organ function. Stem cells are characterized by their capacity to extensively self-renew and give rise to one or more differentiated cell types. These powerful stem cell properties are key to meet the changing demand for tissue replacement during normal lung homeostasis and regeneration after lung injury. Great strides have been made over the last few years to identify and characterize lung epithelial stem cells as well as their lineage relationships. Unfortunately, knowledge on what regulates the behavior and fate specification of lung epithelial stem cells is still limited, but involves communication with their microenvironment or niche, a local tissue environment that hosts and influences the behaviors or characteristics of stem cells and that comprises other cell types and extracellular matrix. As such, an intimate and dynamic epithelial-mesenchymal cross-talk, which is also essential during lung development, is required for normal homeostasis and to mount an appropriate regenerative response after lung injury. Fibroblast growth factor 10 (Fgf10) signaling in particular seems to be a well-conserved signaling pathway governing epithelial-mesenchymal interactions during lung development as well as between different adult lung epithelial stem cells and their niches. On the other hand, disruption of these reciprocal interactions leads to a dysfunctional epithelial stem cell-niche unit, which may culminate in chronic lung diseases such as chronic obstructive pulmonary disease (COPD), chronic asthma and idiopathic pulmonary fibrosis (IPF).

Characterization and zoonotic potential of uropathogenic Escherichia coli isolated from dogs.

PubMed

Nam, Eui-Hwa; Ko, Sungjin; Chae, Joon-Seok; Hwang, Cheol-Yong

2013-03-01

The aim of this study was to investigate the characteristics of canine uropathogenic Escherichia coli (UPEC) and the interaction between canine UPEC and human bladder epithelial cells. Ten E. coli isolates collected from dogs with cystitis were analyzed for antimicrobial resistance patterns, the presence of virulence factors, and biofilm formation. The ability of these isolates to induce cytotoxicity, invade human bladder epithelial cells, and stimulate an immune response was also determined. We observed a high rate of antimicrobial resistance among canine UPEC isolates. All virulence genes tested (including adhesins, iron acquisition, and protectin), except toxin genes, were detected among the canine UPEC isolates. We found that all isolates showed varying degrees of biofilm formation (mean, 0.26; range, 0.07 to 0.82), using a microtiter plate assay to evaluate biofilm formation by the isolates. Cytotoxicity to human bladder epithelial cells by the canine UPEC isolates increased in a time-dependent manner, with a 56.9% and 36.1% reduction in cell viability compared with the control at 6 and 9 h of incubation, respectively. We found that most canine UPEC isolates were able to invade human bladder epithelial cells. The interaction between these isolates and human bladder epithelial cells strongly induced the production of proinflammatory cytokines such as IL-6 and IL-8. We demonstrated that canine UPEC isolates can interact with human bladder epithelial cells, although the detailed mechanisms remain unknown. The results suggest that canine UPEC isolates, rather than dogspecific pathogens, have zoonotic potential.

Interaction of resident sperm with sperm-storage tubule (SST) epithelial cell microvilli in the turkey breeder hen

USDA-ARS?s Scientific Manuscript database

Interaction of resident sperm with sperm-storage tubule (SST) epithelial cell microvilli in the turkey breeder hen M.R. Bakst*1 and C. Murphy2, 1Animal Biosciences and Biotechnology Laboratory, 2Electron & Confocal Microscopy Unit, Beltsville Area, ARS, USDA, Beltsville MD Sustained fertilization o...

Interaction of Mycobacterium leprae with Human Airway Epithelial Cells: Adherence, Entry, Survival, and Identification of Potential Adhesins by Surface Proteome Analysis

PubMed Central

Silva, Carlos A. M.; Danelishvili, Lia; McNamara, Michael; Berredo-Pinho, Márcia; Bildfell, Robert; Biet, Franck; Rodrigues, Luciana S.; Oliveira, Albanita V.

2013-01-01

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control. PMID:23670556

Structure of neuro-endocrine and neuro-epithelial interactions in human foetal pancreas.

PubMed

Krivova, Yuliya; Proshchina, Alexandra; Barabanov, Valeriy; Leonova, Olga; Saveliev, Sergey

2016-12-01

In the pancreas of many mammals including humans, endocrine islet cells can be integrated with the nervous system components into neuro-insular complexes. The mechanism of the formation of such complexes is not clearly understood. The present study evaluated the interactions between the nervous system components, epithelial cells and endocrine cells in the human pancreas. Foetal pancreas, gestational age 19-23 weeks (13 cases) and 30-34 weeks (7 cases), were studied using double immunohistochemical labeling with neural markers (S100 protein and beta III tubulin), epithelial marker (cytokeratin 19 (CK19)) and antibodies to insulin and glucagon. We first analyse the structure of neuro-insular complexes using confocal microscopy and provide immunohistochemical evidences of the presence of endocrine cells within the ganglia or inside the nerve bundles. We showed that the nervous system components contact with the epithelial cells located in ducts or in clusters outside the ductal epithelium and form complexes with separate epithelial cells. We observed CK19-positive cells inside the ganglia and nerve bundles which were located separately or were integrated with the islets. Therefore, we conclude that neuro-insular complexes may forms as a result of integration between epithelial cells and nervous system components at the initial stages of islets formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in human epithelial cancer cells.

PubMed

Piyush, Tushar; Chacko, Anisha R; Sindrewicz, Paulina; Hilkens, John; Rhodes, Jonathan M; Yu, Lu-Gang

2017-11-01

Epidermal growth factor receptor (EGFR) is an important regulator of epithelial cell growth and survival in normal and cancerous tissues and is a principal therapeutic target for cancer treatment. EGFR is associated in epithelial cells with the heavily glycosylated transmembrane mucin protein MUC1, a natural ligand of galectin-3 that is overexpressed in cancer. This study reveals that the expression of cell surface MUC1 is a critical enhancer of EGF-induced EGFR activation in human breast and colon cancer cells. Both the MUC1 extracellular and intracellular domains are involved in EGFR activation but the predominant influence comes from its extracellular domain. Binding of galectin-3 to the MUC1 extracellular domain induces MUC1 cell surface polarization and increases MUC1-EGFR association. This leads to a rapid increase of EGFR homo-/hetero-dimerization and subsequently increased, and also prolonged, EGFR activation and signalling. This effect requires both the galectin-3 C-terminal carbohydrate recognition domain and its N-terminal ligand multi-merization domain. Thus, interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in epithelial cancer cells. As MUC1 and galectin-3 are both commonly overexpressed in most types of epithelial cancers, their interaction and impact on EGFR activation likely makes important contribution to EGFR-associated tumorigenesis and cancer progression and may also influence the effectiveness of EGFR-targeted cancer therapy.

Engineering Functional Epithelium for Regenerative Medicine and In Vitro Organ Models: A Review

PubMed Central

Vrana, Nihal E.; Lavalle, Philippe; Dokmeci, Mehmet R.; Dehghani, Fariba; Ghaemmaghami, Amir M.

2013-01-01

Recent advances in the fields of microfabrication, biomaterials, and tissue engineering have provided new opportunities for developing biomimetic and functional tissues with potential applications in disease modeling, drug discovery, and replacing damaged tissues. An intact epithelium plays an indispensable role in the functionality of several organs such as the trachea, esophagus, and cornea. Furthermore, the integrity of the epithelial barrier and its degree of differentiation would define the level of success in tissue engineering of other organs such as the bladder and the skin. In this review, we focus on the challenges and requirements associated with engineering of epithelial layers in different tissues. Functional epithelial layers can be achieved by methods such as cell sheets, cell homing, and in situ epithelialization. However, for organs composed of several tissues, other important factors such as (1) in vivo epithelial cell migration, (2) multicell-type differentiation within the epithelium, and (3) epithelial cell interactions with the underlying mesenchymal cells should also be considered. Recent successful clinical trials in tissue engineering of the trachea have highlighted the importance of a functional epithelium for long-term success and survival of tissue replacements. Hence, using the trachea as a model tissue in clinical use, we describe the optimal structure of an artificial epithelium as well as challenges of obtaining a fully functional epithelium in macroscale. One of the possible remedies to address such challenges is the use of bottom-up fabrication methods to obtain a functional epithelium. Modular approaches for the generation of functional epithelial layers are reviewed and other emerging applications of microscale epithelial tissue models for studying epithelial/mesenchymal interactions in healthy and diseased (e.g., cancer) tissues are described. These models can elucidate the epithelial/mesenchymal tissue interactions at the microscale and provide the necessary tools for the next generation of multicellular engineered tissues and organ-on-a-chip systems. PMID:23705900

Engineering functional epithelium for regenerative medicine and in vitro organ models: a review.

PubMed

Vrana, Nihal E; Lavalle, Philippe; Dokmeci, Mehmet R; Dehghani, Fariba; Ghaemmaghami, Amir M; Khademhosseini, Ali

2013-12-01

Recent advances in the fields of microfabrication, biomaterials, and tissue engineering have provided new opportunities for developing biomimetic and functional tissues with potential applications in disease modeling, drug discovery, and replacing damaged tissues. An intact epithelium plays an indispensable role in the functionality of several organs such as the trachea, esophagus, and cornea. Furthermore, the integrity of the epithelial barrier and its degree of differentiation would define the level of success in tissue engineering of other organs such as the bladder and the skin. In this review, we focus on the challenges and requirements associated with engineering of epithelial layers in different tissues. Functional epithelial layers can be achieved by methods such as cell sheets, cell homing, and in situ epithelialization. However, for organs composed of several tissues, other important factors such as (1) in vivo epithelial cell migration, (2) multicell-type differentiation within the epithelium, and (3) epithelial cell interactions with the underlying mesenchymal cells should also be considered. Recent successful clinical trials in tissue engineering of the trachea have highlighted the importance of a functional epithelium for long-term success and survival of tissue replacements. Hence, using the trachea as a model tissue in clinical use, we describe the optimal structure of an artificial epithelium as well as challenges of obtaining a fully functional epithelium in macroscale. One of the possible remedies to address such challenges is the use of bottom-up fabrication methods to obtain a functional epithelium. Modular approaches for the generation of functional epithelial layers are reviewed and other emerging applications of microscale epithelial tissue models for studying epithelial/mesenchymal interactions in healthy and diseased (e.g., cancer) tissues are described. These models can elucidate the epithelial/mesenchymal tissue interactions at the microscale and provide the necessary tools for the next generation of multicellular engineered tissues and organ-on-a-chip systems.

The world of epithelial sheets.

PubMed

Honda, Hisao

2017-06-01

An epithelium is a layer of closely connected cells covering the body or lining a body cavity. In this review, several fundamental questions are addressed regarding the epithelium. (i) While an epithelium functions as barrier against the external environment, how is barrier function maintained during its construction? (ii) What determines the apical and basal sides of epithelial layer? (iii) Is there any relationship between the apical side of the epithelium and the apical membrane of an epithelial cell? (iv) Why are hepatocytes (liver cells) called epithelial, even though they differ completely from column-like shape of typical epithelial cells? Keeping these questions in mind, multiple shapes of epithelia were considered, extracting a few of their elemental processes, and constructing a virtual world of epithelia by combining them. Epithelial cells were also classified into several types based on the number of apical domains of each cell. In addition, an intracellular organelle was introduced within epithelial cells, the vacuolar apical compartment (VAC), which is produced within epithelial cells surrounded by external cell matrix (ECM). The VAC interacts with areas of cell-cell contact of the cell surface membrane and is converted to apical membrane. The properties of VACs enable us to answer the initial questions posed above. Finally, the genetic and molecular mechanisms of epithelial morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

Interaction between Fibronectin and β1 Integrin Is Essential for Tooth Development

PubMed Central

Yamada, Aya; Yuasa, Kenji; Yoshizaki, Keigo; Iwamoto, Tsutomu; Saito, Masahiro; Nakamura, Takashi; Fukumoto, Satoshi

2015-01-01

The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fibronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ1lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation. PMID:25830530

Interactions of Aspergillus fumigatus Conidia with Airway Epithelial Cells: A Critical Review

PubMed Central

Croft, Carys A.; Culibrk, Luka; Moore, Margo M.; Tebbutt, Scott J.

2016-01-01

Aspergillus fumigatus is an environmental filamentous fungus that also acts as an opportunistic pathogen able to cause a variety of symptoms, from an allergic response to a life-threatening disseminated fungal infection. The infectious agents are inhaled conidia whose first point of contact is most likely to be an airway epithelial cell (AEC). The interaction between epithelial cells and conidia is multifaceted and complex, and has implications for later steps in pathogenesis. Increasing evidence has demonstrated a key role for the airway epithelium in the response to respiratory pathogens, particularly at early stages of infection; therefore, elucidating the early stages of interaction of conidia with AECs is essential to understand the establishment of infection in cohorts of at-risk patients. Here, we present a comprehensive review of the early interactions between A. fumigatus and AECs, including bronchial and alveolar epithelial cells. We describe mechanisms of adhesion, internalization of conidia by AECs, the immune response of AECs, as well as the role of fungal virulence factors, and patterns of fungal gene expression characteristic of early infection. A clear understanding of the mechanisms involved in the early establishment of infection by A. fumigatus could point to novel targets for therapy and prophylaxis. PMID:27092126

Diverse Profiles of Ricin-Cell Interactions in the Lung Following Intranasal Exposure to Ricin

PubMed Central

Sapoznikov, Anita; Falach, Reut; Mazor, Ohad; Alcalay, Ron; Gal, Yoav; Seliger, Nehama; Sabo, Tamar; Kronman, Chanoch

2015-01-01

Ricin, a plant-derived exotoxin, inhibits protein synthesis by ribosomal inactivation. Due to its wide availability and ease of preparation, ricin is considered a biothreat, foremost by respiratory exposure. We examined the in vivo interactions between ricin and cells of the lungs in mice intranasally exposed to the toxin and revealed multi-phasic cell-type-dependent binding profiles. While macrophages (MΦs) and dendritic cells (DCs) displayed biphasic binding to ricin, monophasic binding patterns were observed for other cell types; epithelial cells displayed early binding, while B cells and endothelial cells bound toxin late after intoxication. Neutrophils, which were massively recruited to the intoxicated lung, were refractive to toxin binding. Although epithelial cells bound ricin as early as MΦs and DCs, their rates of elimination differed considerably; a reduction in epithelial cell counts occurred late after intoxication and was restricted to alveolar type II cells only. The differential binding and cell-elimination patterns observed may stem from dissimilar accessibility of the toxin to different cells in the lung and may also reflect unequal interactions of the toxin with different cell-surface receptors. The multifaceted interactions observed in this study between ricin and the various cells of the target organ should be considered in the future development of efficient post-exposure countermeasures against ricin intoxication. PMID:26593946

Diverse profiles of ricin-cell interactions in the lung following intranasal exposure to ricin.

PubMed

Sapoznikov, Anita; Falach, Reut; Mazor, Ohad; Alcalay, Ron; Gal, Yoav; Seliger, Nehama; Sabo, Tamar; Kronman, Chanoch

2015-11-17

Ricin, a plant-derived exotoxin, inhibits protein synthesis by ribosomal inactivation. Due to its wide availability and ease of preparation, ricin is considered a biothreat, foremost by respiratory exposure. We examined the in vivo interactions between ricin and cells of the lungs in mice intranasally exposed to the toxin and revealed multi-phasic cell-type-dependent binding profiles. While macrophages (MΦs) and dendritic cells (DCs) displayed biphasic binding to ricin, monophasic binding patterns were observed for other cell types; epithelial cells displayed early binding, while B cells and endothelial cells bound toxin late after intoxication. Neutrophils, which were massively recruited to the intoxicated lung, were refractive to toxin binding. Although epithelial cells bound ricin as early as MΦs and DCs, their rates of elimination differed considerably; a reduction in epithelial cell counts occurred late after intoxication and was restricted to alveolar type II cells only. The differential binding and cell-elimination patterns observed may stem from dissimilar accessibility of the toxin to different cells in the lung and may also reflect unequal interactions of the toxin with different cell-surface receptors. The multifaceted interactions observed in this study between ricin and the various cells of the target organ should be considered in the future development of efficient post-exposure countermeasures against ricin intoxication.

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

PubMed Central

Baranwal, Somesh

2015-01-01

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. PMID:25792565

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

PubMed

Lechuga, Susana; Baranwal, Somesh; Ivanov, Andrei I

2015-05-01

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. Copyright © 2015 the American Physiological Society.

Obesity Suppresses Cell-Competition-Mediated Apical Elimination of RasV12-Transformed Cells from Epithelial Tissues.

PubMed

Sasaki, Ayana; Nagatake, Takahiro; Egami, Riku; Gu, Guoqiang; Takigawa, Ichigaku; Ikeda, Wataru; Nakatani, Tomoya; Kunisawa, Jun; Fujita, Yasuyuki

2018-04-24

Recent studies have revealed that newly emerging transformed cells are often eliminated from epithelial tissues via cell competition with the surrounding normal epithelial cells. This cancer preventive phenomenon is termed epithelial defense against cancer (EDAC). However, it remains largely unknown whether and how EDAC is diminished during carcinogenesis. In this study, using a cell competition mouse model, we show that high-fat diet (HFD) feeding substantially attenuates the frequency of apical elimination of RasV12-transformed cells from intestinal and pancreatic epithelia. This process involves both lipid metabolism and chronic inflammation. Furthermore, aspirin treatment significantly facilitates eradication of transformed cells from the epithelial tissues in HFD-fed mice. Thus, our work demonstrates that obesity can profoundly influence competitive interaction between normal and transformed cells, providing insights into cell competition and cancer preventive medicine. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Proteomic analysis of protein interactions between Eimeria maxima sporozoites and chicken jejunal epithelial cells by shotgun LC-MS/MS.

PubMed

Huang, Jingwei; Liu, Tingqi; Li, Ke; Song, Xiaokai; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

2018-04-04

Eimeria maxima initiates infection by invading the jejunal epithelial cells of chicken. However, the proteins involved in invasion remain unknown. The research of the molecules that participate in the interactions between E. maxima sporozoites and host target cells will fill a gap in our understanding of the invasion system of this parasitic pathogen. In the present study, chicken jejunal epithelial cells were isolated and cultured in vitro. Western blot was employed to analyze the soluble proteins of E. maxima sporozoites that bound to chicken jejunal epithelial cells. Co-immunoprecipitation (co-IP) assay was used to separate the E. maxima proteins that bound to chicken jejunal epithelial cells. Shotgun LC-MS/MS technique was used for proteomics identification and Gene Ontology was employed for the bioinformatics analysis. The results of Western blot analysis showed that four proteins bands from jejunal epithelial cells co-cultured with soluble proteins of E. maxima sporozoites were recognized by the positive sera, with molecular weights of 70, 90, 95 and 130 kDa. The co-IP dilutions were analyzed by shotgun LC-MS/MS. A total of 204 proteins were identified in the E. maxima protein database using the MASCOT search engine. Thirty-five proteins including microneme protein 3 and 7 had more than two unique peptide counts and were annotated using Gene Ontology for molecular function, biological process and cellular localization. The results revealed that of the 35 annotated peptides, 22 (62.86%) were associated with binding activity and 15 (42.86%) were involved in catalytic activity. Our findings provide an insight into the interaction between E. maxima and the corresponding host cells and it is important for the understanding of molecular mechanisms underlying E. maxima invasion.

Interactions between Exosomes from Breast Cancer Cells and Primary Mammary Epithelial Cells Leads to Generation of Reactive Oxygen Species Which Induce DNA Damage Response, Stabilization of p53 and Autophagy in Epithelial Cells

PubMed Central

Dutta, Sujoy; Warshall, Case; Bandyopadhyay, Chirosree; Dutta, Dipanjan; Chandran, Bala

2014-01-01

Exosomes are nanovesicles originating from multivesicular bodies and are released by all cell types. They contain proteins, lipids, microRNAs, mRNAs and DNA fragments, which act as mediators of intercellular communications by inducing phenotypic changes in recipient cells. Tumor-derived exosomes have been shown to play critical roles in different stages of tumor development and metastasis of almost all types of cancer. One of the ways by which exosomes affect tumorigenesis is to manipulate the tumor microenvironments to create tumor permissive “niches”. Whether breast cancer cell secreted exosomes manipulate epithelial cells of the mammary duct to facilitate tumor development is not known. To address whether and how breast cancer cell secreted exosomes manipulate ductal epithelial cells we studied the interactions between exosomes isolated from conditioned media of 3 different breast cancer cell lines (MDA-MB-231, T47DA18 and MCF7), representing three different types of breast carcinomas, and normal human primary mammary epithelial cells (HMECs). Our studies show that exosomes released by breast cancer cell lines are taken up by HMECs, resulting in the induction of reactive oxygen species (ROS) and autophagy. Inhibition of ROS by N-acetyl-L-cysteine (NAC) led to abrogation of autophagy. HMEC-exosome interactions also induced the phosphorylation of ATM, H2AX and Chk1 indicating the induction of DNA damage repair (DDR) responses. Under these conditions, phosphorylation of p53 at serine 15 was also observed. Both DDR responses and phosphorylation of p53 induced by HMEC-exosome interactions were also inhibited by NAC. Furthermore, exosome induced autophagic HMECs were found to release breast cancer cell growth promoting factors. Taken together, our results suggest novel mechanisms by which breast cancer cell secreted exosomes manipulate HMECs to create a tumor permissive microenvironment. PMID:24831807

Epithelial GM-CSF induction by Candida glabrata.

PubMed

Li, L; Dongari-Bagtzoglou, A

2009-08-01

The main cytokine induced by the interaction of oral epithelial cells with C. glabrata is granulocyte monocyte colony-stimulating factor (GM-CSF); however, the mechanisms regulating this response are unknown. Based on previously published information on the interactions of C. albicans with oral epithelial cells, we hypothesized that interaction with viable C. glabrata triggers GM-CSF synthesis via NF-kappaB activation. We found that C. glabrata-induced GM-CSF synthesis was adhesion-dependent, enhanced by endocytosis, and required fungal viability. NF-kappaB activation was noted during interaction of epithelial cells with C. glabrata, and pre-treatment with an NF-kappaB inhibitor partly inhibited GM-CSF synthesis. Blocking TLR4 with anti-TLR4 antibody did not inhibit GM-CSF production. In contrast, an anti-CDw17 antibody triggered significant inhibition of NF-kappaB activation and GM-CSF synthesis. beta-glucans did not stimulate GM-CSF synthesis, suggesting that the CDw17/NF-kappaB/GM-CSF pathway may be beta-glucan-independent. This study provides new insights into the mechanism of GM-CSF induction by C. glabrata.

Zinc transport by respiratory epithelial cells and interaction with iron homeostasis.

PubMed

Deng, Zhongping; Dailey, Lisa A; Soukup, Joleen; Stonehuerner, Jacqueline; Richards, Judy D; Callaghan, Kimberly D; Yang, Funmei; Ghio, Andrew J

2009-10-01

Despite recurrent exposure to zinc through inhalation of ambient air pollution particles, relatively little information is known about the homeostasis of this metal in respiratory epithelial cells. We describe zinc uptake and release by respiratory epithelial cells and test the postulate that Zn(2+) transport interacts with iron homeostasis in these same cells. Zn(2+) uptake after 4 and 8 h of exposure to zinc sulfate was concentration- and time-dependent. A majority of Zn(2+) release occurred in the 4 h immediately following cell exposure to ZnSO(4). Regarding metal importers, mRNA for Zip1 and Zip2 showed no change after respiratory epithelial cell exposure to zinc while mRNA for divalent metal transporter (DMT)1 increased. Western blot assay for DMT1 protein supported an elevated expression of this transport protein following zinc exposure. RT-PCR confirmed mRNA for the metal exporters ZnT1 and ZnT4 with the former increasing after ZnSO(4). Cell concentrations of ferritin increased with zinc exposure while oxidative stress, measured as lipid peroxides, was decreased supporting an anti-oxidant function for Zn(2+). Increased DMT1 expression, following pre-incubations of respiratory epithelial cells with TNF-alpha, IFN-gamma, and endotoxin, was associated with significantly decreased intracellular zinc transport. Finally, incubations of respiratory epithelial cells with both zinc sulfate and ferric ammonium citrate resulted in elevated intracellular concentrations of both metals. We conclude that exposure to zinc increases iron uptake by respiratory epithelial cells. Elevations in cell iron can possibly affect an increased expression of DMT1 and ferritin which function to diminish oxidative stress. Comparable to other metal exposures, changes in iron homeostasis may contribute to the biological effects of zinc in specific cells and tissues.

Isolation and Quantitative Estimation of Diesel Exhaust and Carbon Black Particles Ingested by Lung Epithelial Cells and Alveolar Macrophages In Vitro

EPA Science Inventory

A new procedure for isolating and estimating ingested carbonaceous diesel exhaust particles (DEP) or carbon black (CB) particles by lung epithelial cells and macrophages is described. Cells were incubated with DEP or CB to examine cell-particle interaction and ingestion. After va...

Mechanisms of Disease: Host-Pathogen Interactions between Burkholderia Species and Lung Epithelial Cells

PubMed Central

David, Jonathan; Bell, Rachel E.; Clark, Graeme C.

2015-01-01

Members of the Burkholderia species can cause a range of severe, often fatal, respiratory diseases. A variety of in vitro models of infection have been developed in an attempt to elucidate the mechanism by which Burkholderia spp. gain entry to and interact with the body. The majority of studies have tended to focus on the interaction of bacteria with phagocytic cells with a paucity of information available with regard to the lung epithelium. However, the lung epithelium is becoming more widely recognized as an important player in innate immunity and the early response to infections. Here we review the complex relationship between Burkholderia species and epithelial cells with an emphasis on the most pathogenic species, Burkholderia pseudomallei and Burkholderia mallei. The current gaps in knowledge in our understanding are highlighted along with the epithelial host-pathogen interactions that offer potential opportunities for therapeutic intervention. PMID:26636042

A dynamic cellular vertex model of growing epithelial tissues

NASA Astrophysics Data System (ADS)

Lin, Shao-Zhen; Li, Bo; Feng, Xi-Qiao

2017-04-01

Intercellular interactions play a significant role in a wide range of biological functions and processes at both the cellular and tissue scales, for example, embryogenesis, organogenesis, and cancer invasion. In this paper, a dynamic cellular vertex model is presented to study the morphomechanics of a growing epithelial monolayer. The regulating role of stresses in soft tissue growth is revealed. It is found that the cells originating from the same parent cell in the monolayer can orchestrate into clustering patterns as the tissue grows. Collective cell migration exhibits a feature of spatial correlation across multiple cells. Dynamic intercellular interactions can engender a variety of distinct tissue behaviors in a social context. Uniform cell proliferation may render high and heterogeneous residual compressive stresses, while stress-regulated proliferation can effectively release the stresses, reducing the stress heterogeneity in the tissue. The results highlight the critical role of mechanical factors in the growth and morphogenesis of epithelial tissues and help understand the development and invasion of epithelial tumors.

Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellular interferon-gamma (IFN-γ) in peripheral CD8+ lymphocytes co-cultured with intestinal epithelial cells

PubMed Central

Bisping, G; Lügering, N; Lütke-Brintrup, S; Pauels, H -G; Schürmann, G; Domschke, W; Kucharzik, T

2001-01-01

Intestinal epithelial cells seem to play a key role during IBD. The network of cellular interactions between epithelial cells and lamina propria mononuclear cells is still incompletely understood. In the following co-culture model we investigated the influence of intestinal epithelial cells on cytokine expression of T cytotoxic and T helper cells from patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMC) were purified by a Ficoll–Hypaque gradient followed by co-incubation with epithelial cells in multiwell cell culture insert plates in direct contact as well as separated by transwell filters. We used Caco-2 cells as well as freshly isolated colonic epithelia obtained from surgical specimens. Three-colour immunofluorescence flow cytometry was performed after collection, stimulation and staining of PBMC with anti-CD4, anti-CD8, anti-IFN-γ and anti-IL-4. Patients with IBD (Crohn's disease (CD), n = 12; ulcerative colitis (UC), n = 16) and healthy controls (n = 10) were included in the study. After 24 h of co-incubation with Caco-2 cells we found a significant increase of IFN-γ-producing CD8+ lymphocytes in patients with IBD. In contrast, healthy controls did not respond to the epithelial stimulus. No significant differences could be found between CD and UC or active and inactive disease. A significant increase of IFN-γ+/CD8+ lymphocytes in patients with UC was also seen after direct co-incubation with primary cultures of colonic crypt cells. The observed epithelial–lymphocyte interaction seems to be MHC I-restricted. No significant epithelial cell-mediated effects on cytokine expression were detected in the PBMC CD4+ subsets. Patients with IBD—even in an inactive state of disease—exert an increased capacity for IFN-γ induction in CD8+ lymphocytes mediated by intestinal epithelial cells. This mechanism may be important during chronic intestinal inflammation, as in the case of altered mucosal barrier function epithelial cells may become targets for IFN-γ-producing CD8+ lymphocytes. PMID:11167992

Epithelial-stromal interaction via Notch signaling is essential for the full maturation of gut-associated lymphoid tissues.

PubMed

Obata, Yuuki; Kimura, Shunsuke; Nakato, Gaku; Iizuka, Keito; Miyagawa, Yurika; Nakamura, Yutaka; Furusawa, Yukihiro; Sugiyama, Machiko; Suzuki, Keiichiro; Ebisawa, Masashi; Fujimura, Yumiko; Yoshida, Hisahiro; Iwanaga, Toshihiko; Hase, Koji; Ohno, Hiroshi

2014-12-01

Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpj-a gene encoding a transcription factor implicated in Notch signaling-in intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE. © 2014 The Authors.

β2 adrenergic agonist suppresses eosinophil-induced epithelial-to-mesenchymal transition of bronchial epithelial cells.

PubMed

Kainuma, Keigo; Kobayashi, Tetsu; D'Alessandro-Gabazza, Corina N; Toda, Masaaki; Yasuma, Taro; Nishihama, Kota; Fujimoto, Hajime; Kuwabara, Yu; Hosoki, Koa; Nagao, Mizuho; Fujisawa, Takao; Gabazza, Esteban C

2017-05-02

Epithelial-mesenchymal transition is currently recognized as an important mechanism for the increased number of myofibroblasts in cancer and fibrotic diseases. We have already reported that epithelial-mesenchymal transition is involved in airway remodeling induced by eosinophils. Procaterol is a selective and full β 2 adrenergic agonist that is used as a rescue of asthmatic attack inhaler form and orally as a controller. In this study, we evaluated whether procaterol can suppress epithelial-mesenchymal transition of airway epithelial cells induced by eosinophils. Epithelial-mesenchymal transition was assessed using a co-culture system of human bronchial epithelial cells and primary human eosinophils or an eosinophilic leukemia cell line. Procaterol significantly inhibited co-culture associated morphological changes of bronchial epithelial cells, decreased the expression of vimentin, and increased the expression of E-cadherin compared to control. Butoxamine, a specific β 2 -adrenergic antagonist, significantly blocked changes induced by procaterol. In addition, procaterol inhibited the expression of adhesion molecules induced during the interaction between eosinophils and bronchial epithelial cells, suggesting the involvement of adhesion molecules in the process of epithelial-mesenchymal transition. Forskolin, a cyclic adenosine monophosphate-promoting agent, exhibits similar inhibitory activity of procaterol. Overall, these observations support the beneficial effect of procaterol on airway remodeling frequently associated with chronic obstructive pulmonary diseases.

The epithelial-mesenchymal interactions: insights into physiological and pathological aspects of oral tissues.

PubMed

Santosh, Arvind Babu Rajendra; Jones, Thaon Jon

2014-03-17

In the human biological system, the individual cells divide and form tissues and organs. These tissues are hetero-cellular. Basically any tissue consists of an epithelium and the connective tissue. The latter contains mainly mesenchymally-derived tissues with a diversified cell population. The cell continues to grow and differentiate in a pre-programmed manner using a messenger system. The epithelium and the mesenchymal portion of each tissue have two different origins and perform specific functions, but there is a well-defined interaction mechanism, which mediates between them. Epithelial mesenchymal interactions (EMIs) are part of this mechanism, which can be regarded as a biological conversation between epithelial and mesenchymal cell populations involved in the cellular differentiation of one or both cell populations. EMIs represent a process that is essential for cell growth, cell differentiation and cell multiplication. EMIs are associated with normal physiological processes in the oral cavity, such as odontogenesis, dentino-enamel junction formation, salivary gland development, palatogenesis, and also pathological processes, such as oral cancer. This paper focuses the role EMIs in odontogenesis, salivary gland development, palatogenesis and oral cancer.

The adaptor protein Cindr regulates JNK activity to maintain epithelial sheet integrity.

PubMed

Yasin, Hannah W R; van Rensburg, Samuel H; Feiler, Christina E; Johnson, Ruth I

2016-02-15

Epithelia are essential barrier tissues that must be appropriately maintained for their correct function. To achieve this a plethora of protein interactions regulate epithelial cell number, structure and adhesion, and differentiation. Here we show that Cindr (the Drosophila Cin85 and Cd2ap ortholog) is required to maintain epithelial integrity. Reducing Cindr triggered cell delamination and movement. Most delaminating cells died. These behaviors were consistent with JNK activation previously associated with loss of epithelial integrity in response to ectopic oncogene activity. We confirmed a novel interaction between Cindr and Drosophila JNK (dJNK), which when perturbed caused inappropriate JNK signaling. Genetically reducing JNK signaling activity suppressed the effects of reducing Cindr. Furthermore, ectopic JNK signaling phenocopied loss of Cindr and was partially rescued by concomitant cindr over-expression. Thus, correct Cindr-dJNK stoichiometry is essential to maintain epithelial integrity and disturbing this balance may contribute to the pathogenesis of disease states, including cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

TGFβ loss activates ADAMTS-1-mediated EGF-dependent invasion in a model of esophageal cell invasion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Le Bras, Grégoire F.; Taylor, Chase; Koumangoye, Rainelli B.

2015-01-01

The TGFβ signaling pathway is essential to epithelial homeostasis and is often inhibited during progression of esophageal squamous cell carcinoma. Recently, an important role for TGFβ signaling has been described in the crosstalk between epithelial and stromal cells regulating squamous tumor cell invasion in mouse models of head-and-neck squamous cell carcinoma (HNSCC). Loss of TGFβ signaling, in either compartment, leads to HNSCC however, the mechanisms involved are not well understood. Using organotypic reconstruct cultures (OTC) to model the interaction between epithelial and stromal cells that occur in dysplastic lesions, we show that loss of TGFβ signaling promotes an invasive phenotypemore » in both fibroblast and epithelial compartments. Employing immortalized esophageal keratinocytes established to reproduce common mutations of esophageal squamous cell carcinoma, we show that treatment of OTC with inhibitors of TGFβ signaling (A83-01 or SB431542) enhances invasion of epithelial cells into a fibroblast-embedded Matrigel/collagen I matrix. Invasion induced by A83-01 is independent of proliferation but relies on protease activity and expression of ADAMTS-1 and can be altered by matrix density. This invasion was associated with increased expression of pro-inflammatory cytokines, IL1 and EGFR ligands HB-EGF and TGFα. Altering EGF signaling prevented or induced epithelial cell invasion in this model. Loss of expression of the TGFβ target gene ROBO1 suggested that chemorepulsion may regulate keratinocyte invasion. Taken together, our data show increased invasion through inhibition of TGFβ signaling altered epithelial-fibroblasts interactions, repressing markers of activated fibroblasts, and altering integrin-fibronectin interactions. These results suggest that inhibition of TGFβ signaling modulates an array of pathways that combined promote multiple aspects of tumor invasion. - Highlights: • Chemical inhibition of TGFβ signaling advances collective invasion of esophageal keratinocytes. • Collective cell invasion is protease dependent and ADAMTS-1 is upregulated. • Inhibition of TGFβ signaling promotes EGF-mediated invasion. • Loss of TGFβ signaling promotes collagen binding and induces alterations in integrin expression. • The TGFβ target ROBO1 is downregulated in cell invasion implying mechanisms of chemorepulsion.« less

Association of Cell Surface Mucins with Galectin-3 Contributes to the Ocular Surface Epithelial Barrier*

PubMed Central

Argüeso, Pablo; Guzman-Aranguez, Ana; Mantelli, Flavio; Cao, Zhiyi; Ricciuto, Jessica; Panjwani, Noorjahan

2009-01-01

Maintenance of an intact mucosal barrier is critical to preventing damage to and infection of wet-surfaced epithelia. The mechanism of defense has been the subject of much investigation, and there is evidence now implicating O-glycosylated mucins on the epithelial cell surface. Here we investigate a new role for the carbohydrate-binding protein galectin-3 in stabilizing mucosal barriers through its interaction with mucins on the apical glycocalyx. Using the surface of the eye as a model system, we found that galectin-3 colocalized with two distinct membrane-associated mucins, MUC1 and MUC16, on the apical surface of epithelial cells and that both mucins bound to galectin-3 affinity columns in a galactose-dependent manner. Abrogation of the mucin-galectin interaction in four different mucosal epithelial cell types using competitive carbohydrate inhibitors of galectin binding, β-lactose and modified citrus pectin, resulted in decreased levels of galectin-3 on the cell surface with concomitant loss of barrier function, as indicated by increased permeability to rose bengal diagnostic dye. Similarly, down-regulation of mucin O-glycosylation using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1 (T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, resulted in decreased cell surface O-glycosylation, reduced cell surface galectin-3, and increased epithelial permeability. Taken together, these results suggest that galectin-3 plays a key role in maintaining mucosal barrier function through carbohydrate-dependent interactions with cell surface mucins. PMID:19556244

Type I IFN triggers RIG-I/TLR3/NLRP3-dependent inflammasome activation in influenza A virus infected cells.

PubMed

Pothlichet, Julien; Meunier, Isabelle; Davis, Beckley K; Ting, Jenny P-Y; Skamene, Emil; von Messling, Veronika; Vidal, Silvia M

2013-01-01

Influenza A virus (IAV) triggers a contagious and potentially lethal respiratory disease. A protective IL-1β response is mediated by innate receptors in macrophages and lung epithelial cells. NLRP3 is crucial in macrophages; however, which sensors elicit IL-1β secretion in lung epithelial cells remains undetermined. Here, we describe for the first time the relative roles of the host innate receptors RIG-I (DDX58), TLR3, and NLRP3 in the IL-1β response to IAV in primary lung epithelial cells. To activate IL-1β secretion, these cells employ partially redundant recognition mechanisms that differ from those described in macrophages. RIG-I had the strongest effect through a MAVS/TRIM25/Riplet-dependent type I IFN signaling pathway upstream of TLR3 and NLRP3. Notably, RIG-I also activated the inflammasome through interaction with caspase 1 and ASC in primary lung epithelial cells. Thus, NS1, an influenza virulence factor that inhibits the RIG-I/type I IFN pathway, strongly modulated the IL-1β response in lung epithelial cells and in ferrets. The NS1 protein derived from a highly pathogenic strain resulted in increased interaction with RIG-I and inhibited type I IFN and IL-1β responses compared to the least pathogenic virus strains. These findings demonstrate that in IAV-infected lung epithelial cells RIG-I activates the inflammasome both directly and through a type I IFN positive feedback loop.

Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells.

PubMed

de Bessa Garcia, Simone A; Pereira, Michelly C; Nagai, Maria A

2010-12-21

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

Vaccinia Virus Entry, Exit, and Interaction with Differentiated Human Airway Epithelia▿

PubMed Central

Vermeer, Paola D.; McHugh, Julia; Rokhlina, Tatiana; Vermeer, Daniel W.; Zabner, Joseph; Welsh, Michael J.

2007-01-01

Variola virus, the causative agent of smallpox, enters and exits the host via the respiratory route. To better understand the pathogenesis of poxvirus infection and its interaction with respiratory epithelia, we used vaccinia virus and examined its interaction with primary cultures of well-differentiated human airway epithelia. We found that vaccinia virus preferentially infected the epithelia through the basolateral membrane and released viral progeny across the apical membrane. Despite infection and virus production, epithelia retained tight junctions, transepithelial electrical conductance, and a steep transepithelial concentration gradient of virus, indicating integrity of the epithelial barrier. In fact, during the first four days of infection, epithelial height and cell number increased. These morphological changes and maintenance of epithelial integrity required vaccinia virus growth factor, which was released basolaterally, where it activated epidermal growth factor 1 receptors. These data suggest a complex interaction between the virus and differentiated airway epithelia; the virus preferentially enters the cells basolaterally, exits apically, and maintains epithelial integrity by stimulating growth factor receptors. PMID:17581984

Using 3D Culture of Primary Mammary Epithelial Cells to Define Molecular Entities Required for Acinus Formation: Analyzing MAP Kinase Phosphatases.

PubMed

Gajewska, Malgorzata; McNally, Sara

2017-01-01

Three-dimensional (3D) cell cultures on reconstituted basement membrane (rBM) enable the study of complex interactions between extracellular matrix (ECM) components and epithelial cells, which are crucial for the establishment of cell polarity and functional development of epithelia. 3D cultures of mammary epithelial cells (MECs) on Matrigel (a laminin-rich ECM derived from the Engelbreth-Holm-Swarm (EHS) murine tumor) promote interactions of MECs with the matrix via integrins, leading to formation of spherical monolayers of polarized cells surrounding a hollow lumen (acini). Acini closely resemble mammary alveoli found in the mammary gland. Thus, it is possible to study ECM-cell interactions and signalling pathways that regulate formation and maintenance of tissue-specific shape and functional differentiation of MECs in 3D under in vitro conditions. Here we present experimental protocols used to investigate the role of mitogen-activated protein kinase phosphatases (MKPs) during development of the alveoli-like structures by primary mouse mammary epithelial cells (PMMEC) cultured on Matrigel. We present detailed protocols for PMMEC isolation, and establishment of 3D cultures using an "on top" method, use of specific kinase and phosphatases inhibitors (PD98059 and pervanadate, respectively) administered at different stages of acinus development, and give examples of analyses carried out post-culture (Western blot, immunofluorescence staining, and confocal imaging).

A PDZ-interacting domain in CFTR is an apical membrane polarization signal

PubMed Central

Moyer, Bryan D.; Denton, Jerod; Karlson, Katherine H.; Reynolds, Donna; Wang, Shusheng; Mickle, John E.; Milewski, Michal; Cutting, Garry R.; Guggino, William B.; Li, Min; Stanton, Bruce A.

1999-01-01

Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin–binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain–containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain–containing proteins play a key role in the apical polarization of ion channels in epithelial cells. J. Clin. Invest. 104:1353–1361 (1999). PMID:10562297

Validation of a chicken ileal explant culture for measurement of mucosal inflammation induced by lipopolysaccharide

USDA-ARS?s Scientific Manuscript database

Gut mucosa holds a single layer of epithelial cells and the largest mass of lymphoid tissue in the body. While epithelial cell cultures are widely used to assess intestinal barrier functions, they have limitations to study cellular interactions with other cells, in particular those of the immune sys...

Host epithelial-viral interactions as cause and cure for asthma.

PubMed

Holtzman, Michael J; Patel, Dhara A; Zhang, Yong; Patel, Anand C

2011-08-01

Research on the pathogenesis of asthma has concentrated on initial stimuli, genetic susceptibilities, adaptive immune responses, and end-organ alterations (particularly in airway mucous cells and smooth muscle) as critical steps leading to disease. Recent evidence indicates that the innate immune cell response to respiratory viruses also contributes to the development of inflammatory airway disease. We further develop this concept by raising the issue that the interaction between host airway epithelial cells and respiratory viruses is another aspect of innate immunity that is also a critical determinant of asthma. We also introduce a rationale for how antiviral performance at the epithelial cell level might be improved to prevent acute infectious illness and chronic inflammatory disease caused by respiratory viruses. Copyright © 2011 Elsevier Ltd. All rights reserved.

Moderate plasma activated media suppresses proliferation and migration of MDCK epithelial cells

NASA Astrophysics Data System (ADS)

Mohades, Soheila; Laroussi, Mounir; Maruthamuthu, Venkat

2017-05-01

Low-temperature plasma has been shown to have diverse biomedical uses, including its applications in cancer and wound healing. One recent approach in treating mammalian cells with plasma is through the use of plasma activated media (PAM), which is produced by exposing cell culture media to plasma. While the adverse effects of PAM treatment on cancerous epithelial cell lines have been recently studied, much less is known about the interaction of PAM with normal epithelial cells. In this paper, non-cancerous canine kidney MDCK (Madin-Darby Canine Kidney) epithelial cells were treated by PAM and time-lapse microscopy was used to directly monitor their proliferation and random migration upon treatment. While longer durations of PAM treatment led to cell death, we found that moderate levels of PAM treatment inhibited proliferation in these epithelial cells. We also found that PAM treatment reduced random cell migration within epithelial islands. Immunofluorescence staining showed that while there were no major changes in the actin/adhesion apparatus, there was a significant change in the nuclear localization of proliferation marker Ki-67, consistent with our time-lapse results.

The Absence of N-Acetyl-D-glucosamine Causes Attenuation of Virulence of Candida albicans upon Interaction with Vaginal Epithelial Cells In Vitro

PubMed Central

Manczinger, Máté; Bocsik, Alexandra; Kocsis, Gabriella F.; Vörös, Andrea; Hegedűs, Zoltán; Marton, Annamária; Vízler, Csaba; Tubak, Vilmos; Deli, Mária; Kemény, Lajos; Nagy, István; Lakatos, Lóránt

2015-01-01

To better understand the molecular events underlying vulvovaginal candidiasis, we established an in vitro system. Immortalized vaginal epithelial cells were infected with live, yeast form C. albicans and C. albicans cultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes in C. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in our in vitro system revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cells in vitro, thus indicating the key role for this pathway in the virulence of C. albicans upon vulvovaginal candidiasis. PMID:26366412

Induction of proinflammatory cytokines in human lung epithelial cells during Rhodococcus equi infection.

PubMed

Remuzgo-Martínez, Sara; Pilares-Ortega, Lilian; Alvarez-Rodríguez, Lorena; Aranzamendi-Zaldunbide, Maitane; Padilla, Daniel; Icardo, Jose Manuel; Ramos-Vivas, Jose

2013-08-01

Rhodococcus equi is an opportunistic human pathogen associated with immunosuppressed people. While the interaction of R. equi with macrophages has been comprehensively studied, little is known about its interactions with non-phagocytic cells. Here, we characterized the entry process of this bacterium into human lung epithelial cells. The invasion is inhibited by nocodazole and wortmannin, suggesting that the phosphatidylinositol 3-kinase pathway and microtubule cytoskeleton are important for invasion. Pre-incubation of R. equi with a rabbit anti-R. equi polyclonal antiserum resulted in a dramatic reduction in invasion. Also, the invasion process as studied by immunofluorescence and scanning electron microscopy indicates that R. equi make initial contact with the microvilli of the A549 cells, and at the structural level, the entry process was observed to occur via a zipper-like mechanism. Infected lung epithelial cells upregulate the expression of cytokines IL-8 and IL-6 upon infection. The production of these pro-inflammatory cytokines was significantly enhanced in culture supernatants from cells infected with non-mucoid plasmid-less strains when compared with cells infected with mucoid strains. These results demonstrate that human airway epithelial cells produce pro-inflammatory mediators against R. equi isolates.

Oligosaccharide receptor mimics inhibit Legionella pneumophila attachment to human respiratory epithelial cells.

PubMed

Thomas, Richard J; Brooks, Tim J

2004-02-01

Legionnaire's disease is caused by the intracellular pathogen Legionella pneumophila, presenting as an acute pneumonia. Attachment is the key step during infection, often relying on an interaction between host cell oligosaccharides and bacterial adhesins. Inhibition of this interaction by receptor mimics offers possible novel therapeutic treatments. L. pneumophila attachment to the A549 cell line was significantly reduced by treatment with tunicamycin (73.6%) and sodium metaperiodate (63.7%). This indicates the importance of cell surface oligosaccharide chains in adhesion. A number of putative anti-adhesion compounds inhibited attachment to the A549 and U937 cell lines. The most inhibitory compounds were polymeric saccharides, GalNAcbeta1-4Gal, Galbeta1-4GlcNAc and para-nitrophenol. These compounds inhibited adhesion to a range of human respiratory cell lines, including nasal epithelial, bronchial epithelial and alveolar epithelial cell lines and the human monocytic cell line, U937. Some eukaryotic receptors for L. pneumophila were determined to be the glycolipids, asialo-GM1 and asialo-GM2 that contain the inhibitory saccharide moiety, GalNAcbeta1-4Gal. The identified compounds have the potential to be used as novel treatments for Legionnaire's disease.

Engineering stromal-epithelial interactions in vitro for ...

EPA Pesticide Factsheets

Background: Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue function. Epithelial-mesenchymal interactions (EMIs) have been examined using mammalian models, ex vivo tissue recombination, and in vitro co-cultures. Although these approaches have elucidated signaling mechanisms underlying morphogenetic processes and adult mammalian epithelial tissue function, they are limited by the availability of human tissue, low throughput, and human developmental or physiological relevance. Objectives: Bioengineering strategies to promote EMIs using human epithelial and mesenchymal cells have enabled the development of human in vitro models of adult epidermal and glandular tissues. In this review, we describe recent bioengineered models of human epithelial tissue and organs that can instruct the design of organotypic models of human developmental processes.Methods: We reviewed current bioengineering literature and here describe how bioengineered EMIs have enabled the development of human in vitro epithelial tissue models.Discussion: Engineered models to promote EMIs have recapitulated the architecture, phenotype, and function of adult human epithelial tissue, and similar engineering principles could be used to develop models of developmental morphogenesis. We describe how bioengineering strategies including bioprinting and spheroid culture could be implemented to

Embryo-epithelium interactions during implantation at a glance.

PubMed

Aplin, John D; Ruane, Peter T

2017-01-01

At implantation, with the acquisition of a receptive phenotype in the uterine epithelium, an initial tenuous attachment of embryonic trophectoderm initiates reorganisation of epithelial polarity to enable stable embryo attachment and the differentiation of invasive trophoblasts. In this Cell Science at a Glance article, we describe cellular and molecular events during the epithelial phase of implantation in rodent, drawing on morphological studies both in vivo and in vitro, and genetic models. Evidence is emerging for a repertoire of transcription factors downstream of the master steroidal regulators estrogen and progesterone that coordinate alterations in epithelial polarity, delivery of signals to the stroma and epithelial cell death or displacement. We discuss what is known of the cell interactions that occur during implantation, before considering specific adhesion molecules. We compare the rodent data with our much more limited knowledge of the human system, where direct mechanistic evidence is hard to obtain. In the accompanying poster, we represent the embryo-epithelium interactions in humans and laboratory rodents, highlighting similarities and differences, as well as depict some of the key cell biological events that enable interstitial implantation to occur. © 2017. Published by The Company of Biologists Ltd.

The Interplay between Entamoeba and Enteropathogenic Bacteria Modulates Epithelial Cell Damage

PubMed Central

Galván-Moroyoqui, José Manuel; Domínguez-Robles, M. del Carmen; Franco, Elizabeth; Meza, Isaura

2008-01-01

Background Mixed intestinal infections with Entamoeba histolytica, Entamoeba dispar and bacteria with exacerbated manifestations of disease are common in regions where amoebiasis is endemic. However, amoeba–bacteria interactions remain largely unexamined. Methodology Trophozoites of E. histolytica and E. dispar were co-cultured with enteropathogenic bacteria strains Escherichia coli (EPEC), Shigella dysenteriae and a commensal Escherichia coli. Amoebae that phagocytosed bacteria were tested for a cytopathic effect on epithelial cell monolayers. Cysteine proteinase activity, adhesion and cell surface concentration of Gal/GalNAc lectin were analyzed in amoebae showing increased virulence. Structural and functional changes and induction of IL-8 expression were determined in epithelial cells before and after exposure to bacteria. Chemotaxis of amoebae and neutrophils to human IL-8 and conditioned culture media from epithelial cells exposed to bacteria was quantified. Principal Findings E. histolytica digested phagocytosed bacteria, although S. dysenteriae retained 70% viability after ingestion. Phagocytosis of pathogenic bacteria augmented the cytopathic effect of E. histolytica and increased expression of Gal/GalNAc lectin on the amoebic surface and increased cysteine proteinase activity. E. dispar remained avirulent. Adhesion of amoebae and damage to cells exposed to bacteria were increased. Additional increases were observed if amoebae had phagocytosed bacteria. Co-culture of epithelial cells with enteropathogenic bacteria disrupted monolayer permeability and induced expression of IL-8. Media from these co-cultures and human recombinant IL-8 were similarly chemotactic for neutrophils and E. histolytica. Conclusions Epithelial monolayers exposed to enteropathogenic bacteria become more susceptible to E. histolytica damage. At the same time, phagocytosis of pathogenic bacteria by amoebae further increased epithelial cell damage. Significance The in vitro system presented here provides evidence that the Entamoeba/enteropathogenic bacteria interplay modulates epithelial cell responses to the pathogens. In mixed intestinal infections, where such interactions are possible, they could influence the outcome of disease. The results offer insights to continue research on this phenomenon. PMID:18648517

Stromal-epithelial interaction mediates steroidal regulation of metalloproteinase expression in human endometrium.

PubMed Central

Osteen, K G; Rodgers, W H; Gaire, M; Hargrove, J T; Gorstein, F; Matrisian, L M

1994-01-01

The hallmark of the menstrual cycle is extensive steroid-dependent tissue turnover. Estrogen mediates endometrial cell growth and structural remodeling, whereas progesterone suppresses estrogen-dependent proliferation and promotes cellular differentiation. In nonfertile cycles, tissue degradation and menstruation occur as a consequence of steroidal deprivation as the ovarian corpus luteum fails. Stromal-epithelial interactions are recognized as a necessary component in mediating steroid-induced endometrial turnover. Specific mRNAs for metalloproteinases of the stromelysin family are expressed during endometrial growth and menstrual breakdown but are absent in the progestin-dominated secretory phase. This expression pattern suggests involvement of stromelysins in remodeling the extracellular matrix of the endometrium during tissue growth and breakdown and implicates progesterone in the suppression of these enzymes. We examined the regulation of endometrial stromelysins in explant cultures and found no acute effect of estradiol on their expression, whereas progesterone was a potent inhibitor of stromelysin expression. Progesterone also suppressed stromelysin expression in cultures of isolated stromal cells, but epithelial cells were progesterone insensitive. Coculture of recombined stromal and epithelial cells restored steroidal suppression of the epithelial-specific metalloproteinase. Our data confirm that progesterone inhibits endometrial stromelysins and further demonstrate the necessity for a stromal-derived factor(s) as a mediator of steroid suppression of an epithelial metalloproteinase. Images PMID:7937850

A Pathway for the Control of Anoikis Sensitivity by E-Cadherin and Epithelial-to-Mesenchymal Transition▿‡

PubMed Central

Kumar, Sanjeev; Park, Sun Hee; Cieply, Benjamin; Schupp, Jane; Killiam, Elizabeth; Zhang, Fan; Rimm, David L.; Frisch, Steven M.

2011-01-01

Detachment of epithelial cells from matrix or attachment to an inappropriate matrix engages an apoptotic response known as anoikis, which prevents metastasis. Cellular sensitivity to anoikis is compromised during the oncogenic epithelial-to-mesenchymal transition (EMT), through unknown mechanisms. We report here a pathway through which EMT confers anoikis resistance. NRAGE (neurotrophin receptor-interacting melanoma antigen) interacted with a component of the E-cadherin complex, ankyrin-G, maintaining NRAGE in the cytoplasm. Oncogenic EMT downregulated ankyrin-G, enhancing the nuclear localization of NRAGE. The oncogenic transcriptional repressor protein TBX2 interacted with NRAGE, repressing the tumor suppressor gene p14ARF. P14ARF sensitized cells to anoikis; conversely, the TBX2/NRAGE complex protected cells against anoikis by downregulating this gene. This represents a novel pathway for the regulation of anoikis by EMT and E-cadherin. PMID:21746881

Odontogenic epithelial stem cells: hidden sources.

PubMed

Padma Priya, Sivan; Higuchi, Akon; Abu Fanas, Salem; Pooi Ling, Mok; Kumari Neela, Vasantha; Sunil, P M; Saraswathi, T R; Murugan, Kadarkarai; Alarfaj, Abdullah A; Munusamy, Murugan A; Kumar, Suresh

2015-12-01

The ultimate goal of dental stem cell research is to construct a bioengineered tooth. Tooth formation occurs based on the well-organized reciprocal interaction of epithelial and mesenchymal cells. The dental mesenchymal stem cells are the best explored, but because the human odontogenic epithelium is lost after the completion of enamel formation, studies on these cells are scarce. The successful creation of a bioengineered tooth is achievable only when the odontogenic epithelium is reconstructed to produce a replica of natural enamel. This article discusses the untapped sources of odontogenic epithelial stem cells in humans, such as those present in the active dental lamina in postnatal life, in remnants of dental lamina (the gubernaculum cord), in the epithelial cell rests of Malassez, and in reduced enamel epithelium. The possible uses of these stem cells in regenerative medicine, not just for enamel formation, are discussed.

Limited Interactions between Streptococcus Suis and Haemophilus Parasuis in In Vitro Co-Infection Studies

PubMed Central

Mathieu-Denoncourt, Annabelle; Letendre, Corinne; Auger, Jean-Philippe; Segura, Mariela; Aragon, Virginia; Lacouture, Sonia; Gottschalk, Marcelo

2018-01-01

Streptococcus suis and Haemophilus parasuis are normal inhabitants of the porcine upper respiratory tract but are also among the most frequent causes of disease in weaned piglets worldwide, causing inflammatory diseases such as septicemia, meningitis and pneumonia. Using an in vitro model of infection with tracheal epithelial cells or primary alveolar macrophages (PAMs), it was possible to determine the interaction between S. suis serotype 2 and H. parasuis strains with different level of virulence. Within H. parasuis strains, the low-virulence F9 strain showed higher adhesion levels to respiratory epithelial cells and greater association levels to PAMs than the high-virulence Nagasaki strain. Accordingly, the low-virulence F9 strain induced, in general, higher levels of pro-inflammatory cytokines than the virulent Nagasaki strain from both cell types. In general, S. suis adhesion levels to respiratory epithelial cells were similar to H. parasuis Nagasaki strain. Yet, S. suis strains induced a significantly lower level of pro-inflammatory cytokine expression from epithelial cells and PAMs than those observed with both H. parasuis strains. Finally, this study has shown that, overall and under the conditions used in the present study, S. suis and H. parasuis have limited in vitro interactions between them and use probably different host receptors, regardless to their level of virulence. PMID:29316613

REGULATION OF CYTOKINE PRODUCTION IN HUMAN ALVEOLAR MACHROPHAGES AND AIRWAY EPITHELIAL CELLS IN RESPONSE TO AMBIENT AIR POLLUTION PARTICLES: FURTHER MECHANISTIC STUDIES

EPA Science Inventory

In order to better understand how ambient air particulate matter (PM) affect lung health, the two main airway cell types likely to interact with inhaled particles, alveolar macrophages (AM) and airway epithelial cells have been exposed to particles in vitro and followed for endp...

Development of a chicken ileal explant culture model for measurement of gut inflammation induced by lipopolysaccharide

USDA-ARS?s Scientific Manuscript database

Gut mucosa holds a single layer of epithelial cells and the largest mass of lymphoid tissue in the body. While epithelial cell culture is widely used to assess intestinal barrier functions, it has limitations for studying cellular interactions with other cells, in particular those of the immune syst...

Reciprocal changes in gene expression profiles of cocultured breast epithelial cells and primary fibroblasts.

PubMed

Rozenchan, Patricia Bortman; Carraro, Dirce Maria; Brentani, Helena; de Carvalho Mota, Louise Danielle; Bastos, Elen Pereira; e Ferreira, Elisa Napolitano; Torres, Cesar H; Katayama, Maria Lúcia Hirata; Roela, Rosimeire Aparecida; Lyra, Eduardo C; Soares, Fernando Augusto; Folgueira, Maria Aparecida Azevedo Koike; Góes, João Carlos Guedes Sampaio; Brentani, Maria Mitzi

2009-12-15

The importance of epithelial-stroma interaction in normal breast development and tumor progression has been recognized. To identify genes that were regulated by these reciprocal interactions, we cocultured a nonmalignant (MCF10A) and a breast cancer derived (MDA-MB231) basal cell lines, with fibroblasts isolated from breast benign-disease adjacent tissues (NAF) or with carcinoma-associated fibroblasts (CAF), in a transwell system. Gene expression profiles of each coculture pair were compared with the correspondent monocultures, using a customized microarray. Contrariwise to large alterations in epithelial cells genomic profiles, fibroblasts were less affected. In MDA-MB231 highly represented genes downregulated by CAF derived factors coded for proteins important for the specificity of vectorial transport between ER and golgi, possibly affecting cell polarity whereas the response of MCF10A comprised an induction of genes coding for stress responsive proteins, representing a prosurvival effect. While NAF downregulated genes encoding proteins associated to glycolipid and fatty acid biosynthesis in MDA-MB231, potentially affecting membrane biogenesis, in MCF10A, genes critical for growth control and adhesion were altered. NAFs responded to coculture with MDA-MB231 by a decrease in the expression of genes induced by TGFbeta1 and associated to motility. However, there was little change in NAFs gene expression profile influenced by MCF10A. CAFs responded to the presence of both epithelial cells inducing genes implicated in cell proliferation. Our data indicate that interactions between breast fibroblasts and basal epithelial cells resulted in alterations in the genomic profiles of both cell types which may help to clarify some aspects of this heterotypic signaling. Copyright (c) 2009 UICC.

From The Cover: Reconstruction of functionally normal and malignant human breast tissues in mice

NASA Astrophysics Data System (ADS)

Kuperwasser, Charlotte; Chavarria, Tony; Wu, Min; Magrane, Greg; Gray, Joe W.; Carey, Loucinda; Richardson, Andrea; Weinberg, Robert A.

2004-04-01

The study of normal breast epithelial morphogenesis and carcinogenesis in vivo has largely used rodent models. Efforts at studying mammary morphogenesis and cancer with xenotransplanted human epithelial cells have failed to recapitulate the full extent of development seen in the human breast. We have developed an orthotopic xenograft model in which both the stromal and epithelial components of the reconstructed mammary gland are of human origin. Genetic modification of human stromal cells before the implantation of ostensibly normal human mammary epithelial cells resulted in the outgrowth of benign and malignant lesions. This experimental model allows for studies of human epithelial morphogenesis and differentiation in vivo and underscores the critical role of heterotypic interactions in human breast development and carcinogenesis.

Proteases of Sporothrix schenckii: Cytopathological effects on a host-cell model.

PubMed

Sabanero López, Myrna; Flores Villavicencio, Lérida L; Soto Arredondo, Karla; Barbosa Sabanero, Gloria; Villagómez-Castro, Julio César; Cruz Jiménez, Gustavo; Sandoval Bernal, Gerardo; Torres Guerrero, Haydee

Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. The adhesion of the fungus to the host tissue has been considered the key step in the colonization and invasion, but little is known about the early events in the host-parasite interaction. To evaluate the proteolytic activity of S. schenckii on epithelial cells. The proteolytic system (at pH 5 and 7) was evaluated using azocoll and zymograms. The host-parasite interaction and epithelial cell response were also analyzed by examining the microfilament cytoskeleton using phalloidin-FITC and transmission electron microscopy. Finally, the metabolic activity was determined using an XTT assay. The zymograms showed that S. schenckii yeast cells possess high intracellular and extracellular proteolytic activities (Mr≥200, 116, 97, and 70kDa) that are pH dependent and are inhibited by PMSF and E64, which act on serine and cysteine-type proteases. During the epithelial cell-protease interaction, the cells showed alterations in the microfilament distribution, as well as in the plasma membrane structure. Moreover, the metabolic activity of the epithelial cells decreased 60% without a protease inhibitor. Our data demonstrate the complexity of the cellular responses during the infection process. This process is somehow counteracted by the action of proteases inhibitors. Furthermore, the results provide critical information for understanding the nature of host-fungus interactions and for searching a new effective antifungal therapy, which includes protease inhibitors. Copyright © 2017 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

Epithelial-mesenchymal status influences how cells deposit fibrillin microfibrils.

PubMed

Baldwin, Andrew K; Cain, Stuart A; Lennon, Rachel; Godwin, Alan; Merry, Catherine L R; Kielty, Cay M

2014-01-01

Here, we show that epithelial-mesenchymal status influences how cells deposit extracellular matrix. Retinal pigmented epithelial (RPE) cells that expressed high levels of E-cadherin and had cell-cell junctions rich in zona occludens (ZO)-1, β-catenin and heparan sulfate, required syndecan-4 but not fibronectin or protein kinase C α (PKCα) to assemble extracellular matrix (fibrillin microfibrils and perlecan). In contrast, RPE cells that strongly expressed mesenchymal smooth muscle α-actin but little ZO-1 or E-cadherin, required fibronectin (like fibroblasts) and PKCα, but not syndecan-4. Integrins α5β1 and/or α8β1 and actomyosin tension were common requirements for microfibril deposition, as was heparan sulfate biosynthesis. TGFβ, which stimulates epithelial-mesenchymal transition, altered gene expression and overcame the dependency on syndecan-4 for microfibril deposition in epithelial RPE cells, whereas blocking cadherin interactions disrupted microfibril deposition. Renal podocytes had a transitional phenotype with pericellular β-catenin but little ZO-1; they required syndecan-4 and fibronectin for efficient microfibril deposition. Thus, epithelial-mesenchymal status modulates microfibril deposition.

Expression and cytokine regulation of immune recognition elements by normal human biliary epithelial and established liver cell lines in vitro.

PubMed

Cruickshank, S M; Southgate, J; Selby, P J; Trejdosiewicz, L K

1998-10-01

Biliary epithelial cells are targets of immune-mediated attack in conditions such as primary biliary cirrhosis and allograft rejection. This has been attributed to the ability of biliary epithelial cells to express ligands for T cell receptors. We aimed to investigate the expression of immune recognition elements and the effects of pro-inflammatory and anti-inflammatory cytokines on cell surface phenotypes of normal human biliary epithelial cells and established human liver-derived (PLC/PRF/5, HepG2, Hep3B and CC-SW) lines. Cells were cultured in the presence or absence of cytokines for 72 h, and expression of cell surface molecules was assessed by flow cytometry and immunofluorescence. All cell lines expressed MHC class I, ICAM-1 (CD54), LFA-3 (CD58) and EGF receptor, and all but Hep3B expressed Fas/Apo-1 (CD95). Unlike hepatocyte-derived cell lines, biliary epithelial cells and CC-SW expressed CD40 and CD44. As expected, IFNgamma and TNFalpha upregulated expression of ICAM-1, MHC class I and MHC class II, particularly in biliary epithelial cells. TGFbeta downregulated these molecules and downregulated CD95 on biliary epithelial cells, but upregulated LFA-3. The Th2 cytokines had little effect, although IL-4 upregulated CD95 expression on biliary epithelial cells. IFNgamma upregulated CD40 expression on biliary epithelial cells, CC-SW and HepG2. These findings imply that biliary epithelial cells may be capable of interacting with activated T lymphocytes via CD40 and LFA-3, which are thought to be important T cell accessory ligands for T cell activation in a B7-independent manner. Sensitivity to pro-inflammatory cytokines and expression of CD95 may explain why biliary epithelial cells are primary targets for autoimmune attack.

Putting tumours in context

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bissell, Mina J.; Radisky, Derek

2001-10-01

The interactions between cancer cells and their micro- and macroenvironment create a context that promotes tumor growth and protects it from immune attack. The functional association of cancer cells with their surrounding tissues forms a new 'organ' that changes as malignancy progresses. Investigation of this process might provide new insights into the mechanisms of tumorigenesis and could also lead to new therapeutic targets. Under normal conditions, ORGANS are made up of TISSUES that exchange information with other cell types via cell-cell contact, cytokines and the EXTRACELLULAR MATRIX (ECM). The ECM, which is produced by collaboration between STROMAL fibroblasts and EPITHELIALmore » cells, provides structural scaffolding for cells, as well as contextual information. The endothelial vasculature provides nutrients and oxygen, and cells of the immune system combat pathogens and remove apoptotic cells. Epithelial cells associate into intact, polarized sheets. These tissues communicate through a complex network of interactions: physically, through direct contact or through the intervening ECM, and biochemically, through both soluble and insoluble signalling molecules. In combination, these interactions provide the information that is necessary to maintain cellular differentiation and to create complex tissue structures. Occasionally, the intercellular signals that define the normal context become disrupted. Alterations in epithelial tissues can lead to movement of epithelial sheets and proliferation - for example, after activation of mesenchymal fibroblasts due to wounding.Normally, these conditions are temporary and reversible, but when inflammation is sustained, an escalating feedback loop ensues.Under persistent inflammatory conditions, continual upregulation of enzymes such as matrix metalloproteinases (MMPs) by stromal fibroblasts can disrupt the ECM, and invading immune cells can overproduce factors that promote abnormal proliferation. As this process progresses, the normal organization of the organ is replaced by a functional disorder. If there are pre-existing epithelial cells within this changing context that possess tumorigenic potential, they can start to proliferate. Alternatively, the abnormal interactions might lead to genomic instability within the epithelial cells and the acquisition of tumorigenic potential. The proliferating cancer cells can then interact with their microenvironment and enhance the abnormal interactions. At this point, the tumor has become its own organ, with a distinct context that now defines all its cellular responses. Here, we will examine how the mechanisms that contribute to the normal context also act to suppress developing tumors, how disruption of this context initiates and supports the process of tumorigenicity, and how some cells with a tumorigenic genotype can become phenotypically normal if the context is appropriately manipulated.« less

Multimerization of Adenovirus Serotype 3 Fiber Knob Domains Is Required for Efficient Binding of Virus to Desmoglein 2 and Subsequent Opening of Epithelial Junctions▿

PubMed Central

Wang, Hongjie; Li, ZongYi; Yumul, Roma; Lara, Stephanie; Hemminki, Akseli; Fender, Pascal; Lieber, André

2011-01-01

Recently, we identified desmoglein 2 (DSG2) as the main receptor for a group of species B adenoviruses (Ads), including Ad3, a serotype that is widely distributed in the human population (H. Wang et al., Nat. Med. 17:96–104, 2011). In this study, we have attempted to delineate structural details of the Ad3 interaction with DSG2. For CAR- and CD46-interacting Ad serotypes, attachment to cells can be completely blocked by an excess of recombinant fiber knob protein, while soluble Ad3 fiber knob only inefficiently blocks Ad3 infection. We found that the DSG2-interacting domain(s) within Ad3 is formed by several fiber knob domains that have to be in the spatial constellation that is present in viral particles. Based on this finding, we generated a small recombinant, self-dimerizing protein containing the Ad3 fiber knob (Ad3-K/S/Kn). Ad3-K/S/Kn bound to DSG2 with high affinity and blocked Ad3 infection. We demonstrated by confocal immunofluorescence and transmission electron microscopy analyses that Ad3-K/S/Kn, through its binding to DSG2, triggered the transient opening of intercellular junctions in epithelial cells. The pretreatment of epithelial cells with Ad3-K/S/Kn resulted in increased access to receptors that are localized in or masked by epithelial junctions, e.g., CAR or Her2/neu. Ad3-K/S/Kn treatment released CAR from tight junctions and thus increased the transduction of epithelial cells by a serotype Ad5-based vector. Furthermore, the pretreatment of Her2/neu-positive breast cancer cells with Ad3-K/S/Kn increased the killing of cancer cells by the Her2/neu-targeting monoclonal antibody trastuzumab (Herceptin). This study widens our understanding of how Ads achieve high avidity to their receptors and the infection of epithelial tissue. The small recombinant protein Ad3-K/S/Kn has practical implications for the therapy of epithelial cancer and gene/drug delivery to normal epithelial tissues. PMID:21525338

Mucin (MUC1) Expression and Function in Prostate Cancer Cells

DTIC Science & Technology

2001-09-01

Interactions at the Cell Surface of Mouse Uterine Epithelial Cells and Periimplantation -Stage Embryos. Trophoblast Res., 4:211-241, 1990. 37. Dutt...and Julian, J. Heparan Sulfate Proteoglycan Expression by Periimplantation Stage Embryos. Dev. Biol. 155:97-106,1993. 56. Rohde, L.H., and Carson...Modulators of Embryo-Uterine Epithelial Cell Attachment. In: S.K. Dey (ed.), Molecular and Cellular Aspects of Periimplantation Processes, Springer

Molecular Dissection of Mesenchymal–Epithelial Interactions in the Hair Follicle

PubMed Central

Rendl, Michael; Lewis, Lisa

2005-01-01

De novo hair follicle formation in embryonic skin and new hair growth in adult skin are initiated when specialized mesenchymal dermal papilla (DP) cells send cues to multipotent epithelial stem cells. Subsequently, DP cells are enveloped by epithelial stem cell progeny and other cell types to form a niche orchestrating hair growth. Understanding the general biological principles that govern the mesenchymal–epithelial interactions within the DP niche, however, has been hampered so far by the lack of systematic approaches to dissect the complete molecular make-up of this complex tissue. Here, we take a novel multicolor labeling approach, using cell type–specific transgenic expression of red and green fluorescent proteins in combination with immunolabeling of specific antigens, to isolate pure populations of DP and four of its surrounding cell types: dermal fibroblasts, melanocytes, and two different populations of epithelial progenitors (matrix and outer root sheath cells). By defining their transcriptional profiles, we develop molecular signatures characteristic for the DP and its niche. Validating the functional importance of these signatures is a group of genes linked to hair disorders that have been largely unexplored. Additionally, the DP signature reveals novel signaling and transcription regulators that distinguish them from other cell types. The mesenchymal–epithelial signatures include key factors previously implicated in ectodermal-neural fate determination, as well as a myriad of regulators of bone morphogenetic protein signaling. These findings establish a foundation for future functional analyses of the roles of these genes in hair development. Overall, our strategy illustrates how knowledge of the genes uniquely expressed by each cell type residing in a complex niche can reveal important new insights into the biology of the tissue and its associated disease states. PMID:16162033

An Engineered Organoid Culture Model That Incorporates Human Mesenchymal and Epithelial Cells to Model Palatal Fusion.

EPA Science Inventory

During embryonic development, fusion events are critical to morphogenesis of organs and tissues, including the iris, urethra, heart, neural tube, and secondary palate. Modeling this process in vitro is challenging as the interactions of mesenchymal and epithelial cells can be cr...

Adaptor protein 1 B mu subunit does not contribute to the recycling of kAE1 protein in polarized renal epithelial cells.

PubMed

Almomani, Ensaf Y; Touret, Nicolas; Cordat, Emmanuelle

2018-04-13

Mutations in the gene encoding the kidney anion exchanger 1 (kAE1) can lead to distal renal tubular acidosis (dRTA). dRTA mutations reported within the carboxyl (C)-terminal tail of kAE1 result in apical mis-targeting of the exchanger in polarized renal epithelial cells. As kAE1 physically interacts with the μ subunit of epithelial adaptor protein 1 B (AP-1B), we investigated the role of heterologously expressed μ1B subunit of the AP-1B complex for kAE1 retention to the basolateral membrane in polarized porcine LLC-PK1 renal epithelial cells that are devoid of endogenous AP-1B. We confirmed the interaction and close proximity between kAE1 and μ1B using immunoprecipitation and proximity ligation assay, respectively. Expressing the human μ1B subunit in these cells decreased significantly the amount of cell surface kAE1 at the steady state, but had no significant effect on kAE1 recycling and endocytosis. We show that (i) heterologous expression of μ1B displaces the physical interaction of endogenous GAPDH with kAE1 WT supporting that both AP-1B and GAPDH proteins bind to an overlapping site on kAE1 and (ii) phosphorylation of tyrosine 904 within the potential YDEV interaction motif does not alter the kAE1/AP-1B interaction. We conclude that μ1B subunit is not involved in recycling of kAE1.

The regulation of tooth morphogenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog through epithelial-mesenchymal interactions.

PubMed

Ishida, Kentaro; Murofushi, Mayumi; Nakao, Kazuhisa; Morita, Ritsuko; Ogawa, Miho; Tsuji, Takashi

2011-02-18

Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial-mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the number and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth. Copyright © 2011 Elsevier Inc. All rights reserved.

Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

PubMed

Gingerich, Aaron; Pang, Lan; Hanson, Jarod; Dlugolenski, Daniel; Streich, Rebecca; Lafontaine, Eric R; Nagy, Tamás; Tripp, Ralph A; Rada, Balázs

2016-01-01

Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1 h in the presence or absence of lactoperoxidase or thiocyanate. Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.

Epithelial-Stromal Interaction 1 (EPSTI1) Substitutes for Peritumoral Fibroblasts in the Tumor Microenvironment

PubMed Central

de Neergaard, Michala; Kim, Jiyoung; Villadsen, René; Fridriksdottir, Agla J.; Rank, Fritz; Timmermans-Wielenga, Vera; Langerød, Anita; Børresen-Dale, Anne-Lise; Petersen, Ole W.; Rønnov-Jessen, Lone

2010-01-01

Tumor cells can activate stroma, yet the implication of this activation in terms of reciprocal induction of gene expression in tumor cells is poorly understood. Epithelial Stromal Interaction 1 (EPSTI1) is an interferon response gene originally isolated from heterotypic recombinant cultures of human breast cancer cells and activated breast myofibroblasts. Here we describe the first immunolocalization of EPSTI1 in normal and cancerous breast tissue, and we provide evidence for a role of this molecule in the regulation of tumor cell properties and epithelial-mesenchymal transition. In general, no EPSTI1 staining was observed in normal breast epithelial cells from reduction mammoplasties (n = 25). However, in carcinomas, staining was positive in 22 of 40 biopsies and inversely correlated with the level of differentiation. To address the function of EPSTI1, we expressed EPSTI1 ectopically in one cell line and silenced endogenous EPSTI1 by RNA interference in another. Irrespective of the experimental approach, EPSTI1 expression led to an increase in tumorsphere formation—a property associated with breast stem/progenitor cells. Most remarkably, we show that EPSTI1, by conveying spread of tumor cells, can replace peritumoral activated fibroblasts in a tumor environment assay. These observations implicate EPSTI1 as a hitherto unappreciated regulator of tumor cell properties. PMID:20133812

GTP-Binding Proteins Inhibit cAMP Activation of Chloride Channels in Cystic Fibrosis Airway Epithelial Cells

NASA Astrophysics Data System (ADS)

Schwiebert, Erik M.; Kizer, Neil; Gruenert, Dieter C.; Stanton, Bruce A.

1992-11-01

Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl^- secretion by airway epithelial cells. In CF, cAMP does not activate Cl^- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[β-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl^- currents and restore cAMP-activated Cl^- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[γ-thio]triphosphate and AlF^-_4 reduce Cl^- currents and inhibit cAMP from activating Cl^- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[β-thio]diphosphate and in normal cells, cAMP activates a Cl^- conductance that has properties similar to CF transmembrane-conductance regulator Cl^- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl^- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl^- secretion in CF.

Primary and Immortalized Human Respiratory Cells Display Different Patterns of Cytotoxicity and Cytokine Release upon Exposure to Deoxynivalenol, Nivalenol and Fusarenon-X

PubMed Central

Ferreira Lopes, Silvia; Vacher, Gaëlle; Savova-Bianchi, Dessislava

2017-01-01

The type B trichothecene mycotoxins deoxynivalenol (DON), nivalenol (NIV) and fusarenon-X (FX) are structurally related secondary metabolites frequently produced by Fusarium on wheat. Consequently, DON, NIV and FX contaminate wheat dusts, exposing grain workers to toxins by inhalation. Those trichothecenes at low, relevant, exposition concentrations have differential effects on intestinal cells, but whether such differences exist with respiratory cells is mostly unknown, while it is required to assess the combined risk of exposure to mycotoxins. The goal of the present study was to compare the effects of DON, NIV and FX alone or in combination on the viability and IL-6 and IL-8-inducing capacity of human epithelial cells representative of the respiratory tract: primary human airway epithelial cells of nasal (hAECN) and bronchial (hAECB) origin, and immortalized human bronchial (16HBE14o-) and alveolar (A549) epithelial cell lines. We report that A549 cells are particularly resistant to the cytotoxic effects of mycotoxins. FX is more toxic than DON and NIV for all epithelial cell types. Nasal and bronchial primary cells are more sensitive than bronchial and alveolar cell lines to combined mycotoxin mixtures at low concentrations, although they are less sensitive to mycotoxins alone. Interactions between mycotoxins at low concentrations are rarely additive and are observed only for DON/NIV and NIV/FX on hAECB cells and DON/NIV/FX on A549 cells. Most interactions at low mycotoxin concentrations are synergistic, antagonistic interactions being observed only for DON/FX on hAECB, DON/NIV on 16HBE14o- and NIV/FX on A549 cells. DON, NIV and FX induce, albeit at different levels, IL-6 and IL-8 release by all cell types. However, NIV and FX at concentrations of low cytotoxicity induce IL-6 release by hAECB and A549 cells, and IL-8 release by hAECN cells. Overall, these data suggest that combined exposure to mycotoxins at low concentrations have a stronger effect on primary nasal epithelial cells than on bronchial epithelial cells and activate different inflammatory pathways. This information is particularly relevant for future studies about the hazard of occupational exposure to mycotoxins by inhalation and its impact on the respiratory tract. PMID:29068378

Cellular interactions of a water-soluble supramolecular polymer complex of carbon nanotubes with human epithelial colorectal adenocarcinoma cells.

PubMed

Lee, Yeonju; Geckeler, Kurt E

2012-08-01

Water-soluble, PAX-loaded carbon nanotubes are fabricated by employing a synthetic polyampholyte, PDM. To investigate the suitability of the polyampholyte and the nanotubes as drug carriers, different cellular interactions such as the human epithelial Caco-2 cells viability, their effect on the cell growth, and the change in the transepithelial electrical resistance in Caco-2 cells are studied. The resulting complex is found to exhibit an effective anti-cancer effect against colon cancer cells and an increased the reduction of the electrical resistance in the Caco-2 cells when compared to the precursor PAX. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Computational Study of the Development of Epithelial Acini: II. Necessary Conditions for Structure and Lumen Stability

PubMed Central

Rejniak, Katarzyna A.; Anderson, Alexander R.A.

2013-01-01

Simple epithelial tissues are organized as single layers of tightly packed cells that surround hollow lumens and form selective barriers separating different internal compartments of the body. The maintenance of epithelial structure and its function requires tight coordination and control of all the life processes of epithelial cells via cell-to-cell communication and signaling. These well-balanced cellular systems are, however, quite often disturbed by genetic or environmental cues that may lead to the formation of epithelial tumors (carcinomas). In fact, more than a half of all diagnosed tumors are initiated from epithelial cells. It is, therefore, important to gain a greater understanding of the factors that form and maintain the epithelial structure, as well as the features of the acinar structure that are modified during cancer development as observable in experimental and clinical research. We address these questions using the bio-mechanical model of the developing hollow epithelial acini introduced in Rejniak and Anderson (Bull. Math. Biol. 70:677–712, 2008). Here, we propose several scenarios involving various bio-mechanical interactions between neighboring cells that result in abnormal acinar development. Whenever possible, we compare our computational results with known experimental cases of mutant acini. PMID:18401665

Interactions of Entamoeba Histolytica with Host Cells in the Gut Mucosa,

DTIC Science & Technology

1977-01-01

amebae were observed close to the cecal epithelium, the apposing epithelial cell was found to have protruded in the lumen and making contact with the... amebae . Such cells often had become detached from their basal lamina and intercellular tight junctions, affording spaces through which amebae invaded the...mucosa. Other epithelial cells in close proximity to amebae showed degenerative changes characterized by swelling of mitochondria and endoplasmic

Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells.

PubMed

Roubos-van den Hil, P J; Nout, M J R; Beumer, R R; van der Meulen, J; Zwietering, M H

2009-03-01

This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and to clarify the mechanism of action. Tempe was prepared at controlled laboratory scale using Rhizopus microsporus var. microsporus as the inoculum. Extracts of raw, soaked and cooked soya beans reduced ETEC adhesion to brush border cells by 40%. Tempe extracts reduced adhesion by 80% or more. ETEC adhesion to Caco-2 cells reduced by 50% in the presence of tempe extracts. ETEC K88 bacteria were found to interact with soya bean extracts, and this may contribute to the observed decrease of ETEC adhesion to intestinal epithelial cells. Fermented soya beans (tempe) reduce the adhesion of ETEC to intestinal epithelial cells of pig and human origin. This reduced adhesion is caused by an interaction between ETEC K88 bacteria and soya bean compounds. The results strengthen previous observations on the anti-diarrhoeal effect of tempe. This effect indicates that soya-derived compounds may reduce adhesion of ETEC to intestinal cells in pigs as well as in humans and prevent against diarrhoeal diseases.

In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

PubMed

Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

2015-01-01

Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue. © 2014 International Federation for Cell Biology.

Integrative Radiation Biology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barcellos-Hoff, Mary Helen

We plan to study tissue-level mechanisms important to human breast radiation carcinogenesis. We propose that the cell biology of irradiated tissues reveals a coordinated multicellular damage response program in which individual cell contributions are primarily directed towards suppression of carcinogenesis and reestablishment of homeostasis. We identified transforming growth factor β1 (TGFβ) as a pivotal signal. Notably, we have discovered that TGFβ suppresses genomic instability by controlling the intrinsic DNA damage response and centrosome integrity. However, TGFβ also mediates disruption of microenvironment interactions, which drive epithelial to mesenchymal transition in irradiated human mammary epithelial cells. This apparent paradox of positive andmore » negative controls by TGFβ is the topic of the present proposal. First, we postulate that these phenotypes manifest differentially following fractionated or chronic exposures; second, that the interactions of multiple cell types in tissues modify the responses evident in this single cell type culture models. The goals are to: 1) study the effect of low dose rate and fractionated radiation exposure in combination with TGFβ on the irradiated phenotype and genomic instability of non-malignant human epithelial cells; and 2) determine whether stromal-epithelial interactions suppress the irradiated phenotype in cell culture and the humanized mammary mouse model. These data will be used to 3) develop a systems biology model that integrates radiation effects across multiple levels of tissue organization and time. Modeling multicellular radiation responses coordinated via extracellular signaling could have a significant impact on the extrapolation of human health risks from high dose to low dose/rate radiation exposure.« less

From single molecule to single tubules

NASA Astrophysics Data System (ADS)

Guo, Chin-Lin

2012-02-01

Biological systems often make decisions upon conformational changes and assembly of single molecules. In vivo, epithelial cells (such as the mammary gland cells) can respond to extracellular matrix (ECM) molecules, type I collagen (COL), and switch their morphology from a lobular lumen (100-200 micron) to a tubular lumen (1mm-1cm). However, how cells make such a morphogenetic decision through interactions with each other and with COL is unclear. Using a temporal control of cell-ECM interaction, we find that epithelial cells, in response to a fine-tuned percentage of type I collagen (COL) in ECM, develop various linear patterns. Remarkably, these patterns allow cells to self-assemble into a tubule of length ˜ 1cm and diameter ˜ 400 micron in the liquid phase (i.e., scaffold-free conditions). In contrast with conventional thought, the linear patterns arise through bi-directional transmission of traction force, but not through diffusible biochemical factors secreted by cells. In turn, the transmission of force evokes a long-range (˜ 600 micron) intercellular mechanical interaction. A feedback effect is encountered when the mechanical interaction modifies cell positioning and COL alignment. Micro-patterning experiments further reveal that such a feedback is a novel cell-number-dependent, rich-get-richer process, which allows cells to integrate mechanical interactions into long-range (> 1mm) linear coordination. Our results suggest a mechanism cells can use to form and coordinate long-range tubular patterns, independent of those controlled by diffusible biochemical factors, and provide a new strategy to engineer/regenerate epithelial organs using scaffold-free self-assembly methods.

Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner

PubMed Central

Hughes, K. R.; Harnisch, L. C.; Alcon-Giner, C.; Mitra, S.; Wright, C. J.; Ketskemety, J.

2017-01-01

Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi—a process termed ‘cell shedding’. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes. PMID:28123052

Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner.

PubMed

Hughes, K R; Harnisch, L C; Alcon-Giner, C; Mitra, S; Wright, C J; Ketskemety, J; van Sinderen, D; Watson, A J M; Hall, L J

2017-01-01

Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi-a process termed 'cell shedding'. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes. © 2017 The Authors.

Single-cell RNA-seq analysis unveils a prevalent epithelial/mesenchymal hybrid state during mouse organogenesis.

PubMed

Dong, Ji; Hu, Yuqiong; Fan, Xiaoying; Wu, Xinglong; Mao, Yunuo; Hu, Boqiang; Guo, Hongshan; Wen, Lu; Tang, Fuchou

2018-03-14

Organogenesis is crucial for proper organ formation during mammalian embryonic development. However, the similarities and shared features between different organs and the cellular heterogeneity during this process at single-cell resolution remain elusive. We perform single-cell RNA sequencing analysis of 1916 individual cells from eight organs and tissues of E9.5 to E11.5 mouse embryos, namely, the forebrain, hindbrain, skin, heart, somite, lung, liver, and intestine. Based on the regulatory activities rather than the expression patterns, all cells analyzed can be well classified into four major groups with epithelial, mesodermal, hematopoietic, and neuronal identities. For different organs within the same group, the similarities and differences of their features and developmental paths are revealed and reconstructed. We identify mutual interactions between epithelial and mesenchymal cells and detect epithelial cells with prevalent mesenchymal features during organogenesis, which are similar to the features of intermediate epithelial/mesenchymal cells during tumorigenesis. The comprehensive transcriptome at single-cell resolution profiled in our study paves the way for future mechanistic studies of the gene-regulatory networks governing mammalian organogenesis.

Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

PubMed Central

Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

2013-01-01

Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells

PubMed Central

Celià-Terrassa, Toni; Meca-Cortés, Óscar; Mateo, Francesca; Martínez de Paz, Alexia; Rubio, Nuria; Arnal-Estapé, Anna; Ell, Brian J.; Bermudo, Raquel; Díaz, Alba; Guerra-Rebollo, Marta; Lozano, Juan José; Estarás, Conchi; Ulloa, Catalina; ρlvarez-Simón, Daniel; Milà, Jordi; Vilella, Ramón; Paciucci, Rosanna; Martínez-Balbás, Marian; García de Herreros, Antonio; Gomis, Roger R.; Kang, Yibin; Blanco, Jerónimo; Fernández, Pedro L.; Thomson, Timothy M.

2012-01-01

Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs. PMID:22505459

Human Primary Intestinal Epithelial Cells as an Improved In Vitro Model for Cryptosporidium parvum Infection

PubMed Central

Cabada, Miguel M.; Nichols, Joan; Gomez, Guillermo; White, A. Clinton

2013-01-01

The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of host-parasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium. In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens. PMID:23509153

Modelling the structure of a ceRNA-theoretical, bipartite microRNA-mRNA interaction network regulating intestinal epithelial cellular pathways using R programming.

PubMed

Robinson, J M; Henderson, W A

2018-01-12

We report a method using functional-molecular databases and network modelling to identify hypothetical mRNA-miRNA interaction networks regulating intestinal epithelial barrier function. The model forms a data-analysis component of our cell culture experiments, which produce RNA expression data from Nanostring Technologies nCounter ® system. The epithelial tight-junction (TJ) and actin cytoskeleton interact as molecular components of the intestinal epithelial barrier. Upstream regulation of TJ-cytoskeleton interaction is effected by the Rac/Rock/Rho signaling pathway and other associated pathways which may be activated or suppressed by extracellular signaling from growth factors, hormones, and immune receptors. Pathway activations affect epithelial homeostasis, contributing to degradation of the epithelial barrier associated with osmotic dysregulation, inflammation, and tumor development. The complexity underlying miRNA-mRNA interaction networks represents a roadblock for prediction and validation of competing-endogenous RNA network function. We developed a network model to identify hypothetical co-regulatory motifs in a miRNA-mRNA interaction network related to epithelial function. A mRNA-miRNA interaction list was generated using KEGG and miRWalk2.0 databases. R-code was developed to quantify and visualize inherent network structures. We identified a sub-network with a high number of shared, targeting miRNAs, of genes associated with cellular proliferation and cancer, including c-MYC and Cyclin D.

Dynamic Transcription Factor Networks in Epithelial-Mesenchymal Transition in Breast Cancer Models

PubMed Central

Siletz, Anaar; Schnabel, Michael; Kniazeva, Ekaterina; Schumacher, Andrew J.; Shin, Seungjin; Jeruss, Jacqueline S.; Shea, Lonnie D.

2013-01-01

The epithelial-mesenchymal transition (EMT) is a complex change in cell differentiation that allows breast carcinoma cells to acquire invasive properties. EMT involves a cascade of regulatory changes that destabilize the epithelial phenotype and allow mesenchymal features to manifest. As transcription factors (TFs) are upstream effectors of the genome-wide expression changes that result in phenotypic change, understanding the sequential changes in TF activity during EMT provides rich information on the mechanism of this process. Because molecular interactions will vary as cells progress from an epithelial to a mesenchymal differentiation program, dynamic networks are needed to capture the changing context of molecular processes. In this study we applied an emerging high-throughput, dynamic TF activity array to define TF activity network changes in three cell-based models of EMT in breast cancer based on HMLE Twist ER and MCF-7 mammary epithelial cells. The TF array distinguished conserved from model-specific TF activity changes in the three models. Time-dependent data was used to identify pairs of TF activities with significant positive or negative correlation, indicative of interdependent TF activity throughout the six-day study period. Dynamic TF activity patterns were clustered into groups of TFs that change along a time course of gene expression changes and acquisition of invasive capacity. Time-dependent TF activity data was combined with prior knowledge of TF interactions to construct dynamic models of TF activity networks as epithelial cells acquire invasive characteristics. These analyses show EMT from a unique and targetable vantage and may ultimately contribute to diagnosis and therapy. PMID:23593114

Dynamic transcription factor networks in epithelial-mesenchymal transition in breast cancer models.

PubMed

Siletz, Anaar; Schnabel, Michael; Kniazeva, Ekaterina; Schumacher, Andrew J; Shin, Seungjin; Jeruss, Jacqueline S; Shea, Lonnie D

2013-01-01

The epithelial-mesenchymal transition (EMT) is a complex change in cell differentiation that allows breast carcinoma cells to acquire invasive properties. EMT involves a cascade of regulatory changes that destabilize the epithelial phenotype and allow mesenchymal features to manifest. As transcription factors (TFs) are upstream effectors of the genome-wide expression changes that result in phenotypic change, understanding the sequential changes in TF activity during EMT provides rich information on the mechanism of this process. Because molecular interactions will vary as cells progress from an epithelial to a mesenchymal differentiation program, dynamic networks are needed to capture the changing context of molecular processes. In this study we applied an emerging high-throughput, dynamic TF activity array to define TF activity network changes in three cell-based models of EMT in breast cancer based on HMLE Twist ER and MCF-7 mammary epithelial cells. The TF array distinguished conserved from model-specific TF activity changes in the three models. Time-dependent data was used to identify pairs of TF activities with significant positive or negative correlation, indicative of interdependent TF activity throughout the six-day study period. Dynamic TF activity patterns were clustered into groups of TFs that change along a time course of gene expression changes and acquisition of invasive capacity. Time-dependent TF activity data was combined with prior knowledge of TF interactions to construct dynamic models of TF activity networks as epithelial cells acquire invasive characteristics. These analyses show EMT from a unique and targetable vantage and may ultimately contribute to diagnosis and therapy.

Evidence of K+ channel function in epithelial cell migration, proliferation, and repair

PubMed Central

Girault, Alban

2013-01-01

Efficient repair of epithelial tissue, which is frequently exposed to insults, is necessary to maintain its functional integrity. It is therefore necessary to better understand the biological and molecular determinants of tissue regeneration and to develop new strategies to promote epithelial repair. Interestingly, a growing body of evidence indicates that many members of the large and widely expressed family of K+ channels are involved in regulation of cell migration and proliferation, key processes of epithelial repair. First, we briefly summarize the complex mechanisms, including cell migration, proliferation, and differentiation, engaged after epithelial injury. We then present evidence implicating K+ channels in the regulation of these key repair processes. We also describe the mechanisms whereby K+ channels may control epithelial repair processes. In particular, changes in membrane potential, K+ concentration, cell volume, intracellular Ca2+, and signaling pathways following modulation of K+ channel activity, as well as physical interaction of K+ channels with the cytoskeleton or integrins are presented. Finally, we discuss the challenges to efficient, specific, and safe targeting of K+ channels for therapeutic applications to improve epithelial repair in vivo. PMID:24196531

Helminths and intestinal barrier function

PubMed Central

McKay, Derek M.; Shute, Adam; Lopes, Fernando

2017-01-01

ABSTRACT Approximately one-sixth of the worlds' population is infected with helminths and this class of parasite takes a major toll on domestic livestock. The majority of species of parasitic helminth that infect mammals live in the gut (the only niche for tapeworms) where they contact the hosts' epithelial cells. Here, the helminth-intestinal epithelial interface is reviewed in terms of the impact on, and regulation of epithelial barrier function, both intrinsic (epithelial permeability) and extrinsic (mucin, bacterial peptides, commensal bacteria) elements of the barrier. The data available on direct effects of helminths on epithelial permeability are scant, fragmentary and pales in comparison with knowledge of mobilization of immune reactions and effector cells in response to helminth parasites and how these impact intestinal barrier function. The interaction of helminth-host and helminth-host-bacteria is an important determinant of gut form and function and precisely defining these interactions will radically alter our understanding of normal gut physiology and pathophysiological reactions, revealing new approaches to infection with parasitic helminths, bacterial pathogens and idiopathic auto-inflammatory disease. PMID:28452686

CD44S-hyaluronan interactions protect cells resulting from EMT against anoikis

PubMed Central

Cieply, Benjamin; Koontz, Colton; Frisch, Steven M.

2016-01-01

The detachment of normal epithelial cells from matrix triggers an apoptotic response known as anoikis, during homeostatic turnover. Metastatic tumor cells evade anoikis, by mechanisms that are only partly characterized. In particular, the epithelial–mesenchymal transition (EMT) in a subset of invasive tumor cells confers anoikis-resistance. In some cases, EMT up-regulates the cancer stem cell marker CD44S and the enzyme hyaluronic acid synthase-2 (HAS2). CD44S is the major receptor for hyaluronan in the extracellular matrix. Herein, we demonstrate that CD44S, unlike the CD44E isoform expressed in normal epithelial cells, contributes to the protection against anoikis. This protection requires the interaction of CD44S with hyaluronan (HA). CD44S–HA interaction is proposed to play an important role in tumor metastasis through enhanced cell survival under detached conditions. PMID:25937513

Site-specific programming of the host epithelial transcriptome by the gut microbiota.

PubMed

Sommer, Felix; Nookaew, Intawat; Sommer, Nina; Fogelstrand, Per; Bäckhed, Fredrik

2015-03-28

The intestinal epithelium separates us from the microbiota but also interacts with it and thus affects host immune status and physiology. Previous studies investigated microbiota-induced responses in the gut using intact tissues or unfractionated epithelial cells, thereby limiting conclusions about regional differences in the epithelium. Here, we sought to investigate microbiota-induced transcriptional responses in specific fractions of intestinal epithelial cells. To this end, we used microarray analysis of laser capture microdissection (LCM)-harvested ileal and colonic tip and crypt epithelial fractions from germ-free and conventionally raised mice and from mice during the time course of colonization. We found that about 10% of the host's transcriptome was microbially regulated, mainly including genes annotated with functions in immunity, cell proliferation, and metabolism. The microbial impact on host gene expression was highly site specific, as epithelial responses to the microbiota differed between cell fractions. Specific transcriptional regulators were enriched in each fraction. In general, the gut microbiota induced a more rapid response in the colon than in the ileum. Our study indicates that the microbiota engage different regulatory networks to alter host gene expression in a particular niche. Understanding host-microbiota interactions on a cellular level may facilitate signaling pathways that contribute to health and disease and thus provide new therapeutic strategies.

Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium.

PubMed

Amsler, K; Kuwada, S K

1999-01-01

Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-gamma protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.

RUNX1 and FOXP3 interplay regulates expression of breast cancer related genes

PubMed Central

Recouvreux, María Sol; Grasso, Esteban Nicolás; Echeverria, Pablo Christian; Rocha-Viegas, Luciana; Castilla, Lucio Hernán; Schere-Levy, Carolina; Tocci, Johanna Melisa; Kordon, Edith Claudia; Rubinstein, Natalia

2016-01-01

Runx1 participation in epithelial mammary cells is still under review. Emerging data indicates that Runx1 could be relevant for breast tumor promotion. However, to date no studies have specifically evaluated the functional contribution of Runx1 to control gene expression in mammary epithelial tumor cells. It has been described that Runx1 activity is defined by protein context interaction. Interestingly, Foxp3 is a breast tumor suppressor gene. Here we show that endogenous Runx1 and Foxp3 physically interact in normal mammary cells and this interaction blocks Runx1 transcriptional activity. Furthermore we demonstrate that Runx1 is able to bind to R-spondin 3 (RSPO3) and Gap Junction protein Alpha 1 (GJA1) promoters. This binding upregulates Rspo3 oncogene expression and downregulates GJA1 tumor suppressor gene expression in a Foxp3-dependent manner. Moreover, reduced Runx1 transcriptional activity decreases tumor cell migration properties. Collectively, these data provide evidence of a new mechanism for breast tumor gene expression regulation, in which Runx1 and Foxp3 physically interact to control mammary epithelial cell gene expression fate. Our work suggests for the first time that Runx1 could be involved in breast tumor progression depending on Foxp3 availability. PMID:26735887

RUNX1 and FOXP3 interplay regulates expression of breast cancer related genes.

PubMed

Recouvreux, María Sol; Grasso, Esteban Nicolás; Echeverria, Pablo Christian; Rocha-Viegas, Luciana; Castilla, Lucio Hernán; Schere-Levy, Carolina; Tocci, Johanna Melisa; Kordon, Edith Claudia; Rubinstein, Natalia

2016-02-09

Runx1 participation in epithelial mammary cells is still under review. Emerging data indicates that Runx1 could be relevant for breast tumor promotion. However, to date no studies have specifically evaluated the functional contribution of Runx1 to control gene expression in mammary epithelial tumor cells. It has been described that Runx1 activity is defined by protein context interaction. Interestingly, Foxp3 is a breast tumor suppressor gene. Here we show that endogenous Runx1 and Foxp3 physically interact in normal mammary cells and this interaction blocks Runx1 transcriptional activity. Furthermore we demonstrate that Runx1 is able to bind to R-spondin 3 (RSPO3) and Gap Junction protein Alpha 1 (GJA1) promoters. This binding upregulates Rspo3 oncogene expression and downregulates GJA1 tumor suppressor gene expression in a Foxp3-dependent manner. Moreover, reduced Runx1 transcriptional activity decreases tumor cell migration properties. Collectively, these data provide evidence of a new mechanism for breast tumor gene expression regulation, in which Runx1 and Foxp3 physically interact to control mammary epithelial cell gene expression fate. Our work suggests for the first time that Runx1 could be involved in breast tumor progression depending on Foxp3 availability.

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells*

PubMed Central

Agarwal, Vaibhav; Asmat, Tauseef M.; Luo, Shanshan; Jensch, Inga; Zipfel, Peter F.; Hammerschmidt, Sven

2010-01-01

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8–11 and SCR19–20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19–20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase. PMID:20504767

The EhCPADH112 Complex of Entamoeba histolytica Interacts with Tight Junction Proteins Occludin and Claudin-1 to Produce Epithelial Damage

PubMed Central

Betanzos, Abigail; Javier-Reyna, Rosario; García-Rivera, Guillermina; Bañuelos, Cecilia; González-Mariscal, Lorenza; Schnoor, Michael; Orozco, Esther

2013-01-01

Entamoeba histolytica, the protozoan responsible for human amoebiasis, causes between 30,000 and 100,000 deaths per year worldwide. Amoebiasis is characterized by intestinal epithelial damage provoking severe diarrhea. However, the molecular mechanisms by which this protozoan causes epithelial damage are poorly understood. Here, we studied the initial molecular interactions between the E. histolytica EhCPADH112 virulence complex and epithelial MDCK and Caco-2 cells. By confocal microscopy, we discovered that after contact with trophozoites or trophozoite extracts (TE), EhCPADH112 and proteins forming this complex (EhCP112 and EhADH112) co-localize with occludin and claudin-1 at tight junctions (TJ). Immunoprecipitation assays revealed interaction between EhCPADH112 and occludin, claudin-1, ZO-1 and ZO-2. Overlay assays confirmed an interaction of EhCP112 and EhADH112 with occludin and claudin-1, whereas only EhADH112 interacted also with ZO-2. We observed degradation of all mentioned TJ proteins after incubation with TE. Importantly, inhibiting proteolytic activity or blocking the complex with a specific antibody not only prevented TJ protein degradation but also epithelial barrier disruption. Furthermore, we discovered that TE treatment induces autophagy and apoptosis in MDCK cells that could contribute to the observed barrier disruption. Our results suggest a model in which epithelial damage caused by E. histolytica is initiated by the interaction of EhCP112 and EhADH112 with TJ proteins followed by their degradation. Disruption of TJs then induces increased paracellular permeability, thus facilitating the entry of more proteases and other parasite molecules leading eventually to tissue destruction. PMID:23762290

Self-organisation after embryonic kidney dissociation is driven via selective adhesion of ureteric epithelial cells.

PubMed

Lefevre, James G; Chiu, Han S; Combes, Alexander N; Vanslambrouck, Jessica M; Ju, Ali; Hamilton, Nicholas A; Little, Melissa H

2017-03-15

Human pluripotent stem cells, after directed differentiation in vitro , can spontaneously generate complex tissues via self-organisation of the component cells. Self-organisation can also reform embryonic organ structure after tissue disruption. It has previously been demonstrated that dissociated embryonic kidneys can recreate component epithelial and mesenchymal relationships sufficient to allow continued kidney morphogenesis. Here, we investigate the timing and underlying mechanisms driving self-organisation after dissociation of the embryonic kidney using time-lapse imaging, high-resolution confocal analyses and mathematical modelling. Organotypic self-organisation sufficient for nephron initiation was observed within a 24 h period. This involved cell movement, with structure emerging after the clustering of ureteric epithelial cells, a process consistent with models of random cell movement with preferential cell adhesion. Ureteric epithelialisation rapidly followed the formation of ureteric cell clusters with the reformation of nephron-forming niches representing a later event. Disruption of P-cadherin interactions was seen to impair this ureteric epithelial cell clustering without affecting epithelial maturation. This understanding could facilitate improved regulation of patterning within organoids and facilitate kidney engineering approaches guided by cell-cell self-organisation. © 2017. Published by The Company of Biologists Ltd.

Neisseria gonorrhoeae breaches the apical junction of polarized epithelial cells for transmigration by activating EGFR

PubMed Central

Edwards, Vonetta L.; Wang, Liang-Chun; Dawson, Valerie; Stein, Daniel C.; Song, Wenxia

2017-01-01

Summary Neisseria gonorrhoeae initiates infection at the apical surface of columnar endocervical epithelial cells in the female reproductive tract. These cells provide a physical barrier against pathogens by forming continuous apical junctional complexes between neighbouring cells. This study examines the interaction of gonococci (GC) with polarized epithelial cells. We show that viable GC preferentially localize at the apical side of the cell–cell junction in polarized endometrial and colonic epithelial cells, HEC-1-B and T84. In GC-infected cells, continuous apical junctional complexes are disrupted, and the junction-associated protein β-catenin is redistributed from the apical junction to the cytoplasm and to GC adherent sites; however, overall cellular levels remain unchanged. This redistribution of junctional proteins is associated with a decrease in the ‘fence’ function of the apical junction but not its ‘gate’ function. Disruption of the apical junction by removing calcium increases GC transmigration across the epithelial monolayer. GC inoculation induces the phosphorylation of both epidermal growth factor receptor (EGFR) and β-catenin, while inhibition of EGFR kinase activity significantly reduces both GC-induced β-catenin redistribution and GC transmigration. Therefore, the gonococcus is capable of weakening the apical junction and polarity of epithelial cells by activating EGFR, which facilitates GC transmigration across the epithelium. PMID:23279089

Cooperative Interactions During Human Mammary Epithelial Cell Immortalization

DTIC Science & Technology

2005-07-01

papilloma virus 16 E6 or E7. Proc. Nat. Acad. Sci. USA, 92: 3687-3691, 1995. 6. Huschtscha, L. I., Neumann, A. A., Noble, J. R., and Reddel, R. R. Effects...Oncology, In press. 5. Wazer, D. E., Liu, X.-L., Chu, Q., Gao, Q., and Band, V. Immortalization of distinct human mammary epithelial cell types by human

Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury.

PubMed

Chandrakesan, Parthasarathy; May, Randal; Weygant, Nathaniel; Qu, Dongfeng; Berry, William L; Sureban, Sripathi M; Ali, Naushad; Rao, Chinthalapally; Huycke, Mark; Bronze, Michael S; Houchen, Courtney W

2016-11-23

Crypt epithelial survival and regeneration after injury require highly coordinated complex interplay between resident stem cells and diverse cell types. The function of Dclk1 expressing tuft cells regulating intestinal epithelial DNA damage response for cell survival/self-renewal after radiation-induced injury is unclear. Intestinal epithelial cells (IECs) were isolated and purified and utilized for experimental analysis. We found that small intestinal crypts of Villin Cre ;Dclk1 f/f mice were hypoplastic and more apoptotic 24 h post-total body irradiation, a time when stem cell survival is p53-independent. Injury-induced ATM mediated DNA damage response, pro-survival genes, stem cell markers, and self-renewal ability for survival and restitution were reduced in the isolated intestinal epithelial cells. An even greater reduction in these signaling pathways was observed 3.5 days post-TBI, when peak crypt regeneration occurs. We found that interaction with Dclk1 is critical for ATM and COX2 activation in response to injury. We determined that Dclk1 expressing tuft cells regulate the whole intestinal epithelial cells following injury through paracrine mechanism. These findings suggest that intestinal tuft cells play an important role in regulating the ATM mediated DNA damage response, for epithelial cell survival/self-renewal via a Dclk1 dependent mechanism, and these processes are indispensable for restitution and function after severe radiation-induced injury.

Cadherin-23 Mediates Heterotypic Cell-Cell Adhesion between Breast Cancer Epithelial Cells and Fibroblasts

PubMed Central

Apostolopoulou, Maria; Ligon, Lee

2012-01-01

In the early stages of breast cancer metastasis, epithelial cells penetrate the basement membrane and invade the surrounding stroma, where they encounter fibroblasts. Paracrine signaling between fibroblasts and epithelial tumor cells contributes to the metastatic cascade, but little is known about the role of adhesive contacts between these two cell types in metastasis. Here we show that MCF-7 breast cancer epithelial cells and normal breast fibroblasts form heterotypic adhesions when grown together in co-culture, as evidenced by adhesion assays. PCR and immunoblotting show that both cell types express multiple members of the cadherin superfamily, including the atypical cadherin, cadherin-23, when grown in isolation and in co-culture. Immunocytochemistry experiments show that cadherin-23 localizes to homotypic adhesions between MCF-7 cells and also to heterotypic adhesions between the epithelial cells and fibroblasts, and antibody inhibition and RNAi experiments show that cadherin-23 plays a role in mediating these adhesive interactions. Finally, we show that cadherin-23 is upregulated in breast cancer tissue samples, and we hypothesize that heterotypic adhesions mediated by this atypical cadherin may play a role in the early stages of metastasis. PMID:22413011

SERS and integrative imaging upon internalization of quantum dots into human oral epithelial cells.

PubMed

Cepeda-Pérez, Elisa; López-Luke, Tzarara; Plascencia-Villa, Germán; Perez-Mayen, Leonardo; Ceja-Fdez, Andrea; Ponce, Arturo; Vivero-Escoto, Juan; de la Rosa, Elder

2016-07-01

CdTe quantum dots (QDs) are widely used in bio-applications due to their size and highly efficient optical properties. However internalization mechanisms thereof for the variety of freshly extracted, not cultivated human cells and their specific molecular interactions remains an open topic for discussion. In this study, we assess the internalization mechanism of CdTe quantum dots (3.3 nm) capped with thioglycolic acid using non cultivated oral epithelial cells obtained from healthy donors. Naked gold nanoparticles (40 nm) were successfully used as nanosensors for surface-enhanced Raman spectroscopy to efficiently identify characteristic Raman peaks, providing new evidence indicating that the first interactions of these QDs with epithelial cells occurred preferentially with aromatic rings and amine groups of amino acid residues and glycans from trans-membrane proteins and cytoskeleton. Using an integrative combination of advanced imaging techniques, including ultra-high resolution SEM, high resolution STEM coupled with EDX spectroscopy together with the results obtained by Raman spectroscopy, it was determined that thioglycolic acid capped CdTe QDs are efficiently internalized into freshly extracted oral epithelial cells only by facilitated diffusion, distributed into cytoplasm and even within the cell nucleus in three minutes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Putting on the brakes: Bacterial impediment of wound healing

PubMed Central

Brothers, Kimberly M.; Stella, Nicholas A.; Hunt, Kristin M.; Romanowski, Eric G.; Liu, Xinyu; Klarlund, Jes K.; Shanks, Robert M. Q.

2015-01-01

The epithelium provides a crucial barrier to infection, and its integrity requires efficient wound healing. Bacterial cells and secretomes from a subset of tested species of bacteria inhibited human and porcine corneal epithelial cell migration in vitro and ex vivo. Secretomes from 95% of Serratia marcescens, 71% of Pseudomonas aeruginosa, 29% of Staphylococcus aureus strains, and other bacterial species inhibited epithelial cell migration. Migration of human foreskin fibroblasts was also inhibited by S. marcescens secretomes indicating that the effect is not cornea specific. Transposon mutagenesis implicated lipopolysaccharide (LPS) core biosynthetic genes as being required to inhibit corneal epithelial cell migration. LPS depletion of S. marcescens secretomes with polymyxin B agarose rendered secretomes unable to inhibit epithelial cell migration. Purified LPS from S. marcescens, but not from Escherichia coli or S. marcescens strains with mutations in the waaG and waaC genes, inhibited epithelial cell migration in vitro and wound healing ex vivo. Together these data suggest that S. marcescens LPS is sufficient for inhibition of epithelial wound healing. This study presents a novel host-pathogen interaction with implications for infections where bacteria impact wound healing and provides evidence that secreted LPS is a key factor in the inhibitory mechanism. PMID:26365869

High LET Radiation Can Enhance TGF(Beta) Induced EMT and Cross-Talk with ATM Pathways

NASA Technical Reports Server (NTRS)

Wang, Minli; Hada, Megumi; Huff, Janice; Pluth, Janice M.; Anderson, Janniffer; ONeill, Peter; Cucinotta, Francis A.

2010-01-01

The TGF(Beta) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation in mammary epithelial cells. We investigated possible interactions between the TGF(Beta) and ATM pathways following simulated space radiation using hTERT immortalized human esophageal epithelial cells (EPC-hTERT), mink lung epithelial cells (Mv1lu), and several human fibroblast cell lines. TGF(Beta) is a key modulator of the Epithelial-Mesenchymal Transition (EMT), important in cancer progression and metastasis. The implication of EMT by radiation also has several lines of developing evidence, however is poorly understood. The identification of TGF(Beta) induced EMT can be shown in changes to morphology, related gene over expression or down regulation, which can be detected by RT-PCR, and immunostaining and western blotting. In this study, we have observed morphologic and molecular alternations consistent with EMT after Mv1lu cells were treated with TGF(Beta) High LET radiation enhanced TGF(Beta) mediated EMT with a dose as low as 0.1Gy. In order to consider the TGF(Beta) interaction with ATM we used a potent ATM inhibitor Ku55933 and investigated gene expression changes and Smad signaling kinetics. Ku559933 was observed to reverse TGF(Beta) induced EMT, while this was not observed in dual treated cells (radiation+TGF(Beta)). In EPC-hTERT cells, TGF(Beta) alone was not able to induce EMT after 3 days of application. A combined treatment with high LET, however, significantly caused the alteration of EMT markers. To study the function of p53 in the process of EMT, we knocked down P53 through RNA interference. Morphology changes associated with EMT were observed in epithelial cells with silenced p53. Our study indicates: high LET radiation can enhance TGF(Beta) induced EMT; while ATM is triggering the process of TGF(Beta)-induced EMT, p53 might be an essential repressor for EMT phenotypes.

Potential role of chitinases and chitin-binding proteins in host-microbial interactions during the development of intestinal inflammation

PubMed Central

Tran, Hoa T.; Barnich, Nicolas; Mizoguchi, Emiko

2011-01-01

Summary The small and large intestines contain an abundance of luminal antigens derived from food products and enteric microorganisms. The function of intestinal epithelial cells is tightly regulated by several factors produced by enteric bacteria and the epithelial cells themselves. Epithelial cells actively participate in regulating the homeostasis of intestine, and failure of this function leads to abnormal and host-microbial interactions resulting in the development of intestinal inflammation. Major determinants of host susceptibility against luminal commensal bacteria include genes regulating mucosal immune responses, intestinal barrier function and microbial defense. Of note, it has been postulated that commensal bacterial adhesion and invasion on/into host cells may be strongly involved in the pathogenesis of inflammatory bowel disease (IBD). During the intestinal inflammation, the composition of the commensal flora is altered, with increased population of aggressive and detrimental bacteria and decreased populations of protective bacteria. In fact, some pathogenic bacteria, including Adherent Invasive Escherichia coli, Listeria monocytogenes and Vibrio cholerae are likely to initiate their adhesion to the host cells by expressing accessory molecules such as chitinases and/or chitin-binding proteins on themselves. In addition, several inducible molecules (e.g., chitinase 3-like-1, CEACAM6) are also induced on the host cells (e.g. epithelial cells, lamina proprial macrophages) under inflammatory conditions, and are actively participated in the host-microbial interactions. In this review, we will summarize and discuss the potential roles of these important molecules during the development of acute and chronic inflammatory conditions. PMID:21938682

Fibroblast-mediated in vivo and in vitro growth promotion of tumorigenic rat thyroid carcinoma cells but not normal Fisher rat thyroid follicular cells.

PubMed

Saitoh, Ohki; Mitsutake, Norisato; Nakayama, Toshiyuki; Nagayama, Yuji

2009-07-01

It is known that genetic abnormalities in oncogenes and/or tumor suppressor genes promote carcinogenesis. Numerous recent articles, however, have demonstrated that epithelial-stromal interaction also plays a critical role for initiation and progression of carcinoma cells. Furthermore, ionizing radiation induces alterations in the tissue microenvironments that promote carcinogenesis. There is little or no information on epithelial-stromal interaction in thyroid carcinoma cells. The objective of this study was to determine if epithelial-stromal interaction influenced the growth of thyroid carcinoma cells in vivo and in vitro and to determine if radiation had added or interacting effects. Normal Fisher rat thyroid follicular cells (FRTL5 cells) and tumorigenic rat thyroid carcinoma cells (FRTL-Tc cells) derived from FRTL5 cells were employed. The cells were injected into thyroids or subcutaneously into left flanks of rats alone or in combination with skin-derived fibroblasts. In groups of rats, fibroblasts were irradiated with 0.1 or 4 Gy x-ray 3 days before inoculation. In vitro growth of FRTL-Tc and FRTL-5 cells were evaluated using the fibroblast-conditioned medium and in a co-culture system with fibroblasts. The in vivo experiments demonstrated that FRTL-Tc cells injected intrathyroidally grew faster than those injected subcutaneously, and that admixed fibroblasts enhanced growth of subcutaneous FRTL-Tc tumors, indicating that the intrathyroidal milieu, particularly in the presence of fibroblasts, confer growth-promoting advantage to thyroid carcinoma cells. This in vivo growth-promoting effect of fibroblasts on FRTL-Tc cells was duplicated in the in vitro experiments using the fibroblast-conditioned medium. Thus, our data demonstrate that this effect is mediated by soluble factor(s), is reversible, and is comparable to that of 10% fetal bovine serum. However, normal FRTL5 cells did not respond to the fibroblast-conditioned medium. Furthermore, high- and low-dose irradiation enhanced and suppressed, respectively, the in vivo fibroblast-mediated growth promotion. This effect was, however, not observed in the in vitro experiment with conditioned medium or even that allowing cell-cell contact. The intrathyroidal stromal microenvironments, particularly fibroblasts, appear to enhance the growth of thyroid carcinomas through soluble factor(s), which is modulated differently by high- and low-dose irradiation. To our knowledge this is the first study to show epithelial-stromal interaction in thyroid carcinoma.

Fibrosis in connective tissue disease: the role of the myofibroblast and fibroblast-epithelial cell interactions

PubMed Central

Krieg, Thomas; Abraham, David; Lafyatis, Robert

2007-01-01

Fibrosis, characterized by excessive extracellular matrix accumulation, is a common feature of many connective tissue diseases, notably scleroderma (systemic sclerosis). Experimental studies suggest that a complex network of intercellular interactions involving endothelial cells, epithelial cells, fibroblasts and immune cells, using an array of molecular mediators, drives the pathogenic events that lead to fibrosis. Transforming growth factor-β and endothelin-1, which are part of a cytokine hierarchy with connective tissue growth factor, are key mediators of fibrogenesis and are primarily responsible for the differentiation of fibroblasts toward a myofibroblast phenotype. The tight skin mouse (Tsk-1) model of cutaneous fibrosis suggests that numerous other genes may also be important. PMID:17767742

Candida albicans triggers interleukin-8 secretion by oral epithelial cells.

PubMed

Dongari-Bagtzoglou, A; Kashleva, H

2003-04-01

Oropharyngeal candidiasis is a frequent opportunistic infection associated with immunocompromised hosts. Candida albicans is the principal species responsible for this infection. Production of interleukin-8 (IL-8), by oral epithelial cells can be expected to play a major role in the recruitment and activation of professional phagocytes at the infected site. The purpose of this study was to determine whether C. albicans triggers secretion of IL-8 by oral epithelial cells in vitro and investigate mechanisms of host cell-fungal interactions that trigger such responses. Oral epithelial cell lines (SCC4, SCC15, and OKF6/TERT-2) as well as primary gingival epithelial cells were used. Epithelial cells were cocultured with C. albicans, strains SC5314, ATCC28366 or ATCC32077, for 24-48 hr, and supernatants were analyzed for IL-8 content by ELISA. A germination-deficient mutant (efg1/efg1 cph1/cph1), otherwise isogenic to strain SC5314, was used to assess the requirement for germination in triggering IL-8 responses. In order to ascertain whether direct contact of yeast with host cells is required to trigger cytokine production, epithelial cells were separated from yeast using cell culture inserts. To test whether IL-8 secretion is dependent on IL-1alpha activity, epithelial cells were challenged with viable C. albicans in the presence or absence of neutralizing anti-IL-1alpha antibody or IL-1ra, and IL-8 secretion was measured in the supernatants. All cell lines and primary cultures responded to C. albicans with an increase in IL-8 secretion. IL-8 responses were contact-dependent, strain-specific, required yeast viability and germination into hyphae, and were in part autoregulated by IL-1alpha. Copyright 2003 Elsevier Science Ltd.

Crossroads of integrins and cadherins in epithelia and stroma remodeling

PubMed Central

Epifano, Carolina; Perez-Moreno, Mirna

2012-01-01

Adhesion events mediated by cadherin and integrin adhesion receptors have fundamental roles in the maintenance of the physiological balance of epithelial tissues, and it is well established that perturbations in their normal functional activity and/or changes in their expression are associated with tumorigenesis. Over the last decades, increasing evidence of a dynamic collaborative interaction between these complexes through their shared interactions with cytoskeletal proteins and common signaling pathways has emerged not only as an important regulator of several aspects of epithelial cell behavior, but also as a coordinated adhesion module that senses and transmits signals from and to the epithelia surrounding microenvironment. The tight regulation of their crosstalk is particularly important during epithelial remodeling events that normally take place during morphogenesis and tissue repair, and when defective it leads to cell transformation and aggravated responses of the tumor microenvironment that contribute to tumorigenesis. In this review we highlight some of the interactions that regulate their crosstalk and how this could be implicated in regulating signals across epithelial tissues to sustain homeostasis. PMID:22568988

Hierarchy of stroma-derived factors in supporting growth of stroma-dependent hemopoietic cells: membrane-bound SCF is sufficient to confer stroma competence to epithelial cells.

PubMed

Friel, Jutta; Itoh, Katsuhiko; Bergholz, Ulla; Jücker, Manfred; Stocking, Carol; Harrison, Paul; Ostertag, Wolfram

2002-03-01

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34+ cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34+ hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.

Cigarette smoke disrupts the integrity of airway adherens junctions through the aberrant interaction of p120-catenin with the cytoplasmic tail of MUC1

PubMed Central

Zhang, Lili; Gallup, Marianne; Zlock, Lorna; Basbaum, Carol; Finkbeiner, Walter E.; McNamara, Nancy A.

2014-01-01

Adherens junctions (AJs) containing epithelial cadherin (E-cad) bound to p120-catenin (p120ctn) and β-catenin (β-ctn) play a crucial role in regulating cell–cell adhesion. Cigarette smoke abrogates cell–cell adhesion between epithelial cells by disrupting E-cad, a hallmark of epithelial–mesenchymal transition (EMT), yet the underlying mechanism remains unknown. We used an organotypic culture of primary human bronchial epithelial (HBE) cells treated with smoke-concentrated medium (Smk) to establish an essential role for the interaction between p120ctn and the cytoplasmic tail of MUC1 (MUC1-CT) in regulating E-cad disruption. Within the first 4 h of smoke exposure, apical MUC1-CT repositioned to the basolateral membrane of pseudo-stratified HBE cells, where it interacted with p120ctn. A time-dependent increase in MUC1-CT/p120ctn complexes occurred in conjunction with a time-dependent dissociation of p120ctn/E-cad/β-ctn complexes, as well as the coordinated degradation of p120ctn and E-cad. Interestingly, Smk induced a similar interaction between MUC1-CT and β-ctn, but this occurred 44 h after MUC1-CT’s initial interaction with p120ctn, and well after the AJs were destroyed. Blocking MUC1-CT’s interaction with p120ctn using a MUC1-CT dominant-negative peptide, PMIP, successfully abolished Smk’s disruptive effects on AJs and recovered apical-basolateral polarity of HBE cells. The MUC1-CT/p120ctn interaction was highly dependent on EGFR/Src/Jnk-mediated tyrosine phosphorylation (TyrP) of MUC1-CT. Accordingly, EGFR, Src or Jnk inhibitors (AG1478, PP2, SP600125, respectively) abrogated Smk-induced MUC1-CT-TyrP, MUC1-CT/p120ctn interaction, AJ disruption, and loss of cellular polarity. Our work identified MUC1-CT and p120ctn as important regulators of epithelial polarity and cell-cell adhesion during a smoke-induced EMT-like process. Novel therapeutics designed to inhibit MUC1-CT/p120ctn complex formation may prevent EMT in the smoker’s airway. PMID:22833523

Epithelial cell kinase-B61: an autocrine loop modulating intestinal epithelial migration and barrier function.

PubMed

Rosenberg, I M; Göke, M; Kanai, M; Reinecker, H C; Podolsky, D K

1997-10-01

Epithelial cell kinase (Eck) is a member of a large family of receptor tyrosine kinases whose functions remain largely unknown. Expression and regulation of Eck and its cognate ligand B61 were analyzed in the human colonic adenocarcinoma cell line Caco-2. Immunocytochemical staining demonstrated coexpression of Eck and B61 in the same cells, suggestive of an autocrine loop. Eck levels were maximal in preconfluent cells. In contrast, B61 levels were barely detectable in preconfluent cells and increased progressively after the cells reached confluence. Caco-2 cells cultured in the presence of added B61 showed a significant reduction in the levels of dipeptidyl peptidase and sucrase-isomaltase mRNA, markers of Caco-2 cell differentiation. Cytokines interleukin-1beta (IL-1beta), basic fibroblast growth factor, IL-2, epidermal growth factor, and transforming growth factor-beta modulated steady-state levels of Eck and B61 mRNA and regulated Eck activation as assessed by tyrosine phosphorylation. Functionally, stimulation of Eck by B61 resulted in increased proliferation, enhanced barrier function, and enhanced restitution of injured epithelial monolayers. These results suggest that the Eck-B61 interaction, a target of regulatory peptides, plays a role in intestinal epithelial cell development, migration, and barrier function, contributing to homeostasis and preservation of continuity of the epithelial barrier.

LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts.

PubMed

Zhang, Hui; Sweezey, Neil B; Kaplan, Feige

2015-02-15

Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1(+/-) mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5(LGL1KD)). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5(LGL1KD) suppressed cell growth and increased apoptosis of annexin V(+) staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5(LGL1KD) cells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development. Copyright © 2015 the American Physiological Society.

Alk5-Mediated Transforming Growth Factor β Signaling Acts Upstream of Fibroblast Growth Factor 10 To Regulate the Proliferation and Maintenance of Dental Epithelial Stem Cells▿

PubMed Central

Zhao, Hu; Li, Sha; Han, Dong; Kaartinen, Vesa; Chai, Yang

2011-01-01

Mouse incisors grow continuously throughout life. This growth is supported by the division of dental epithelial stem cells that reside in the cervical loop region. Little is known about the maintenance and regulatory mechanisms of dental epithelial stem cells. In the present study, we investigated how transforming growth factor β (TGF-β) signaling-mediated mesenchymal-epithelial cell interactions control dental epithelial stem cells. We designed two approaches using incisor organ culture and bromodeoxyuridine (BrdU) pulse-chase experiments to identify and evaluate stem cell functions. We show that the loss of the TGF-β type I receptor (Alk5) in the cranial neural crest-derived dental mesenchyme severely affects the proliferation of TA (transit-amplifying) cells and the maintenance of dental epithelial stem cells. Incisors of Wnt1-Cre; Alk5fl/fl mice lost their ability to continue to grow in vitro. The number of BrdU label-retaining cells (LRCs) was dramatically reduced in Alk5 mutant mice. Fgf10, Fgf3, and Fgf9 signals in the dental mesenchyme were downregulated in Wnt1-Cre; Alk5fl/fl incisors. Strikingly, the addition of exogenous fibroblast growth factor 10 (FGF10) into cultured incisors rescued dental epithelial stem cells in Wnt1-Cre; Alk5fl/fl mice. Therefore, we propose that Alk5 functions upstream of Fgf10 to regulate TA cell proliferation and stem cell maintenance and that this signaling mechanism is crucial for stem cell-mediated tooth regeneration. PMID:21402782

Melanin dependent survival of Apergillus fumigatus conidia in lung epithelial cells.

PubMed

Amin, Shayista; Thywissen, Andreas; Heinekamp, Thorsten; Saluz, Hans Peter; Brakhage, Axel A

2014-07-01

Aspergillus fumigatus is the most important air-borne pathogenic fungus of humans. Upon inhalation of conidia, the fungus makes close contact with lung epithelial cells, which only possess low phagocytic activity. These cells are in particular interesting to address the question whether there is some form of persistence of conidia of A. fumigatus in the human host. Therefore, by also using uracil-auxotrophic mutant strains, we were able to investigate the interaction of A549 lung epithelial cells and A. fumigatus conidia in detail for long periods. Interestingly, unlike professional phagocytes, our study showed that the presence of conidial dihydroxynaphthalene (DHN) melanin enhanced the uptake of A. fumigatus conidia by epithelial cells when compared with non-pigmented pksP mutant conidia. Furthermore, conidia of A. fumigatus were able to survive within epithelial cells. This was due to the presence of DHN melanin in the cell wall of conidia, because melanised wild-type conidia showed a higher survival rate inside epithelial cells and led to inhibition of acidification of phagolysosomes. Both effects were not observed for white (non-melanised) conidia of the pksP mutant strain. Moreover, in contrast to pksP mutant conidia, melanised wild-type conidia were able to inhibit the extrinsic apoptotic pathway in A549 lung epithelial cells even for longer periods. The anti-apoptotic effect was not restricted to conidia, because both conidia-derived melanin ghosts (cell-free DHN melanin) and a different type of melanin, dihydroxyphenylalanine (DOPA) melanin, acted anti-apoptotically. Taken together, these data indicate the possibility of melanin-dependent persistence of conidia in lung epithelial cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

Epithelial organization and cyst lumen expansion require efficient Sec13-Sec31-driven secretion.

PubMed

Townley, Anna K; Schmidt, Katy; Hodgson, Lorna; Stephens, David J

2012-02-01

Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPII-dependent secretion, notably assembly of Sec13-Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture.

Epithelial organization and cyst lumen expansion require efficient Sec13–Sec31-driven secretion

PubMed Central

Townley, Anna K.; Schmidt, Katy; Hodgson, Lorna; Stephens, David J.

2012-01-01

Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPII-dependent secretion, notably assembly of Sec13–Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture. PMID:22331354

Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

PubMed Central

Navarro-Garcia, Fernando; Serapio-Palacios, Antonio; Ugalde-Silva, Paul; Tapia-Pastrana, Gabriela; Chavez-Dueñas, Lucia

2013-01-01

The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology. PMID:23509714

Spatial Distribution of Niche and Stem Cells in Ex Vivo Human Limbal Cultures

PubMed Central

Kacham, Santhosh; Purushotham, Jyothi; Maddileti, Savitri; Siamwala, Jamila; Sangwan, Virender Singh

2014-01-01

Stem cells at the limbus mediate corneal epithelial regeneration and regulate normal tissue homeostasis. Ex vivo cultured limbal epithelial transplantations are being widely practiced in the treatment of limbal stem cell deficiency. In this report, we examined whether the limbal niche cells that nurture and regulate epithelial stem cells coexist in ex vivo limbal cultures. We also compared the inherent differences between explant and suspension culture systems in terms of spatial distribution of niche cells and their effect on epithelial stem cell proliferation, migration, and differentiation in vitro. We report that the stem cell content of both culture systems was similar, explaining the comparable clinical outcomes reported using these two methods. We also showed that the niche cells get expanded in culture and the nestin-positive cells migrate at the leading edges to direct epithelial cell migration in suspension cultures, whereas they are limited to the intact niche in explant cultures. We provide evidence that C/EBPδ-positive, p15-positive, and quiescent, label-retaining, early activated stem cells migrate at the leading edges to regulate epithelial cell proliferation in explant cultures, and this position effect is lost in early suspension cultures. However, in confluent suspension cultures, the stem cells and niche cells interact with each another, migrate in spiraling patterns, and self-organize to form three-dimensional niche-like compartments resembling the limbal crypts and thereby reestablish the position effect. These 3D-sphere clusters are enriched with nestin-, vimentin-, S100-, and p27-positive niche cells and p15-, p21-, p63α-, C/EBPδ-, ABCG2-, and Pax6-positive quiescent epithelial stem cells. PMID:25232182

Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

PubMed

Danelishvili, Lia; McGarvey, Jeffery; Li, Yong-Jun; Bermudez, Luiz E

2003-09-01

Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the expression of caspase-1, caspase-3 and caspase-10, was inhibited. Inhibition of replication of intracellular bacteria resulted in an increase in apoptosis in both cell types. Our results showed that the differential induction of apoptosis between macrophages and alveolar epithelial cells represents specific strategies of M. tuberculosis for survival in the host.

Smooth muscle cell-specific knockout of androgen receptor: a new model for prostatic disease.

PubMed

Welsh, Michelle; Moffat, Lindsey; McNeilly, Alan; Brownstein, David; Saunders, Philippa T K; Sharpe, Richard M; Smith, Lee B

2011-09-01

Androgen-driven stromal-epithelial interactions play a key role in normal prostate development and function as well as in the progression of common prostatic diseases such as benign prostatic hyperplasia and prostate cancer. However, exactly how, and via which cell type, androgens mediate their effects in the adult prostate remains unclear. This study investigated the role for smooth muscle (SM) androgen signaling in normal adult prostate homeostasis and function using mice in which androgen receptor was selectively ablated from prostatic SM cells. In adulthood the knockout (KO) mice displayed a 44% reduction in prostate weight and exhibited histological abnormalities such as hyperplasia, inflammation, fibrosis, and reduced expression of epithelial, SM, and stem cell identify markers (e.g. p63 reduced by 27% and Pten by 31%). These changes emerged beyond puberty and were not explained by changes in serum hormones. Furthermore, in response to exogenous estradiol, adult KO mice displayed an 8.5-fold greater increase in prostate weight than controls and developed urinary retention. KO mice also demonstrated a reduced response to castration compared with controls. Together these results demonstrate that prostate SM cells are vital in mediating androgen-driven stromal-epithelial interactions in adult mouse prostates, determining cell identity and function and limiting hormone-dependent epithelial cell proliferation. This novel mouse model provides new insight into the possible role for SM androgen action in prostate disease.

Critical Role of HAX-1 in Promoting Avian Influenza Virus Replication in Lung Epithelial Cells

PubMed Central

He, Ganlin; Cardona, Carol J.

2018-01-01

The PB1-F2 protein of influenza A virus has been considered a virulence factor, but its function in inducing apoptosis may be of disadvantage to viral replication. Host mechanisms to regulate PB1-F2-induced apoptosis remain unknown. We generated a PB1-F2-deficient avian influenza virus (AIV) H9N2 and found that the mutant virus replicated less efficiently in human lung epithelial cells. The PB1-F2-deficient virus produced less apoptotic cells, indicating that PB1-F2 of the H9N2 virus promotes apoptosis, occurring at the early stage of infection, in the lung epithelial cells. To understand how host cells regulate PB1-F2-induced apoptosis, we explored to identify cellular proteins interacting with PB1-F2 and found that HCLS1-associated protein X-1 (HAX-1), located mainly in the mitochondria as an apoptotic inhibitor, interacted with PB1-F2. Increased procaspase-9 activations, induced by PB1-F2, could be suppressed by HAX-1. In HAX-1 knockdown A549 cells, the replication of AIV H9N2 was suppressed in parallel to the activation of caspase-3 activation, which increased at the early stage of infection. We hypothesize that HAX-1 promotes AIV replication by interacting with PB1-F2, resulting in the suppression of apoptosis, prolonged cell survival, and enhancement of viral replication. Our data suggest that HAX-1 may be a promoting factor for AIV H9N2 replication through desensitizing PB1-F2 from its apoptotic induction in human lung epithelial cells. PMID:29576744

Tenascin-X promotes epithelial-to-mesenchymal transition by activating latent TGF-β

PubMed Central

Alcaraz, Lindsay B.; Exposito, Jean-Yves; Chuvin, Nicolas; Pommier, Roxane M.; Cluzel, Caroline; Martel, Sylvie; Sentis, Stéphanie; Bartholin, Laurent; Lethias, Claire

2014-01-01

Transforming growth factor β (TGF-β) isoforms are secreted as inactive complexes formed through noncovalent interactions between the bioactive TGF-β entity and its N-terminal latency-associated peptide prodomain. Extracellular activation of the latent TGF-β complex is a crucial step in the regulation of TGF-β function for tissue homeostasis. We show that the fibrinogen-like (FBG) domain of the matrix glycoprotein tenascin-X (TNX) interacts physically with the small latent TGF-β complex in vitro and in vivo, thus regulating the bioavailability of mature TGF-β to cells by activating the latent cytokine into an active molecule. Activation by the FBG domain most likely occurs through a conformational change in the latent complex and involves a novel cell adhesion–dependent mechanism. We identify α11β1 integrin as a cell surface receptor for TNX and show that this integrin is crucial to elicit FBG-mediated activation of latent TGF-β and subsequent epithelial-to-mesenchymal transition in mammary epithelial cells. PMID:24821840

BHC80 is Critical in Suppression of Snail-LSD1 Interaction and Breast Cancer Metastasis

DTIC Science & Technology

2014-04-01

Epithelial-mesenchymal transitions in development and disease . Cell 2009; 139:871-90. 9. Wu Y, Zhou BP. Snail: More than EMT. Cell Adh Migr 2010; 4:199...Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease . Cell 2009; 139:871-90. 9. Wu Y, Zhou BP. Snail: More than EMT. Cell...QSRKAFNCKYC Snail ( Canine ) QTRKAFNCKYC Snail (Monkey) QSRKAFNCKYC Snail (Opossum) QPRKAFICKVC A D E F CHX 0 1 3 5 7 0 1 3 5 7 WT Snail

Co-culture of Gastric Organoids and Immortalized Stomach Mesenchymal Cells.

PubMed

Bertaux-Skeirik, Nina; Centeno, Jomaris; Feng, Rui; Schumacher, Michael A; Shivdasani, Ramesh A; Zavros, Yana

2016-01-01

Three-dimensional primary epithelial-derived gastric organoids have recently been established as an important tool to study gastric development, physiology, and disease. Specifically, mouse-derived fundic gastric organoids (mFGOs) co-cultured with Immortalized Stomach Mesenchymal Cells (ISMCs) reflect expression patterns of mature fundic cell types seen in vivo, thus allowing for long-term in vitro studies of gastric epithelial cell physiology, regeneration, and bacterial-host interactions. Here, we describe the development and culture of mFGOs, co-cultured with ISMCs.

Wnt1a maintains characteristics of dermal papilla cells that induce mouse hair regeneration in a 3D preculture system.

PubMed

Dong, Liang; Hao, Haojie; Liu, Jiejie; Tong, Chuan; Ti, Dongdong; Chen, Deyun; Chen, Li; Li, Meirong; Liu, Huiling; Fu, Xiaobing; Han, Weidong

2017-05-01

Hair follicle morphogenesis and regeneration depend on intensive but well-orchestrated interactions between epithelial and mesenchymal components. Therefore, an alternative strategy to reproduce the process of epithelial-mesenchymal interaction in vitro could use a 3D system containing appropriate cell populations. The 3D air-liquid culture system for reproducibly generating hair follicles from dissociated epithelial and dermal papilla (DP) cells combined with a collagen-chitosan scaffold is described in this study. Wnt-CM was prepared from the supernatant of Wnt1a-expressing bone marrow mesenchymal stem cells (BM-MSCs) that maintain the hair-inducing gene expression of DP cells. The collagen-chitosan scaffold cells (CCS cells) were constructed using a two-step method by inoculating the Wnt-CM-treated DP cells and epidermal (EP) cells into the CCS. The cells in the air-liquid culture formed dermal condensates and a proliferative cell layer in vitro. The CCS cells were able to induce hair regeneration in nude mice. The results demonstrate that Wnt-CM can maintain the hair induction ability of DP cells in expansion cultures, and this approach can be used for large-scale preparation of CCS cells in vitro to treat hair loss. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Alpha-catenin-dependent recruitment of the centrosomal protein CAP350 to adherens junctions allows epithelial cells to acquire a columnar shape.

PubMed

Gavilan, Maria P; Arjona, Marina; Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R; Bornens, Michel; Rios, Rosa M

2015-03-01

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.

Alpha-catenin-Dependent Recruitment of the Centrosomal Protein CAP350 to Adherens Junctions Allows Epithelial Cells to Acquire a Columnar Shape

PubMed Central

Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R.; Bornens, Michel; Rios, Rosa M.

2015-01-01

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis. PMID:25764135

Granulocyte-macrophage colony-stimulating factor responses of oral epithelial cells to Candida albicans.

PubMed

Dongari-Bagtzoglou, A; Kashleva, H

2003-06-01

Candida albicans is the principal fungal species responsible for oropharyngeal candidiasis, the most frequent opportunistic infection associated with immune deficiencies. Cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), are important in the generation of effective immunity to C. albicans. The purposes of this investigation were to determine whether C. albicans triggers secretion of GM-CSF by oral epithelial cells in vitro and to investigate mechanisms of host cell-fungal interactions that trigger such responses. Oral epithelial cell lines as well as primary oral mucosal epithelial cells were challenged with stationary phase viable C. albicans, added to human cell cultures at varying yeast:oral cell ratios. Yeast were allowed to germinate for up to 48 h and supernatants were analyzed for GM-CSF by ELISA. Fixed organisms, germination-deficient mutants and separation of yeast from epithelial cells using cell culture inserts were used to assess the effects of viability, germination and physical contact, respectively, on the GM-CSF responses of these cells. Two out of three cell lines and three out of six primary cultures responded to C. albicans with an increase in GM-CSF secretion. GM-CSF responses were contact-dependent, strain-dependent, required yeast viability and were optimal when the yeast germinated into hyphae.

Targeting the complex interactions between microbiota, host epithelial and immune cells in inflammatory bowel disease.

PubMed

Hirata, Yoshihiro; Ihara, Sozaburo; Koike, Kazuhiko

2016-11-01

Inflammatory bowel disease (IBD) is a chronic inflammatory intestinal disorder that includes two distinct disease categories: ulcerative colitis and Crohn's disease. Epidemiological, genetic, and experimental studies have revealed many important aspects of IBD. Genetic susceptibility, inappropriate immune responses, environmental changes, and intestinal microbiota are all associated with the development of IBD. However, the exact mechanisms of the disease and the interactions among these pathogenic factors are largely unknown. Here we introduce recent findings from experimental colitis models that investigated the interactions between host genetic susceptibility and gut microbiota. In addition, we discuss new strategies for the treatment of IBD, focusing on the complex interactions between microbiota and host epithelial and immune cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Interaction between Trichomonas vaginalis and the Prostate Epithelium.

PubMed

Kim, Jung-Hyun; Han, Ik-Hwan; Kim, Sang-Su; Park, Soon-Jung; Min, Duk-Young; Ahn, Myoung-Hee; Ryu, Jae-Sook

2017-04-01

Most men infected with Trichomonas vaginalis are asymptomatic and can remain undiagnosed and untreated. This has been hypothesized to result in chronic persistent prostatic infection. Adhesion of the protozoan organisms to mucosal cells is considered a first and prerequisite step for T. vaginalis infection. Adhesion of T. vaginalis to prostate epithelial cells has not yet been observed; however, there are several reports about inflammation of prostate epithelial cells induced by T. vaginalis . The aim of this study was to investigate whether adhesion and cytotoxicity of T. vaginalis are involved in inflammation of prostate epithelial cells. When RWPE-1 cells were infected with T. vaginalis (1:0.4 or 1:4), adhesion of T. vaginalis continuously increased for 24 hr or 3 hr, respectively. The cytotoxicity of prostate epithelial cells infected with T. vaginalis (RWPE-1: T. vaginalis =1:0.4) increased at 9 hr; at an infection ratio of 1:4, cytotoxicity increased after 3 hr. When the RWPE-1 to T. vaginalis ratio was 1:0.4 or 1:4, production of IL-1β, IL-6, CCL2, and CXCL8 also increased. Epithelial-mesenchymal transition (EMT) was verified by measuring decreased E-cadherin and increased vimentin expression at 24 hr and 48 hr. Taken together, the results indicate that T. vaginalis adhered to prostate epithelial cells, causing cytotoxicity, pro-inflammatory cytokine production, and EMT. Our findings suggest for the first time that T. vaginalis may induce inflammation via adhesion to normal prostate epithelial cells.

Epithelial Microvilli Establish an Electrostatic Barrier to Microbial Adhesion

PubMed Central

Bennett, Kaila M.; Walker, Sharon L.

2014-01-01

Microvilli are membrane extensions on the apical surface of polarized epithelia, such as intestinal enterocytes and tubule and duct epithelia. One notable exception in mucosal epithelia is M cells, which are specialized for capturing luminal microbial particles; M cells display a unique apical membrane lacking microvilli. Based on studies of M cell uptake under different ionic conditions, we hypothesized that microvilli may augment the mucosal barrier by providing an increased surface charge density from the increased membrane surface and associated glycoproteins. Thus, electrostatic charges may repel microbes from epithelial cells bearing microvilli, while M cells are more susceptible to microbial adhesion. To test the role of microvilli in bacterial adhesion and uptake, we developed polarized intestinal epithelial cells with reduced microvilli (“microvillus-minus,” or MVM) but retaining normal tight junctions. When tested for interactions with microbial particles in suspension, MVM cells showed greatly enhanced adhesion and uptake of particles compared to microvillus-positive cells. This preference showed a linear relationship to bacterial surface charge, suggesting that microvilli resist binding of microbes by using electrostatic repulsion. Moreover, this predicts that pathogen modification of electrostatic forces may contribute directly to virulence. Accordingly, the effacement effector protein Tir from enterohemorrhagic Escherichia coli O157:H7 expressed in epithelial cells induced a loss of microvilli with consequent enhanced microbial binding. These results provide a new context for microvillus function in the host-pathogen relationship, based on electrostatic interactions. PMID:24778113

Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes

PubMed Central

Han, Yeon Soo; Thompson, Joanne; Kafatos, Fotis C.; Barillas-Mury, Carolina

2000-01-01

We present a detailed analysis of the interactions between Anopheles stephensi midgut epithelial cells and Plasmodium berghei ookinetes during invasion of the mosquito by the parasite. In this mosquito, P.berghei ookinetes invade polarized columnar epithelial cells with microvilli, which do not express high levels of vesicular ATPase. The invaded cells are damaged, protrude towards the midgut lumen and suffer other characteristic changes, including induction of nitric oxide synthase (NOS) expression, a substantial loss of microvilli and genomic DNA fragmentation. Our results indicate that the parasite inflicts extensive damage leading to subsequent death of the invaded cell. Ookinetes were found to be remarkably plastic, to secrete a subtilisin-like serine protease and the GPI-anchored surface protein Pbs21 into the cytoplasm of invaded cells, and to be capable of extensive lateral movement between cells. The epithelial damage inflicted is repaired efficiently by an actin purse-string-mediated restitution mechanism, which allows the epithelium to ‘bud off’ the damaged cells without losing its integrity. A new model, the time bomb theory of ookinete invasion, is proposed and its implications are discussed. PMID:11080150

Stromal loss of TGFβ drives cancer growth in the epithelium via inflammation | Center for Cancer Research

Cancer.gov

Interactions between epithelial and stromal cells play an important role in cancer development and progression. Epithelial cancers develop when changes occur to tumor suppressor genes in stromal fibroblast cells. For example, loss of tumor suppressor, p53, in stromal fibroblasts leads to p53 inactivation in the epithelium in a prostate cancer model, and disruption of the

Foot-and-mouth disease virus replicates only transiently in well-differentiated porcine nasal epithelial cells.

PubMed

Dash, Pradyot; Barnett, Paul V; Denyer, Michael S; Jackson, Terry; Stirling, Catrina M A; Hawes, Philippa C; Simpson, Jennifer L; Monaghan, Paul; Takamatsu, Haru-H

2010-09-01

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo. FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin alphav beta6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-beta) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-beta mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-alpha mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo. In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.

The effect of LHRH antagonist cetrorelix in crossover conditioned media from epithelial (BPH-1) and stromal (WPMY-1) prostate cells.

PubMed

Siejka, A; Schally, A V; Barabutis, N

2014-01-01

Stromal cells strictly modulate the differentiation of the normal prostate epithelium. In benign prostatic hyperplasia (BPH) tissue, the ratio of stromal to epithelial cells reaches a 5:1 ratio. In this study, we evaluated the effects of crossover conditioned media (CM) of stromal and epithelial prostate cells before and after treatment with LHRH antagonist Cetrorelix. WPMY-1 human prostate stromal cells and BPH-1 human benign prostatic hyperplasia cells were cultured in vitro and the effects of crossover conditioned media (CM) from those cells were studied. We evaluated the effect of Cetrorelix on the expression of PCNA and p53 in those cells. We then studied the effect of Cetrorelix on BPH-1 cells cultured with the CM from WPMY-1 cells, as well as the mechanisms which govern these interactions. CM from WPMY-1 cells strongly stimulated the proliferation of BPH-1 cells in a dose dependent manner, while CM from BPH-1 cells only slightly increased the proliferation of WPMY-1 cells. Cetrorelix inhibited the proliferation of both cell lines and the expression of PCNA, while the expression of p53 was increased. Cetrorelix also inhibited the proliferation of BPH-1 cells stimulated with the CM from WPMY-1 cells. In the crossover experiment, conditioned media from WPMY-1 and BPH-1 cells increased the expression of phosphorylated ERK1/2 and STAT3. Our results support previous observations on the bidirectional stromal-epithelial interactions in prostate gland and shed more light on the mechanistic action of those effects. Our study strongly supports the hypothesis that LHRH antagonists may be beneficial for BPH prevention and treatment. © Georg Thieme Verlag KG Stuttgart · New York.

The Interactive Roles of Lipopolysaccharides and dsRNA/Viruses on Respiratory Epithelial Cells and Dendritic Cells in Allergic Respiratory Disorders: The Hygiene Hypothesis.

PubMed

Lin, Tsang-Hsiung; Su, Hsing-Hao; Kang, Hong-Yo; Chang, Tsung-Hsien

2017-10-23

The original hygiene hypothesis declares "more infections in early childhood protect against later atopy". According to the hygiene hypothesis, the increased incidence of allergic disorders in developed countries is explained by the decrease of infections. Epithelial cells and dendritic cells play key roles in bridging the innate and adaptive immune systems. Among the various pattern-recognition receptor systems of epithelial cells and dendritic cells, including toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and others, TLRs are the key systems of immune response regulation. In humans, TLRs consist of TLR1 to TLR10. They regulate cellular responses through engagement with TLR ligands, e.g., lipopolysaccharides (LPS) acts through TLR4 and dsRNA acts through TLR3, but there are certain common components between these two TLR pathways. dsRNA activates epithelial cells and dendritic cells in different directions, resulting in allergy-related Th2-skewing tendency in epithelial cells, and Th1-skewing tendency in dendritic cells. The Th2-skewing effect by stimulation of dsRNA on epithelial cells could be suppressed by the presence of LPS above some threshold. When LPS level decreases, the Th2-skewing effect increases. It may be via these interrelated networks and related factors that LPS modifies the allergic responses and provides a plausible mechanism of the hygiene hypothesis. Several hygiene hypothesis-related phenomena, seemingly conflicting, are also discussed in this review, along with their proposed mechanisms.

Regulation of exosome release from mammary epithelial and breast cancer cells - a new regulatory pathway.

PubMed

Riches, Andrew; Campbell, Elaine; Borger, Eva; Powis, Simon

2014-03-01

Exosomes are small 50-100nm sized extracellular vesicles released from normal and tumour cells and are a source of a new intercellular communication pathway. Tumour exosomes promote tumour growth and progression. What regulates the release and homoeostatic levels of exosomes, in cancer, in body fluids remains undefined. We utilised a human mammary epithelial cell line (HMEC B42) and a breast cancer cell line derived from it (B42 clone 16) to investigate exosome production and regulation. Exosome numbers were quantified using a Nanosight LM10 and measured in culture supernatants in the absence and presence of exosomes in the medium. Concentrated suspensions of exosomes from the normal mammary epithelial cells, the breast cancer cells and bladder cancer cells were used. The interaction of exosomes with tumour cells was also investigated using fluorescently labelled exosomes. Exosome release from normal human mammary epithelial cells and breast cancer cells is regulated by the presence of exosomes, derived from their own cells, in the extracellular environment of the cells. Exosomes from normal mammary epithelial cells also inhibit exosome secretion by breast cancer cells, which occurs in a tissue specific manner. Labelled exosomes from mammary epithelial cells are internalised into the tumour cells implicating a dynamic equilibrium and suggesting a mechanism for feedback control. These data suggest a previously unknown novel feedback regulatory mechanism for controlling exosome release, which may highlight a new therapeutic approach to controlling the deleterious effects of tumour exosomes. This regulatory mechanism is likely to be generic to other tumours. Copyright © 2014 Elsevier Ltd. All rights reserved.

Interleukin-8, CXCL1, and MicroRNA miR-146a Responses to Probiotic Escherichia coli Nissle 1917 and Enteropathogenic E. coli in Human Intestinal Epithelial T84 and Monocytic THP-1 Cells after Apical or Basolateral Infection.

PubMed

Sabharwal, Harshana; Cichon, Christoph; Ölschläger, Tobias A; Sonnenborn, Ulrich; Schmidt, M Alexander

2016-09-01

Bacterium-host interactions in the gut proceed via directly contacted epithelial cells, the host's immune system, and a plethora of bacterial factors. Here we characterized and compared exemplary cytokine and microRNA (miRNA) responses of human epithelial and THP-1 cells toward the prototype enteropathogenic Escherichia coli (EPEC) strain E2348/69 (O127:H6) and the probiotic strain Escherichia coli Nissle 1917 (EcN) (O6:K5:H1). Human T84 and THP-1 cells were used as cell culture-based model systems for epithelial and monocytic cells. Polarized T84 monolayers were infected apically or basolaterally. Bacterial challenges from the basolateral side resulted in more pronounced cytokine and miRNA responses than those observed for apical side infections. Interestingly, the probiotic EcN also caused a pronounced transcriptional increase of proinflammatory CXCL1 and interleukin-8 (IL-8) levels when human T84 epithelial cells were infected from the basolateral side. miR-146a, which is known to regulate adaptor molecules in Toll-like receptor (TLR)/NF-κB signaling, was found to be differentially regulated in THP-1 cells between probiotic and pathogenic bacteria. To assess the roles of flagella and flagellin, we employed several flagellin mutants of EcN. EcN flagellin mutants induced reduced IL-8 as well as CXCL1 responses in T84 cells, suggesting that flagellin is an inducer of this cytokine response. Following infection with an EPEC type 3 secretion system (T3SS) mutant, we observed increased IL-8 and CXCL1 transcription in T84 and THP-1 cells compared to that in wild-type EPEC. This study emphasizes the differential induction of miR-146a by pathogenic and probiotic E. coli strains in epithelial and immune cells as well as a loss of probiotic properties in EcN interacting with cells from the basolateral side. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Disruption of lipid rafts interferes with the interaction of Toxoplasma gondii with macrophages and epithelial cells.

PubMed

Cruz, Karla Dias; Cruz, Thayana Araújo; Veras de Moraes, Gabriela; Paredes-Santos, Tatiana Christina; Attias, Marcia; de Souza, Wanderley

2014-01-01

The intracellular parasite Toxoplasma gondii can penetrate any warm-blooded animal cell. Conserved molecular assemblies of host cell plasma membranes should be involved in the parasite-host cell recognition. Lipid rafts are well-conserved membrane microdomains that contain high concentrations of cholesterol, sphingolipids, glycosylphosphatidylinositol, GPI-anchored proteins, and dually acylated proteins such as members of the Src family of tyrosine kinases. Disturbing lipid rafts of mouse peritoneal macrophages and epithelial cells of the lineage LLC-MK2 with methyl-beta cyclodextrin (M β CD) and filipin, which interfere with cholesterol or lidocaine, significantly inhibited internalization of T. gondii in both cell types, although adhesion remained unaffected in macrophages and decreased only in LLC-MK2 cells. Scanning and transmission electron microscopy confirmed these observations. Results are discussed in terms of the original role of macrophages as professional phagocytes versus the LLC-MK2 cell lineage originated from kidney epithelial cells.

Distinct cytoprotective roles of pyruvate and ATP by glucose metabolism on epithelial necroptosis and crypt proliferation in ischaemic gut

PubMed Central

Huang, Ching‐Ying; Kuo, Wei‐Ting; Huang, Chung‐Yen; Lee, Tsung‐Chun; Chen, Chin‐Tin; Peng, Wei‐Hao; Lu, Kuo‐Shyan; Yang, Chung‐Yi

2016-01-01

Key points Intestinal ischaemia causes epithelial death and crypt dysfunction, leading to barrier defects and gut bacteria‐derived septic complications.Enteral glucose protects against ischaemic injury; however, the roles played by glucose metabolites such as pyruvate and ATP on epithelial death and crypt dysfunction remain elusive.A novel form of necrotic death that involves the assembly and phosphorylation of receptor interacting protein kinase 1/3 complex was found in ischaemic enterocytes.Pyruvate suppressed epithelial cell death in an ATP‐independent manner and failed to maintain crypt function. Conversely, replenishment of ATP partly restored crypt proliferation but had no effect on epithelial necroptosis in ischaemic gut.Our data argue against the traditional view of ATP as the main cytoprotective factor by glucose metabolism, and indicate a novel anti‐necroptotic role of glycolytic pyruvate under ischaemic stress. Abstract Mesenteric ischaemia/reperfusion induces epithelial death in both forms of apoptosis and necrosis, leading to villus denudation and gut barrier damage. It remains unclear whether programmed cell necrosis [i.e. receptor‐interacting protein kinase (RIP)‐dependent necroptosis] is involved in ischaemic injury. Previous studies have demonstrated that enteral glucose uptake by sodium‐glucose transporter 1 ameliorated ischaemia/reperfusion‐induced epithelial injury, partly via anti‐apoptotic signalling and maintenance of crypt proliferation. Glucose metabolism is generally assumed to be cytoprotective; however, the roles played by glucose metabolites (e.g. pyruvate and ATP) on epithelial cell death and crypt dysfunction remain elusive. The present study aimed to investigate the cytoprotective effects exerted by distinct glycolytic metabolites in ischaemic gut. Wistar rats subjected to mesenteric ischaemia were enterally instilled glucose, pyruvate or liposomal ATP. The results showed that intestinal ischaemia caused RIP1‐dependent epithelial necroptosis and villus destruction accompanied by a reduction in crypt proliferation. Enteral glucose uptake decreased epithelial cell death and increased crypt proliferation, and ameliorated mucosal histological damage. Instillation of cell‐permeable pyruvate suppressed epithelial cell death in an ATP‐independent manner and improved the villus morphology but failed to maintain crypt function. Conversely, the administration of liposomal ATP partly restored crypt proliferation but did not reduce epithelial necroptosis and histopathological injury. Lastly, glucose and pyruvate attenuated mucosal‐to‐serosal macromolecular flux and prevented enteric bacterial translocation upon blood reperfusion. In conclusion, glucose metabolites protect against ischaemic injury through distinct modes and sites, including inhibition of epithelial necroptosis by pyruvate and the promotion of crypt proliferation by ATP. PMID:27121603

The role of gut microbiota in health and disease: In vitro modeling of host-microbe interactions at the aerobe-anaerobe interphase of the human gut.

PubMed

von Martels, Julius Z H; Sadaghian Sadabad, Mehdi; Bourgonje, Arno R; Blokzijl, Tjasso; Dijkstra, Gerard; Faber, Klaas Nico; Harmsen, Hermie J M

2017-04-01

The microbiota of the gut has many crucial functions in human health. Dysbiosis of the microbiota has been correlated to a large and still increasing number of diseases. Recent studies have mostly focused on analyzing the associations between disease and an aberrant microbiota composition. Functional studies using (in vitro) gut models are required to investigate the precise interactions that occur between specific bacteria (or bacterial mixtures) and gut epithelial cells. As most gut bacteria are obligate or facultative anaerobes, studying their effect on oxygen-requiring human gut epithelial cells is technically challenging. Still, several (anaerobic) bacterial-epithelial co-culture systems have recently been developed that mimic host-microbe interactions occurring in the human gut, including 1) the Transwell "apical anaerobic model of the intestinal epithelial barrier", 2) the Host-Microbiota Interaction (HMI) module, 3) the "Human oxygen-Bacteria anaerobic" (HoxBan) system, 4) the human gut-on-a-chip and 5) the HuMiX model. This review discusses the role of gut microbiota in health and disease and gives an overview of the characteristics and applications of these novel host-microbe co-culture systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

Marrow-derived mesenchymal stem cells: role in epithelial tumor cell determination.

PubMed

Fierro, Fernando A; Sierralta, Walter D; Epuñan, Maria J; Minguell, José J

2004-01-01

Marrow stroma represents an advantageous environment for development of micrometastatic cells. Within the cellular structure of marrow stroma, mesenchymal stem cells (MSC) have been postulated as an interacting target for disseminated cancer cells. The studies reported here were performed to gain more information on the interaction of the human breast cancer cell line MCF-7 with human bone marrow-derived MSC cells and to investigate whether this interaction affects tumor cell properties. The results showed that after co-culture with MSC, changes were detected in the morphology, proliferative capacity and aggregation pattern of MCF-7 cells, but these parameters were not affected after the co-culture of MSC cells with a non-tumorigenic breast epithelial cell line, MCF-10. Since the indirect culture of MCF-7 with MSC or its products also resulted in functional changes in the tumor cells, we evaluated whether these effects could be attributed to growth factors produced by MSC cells. It was found that VEGF and IL-6 mimic the effects produced by MSC or its products on the proliferation and aggregation properties of MCF-7, cells, respectively. Thus, it seems that after entry of disseminated tumor cells into the marrow space, their proliferative and morphogenetic organization patterns are modified after interaction with distinct stromal cells and/or with specific signals from the marrow microenvironment.

A multicellular view of cytokinesis in epithelial tissue.

PubMed

Herszterg, Sophie; Pinheiro, Diana; Bellaïche, Yohanns

2014-05-01

The study of cytokinesis in single-cell systems provided a wealth of knowledge on the molecular and biophysical mechanisms controlling daughter cell separation. In this review, we outline recent advances in the understanding of cytokinesis in epithelial tissues. These findings provide evidence for how the cytokinetic machinery adapts to a multicellular context and how the cytokinetic machinery is itself exploited by the tissue for the preservation of tissue function and architecture during proliferation. We propose that cytokinesis in epithelia should be viewed as a multicellular process, whereby the biochemical and mechanical interactions between the dividing cell and its neighbors are essential for successful daughter cell separation while defining epithelial tissue organization and preserving tissue integrity. Copyright © 2013 Elsevier Ltd. All rights reserved.

The regulation of tooth morphogenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog through epithelial-mesenchymal interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishida, Kentaro; Murofushi, Mayumi; Nakao, Kazuhisa

2011-02-18

Research highlights: {yields} Bioengineered teeth regulated the contact area of epithelium and mesenchyme. {yields} The crown width is regulated by the contact area of the epithelium and mesenchyme. {yields} This regulation is associated with cell proliferation and Sonic hedgehog expression. {yields} The cusp number is correlated with the crown width of the bioengineered tooth. {yields} Cell proliferation and Shh expression areas regulate the tooth morphogenesis. -- Abstract: Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial-mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the numbermore » and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth.« less

Fusobacterium nucleatum binding to complement regulatory protein CD46 modulates the expression and secretion of cytokines and matrix metalloproteinases by oral epithelial cells.

PubMed

Mahtout, Hayette; Chandad, Fatiha; Rojo, Jose M; Grenier, Daniel

2011-02-01

Periodontitis is a chronic inflammatory disease that results in the destruction of the supporting tissues of the teeth. Gingival epithelial cells are an important mechanical barrier and participate in the host inflammatory response to periodontopathogens. The aim of the present study is to investigate the capacity of Fusobacterium nucleatum to bind to the complement regulatory protein CD46 expressed by oral epithelial cells and to determine the impact of the binding on the gene expression and protein secretion of interleukin (IL)-6, IL-8, and matrix metalloproteinase (MMP)-9 by oral epithelial cells. Binding of recombinant human CD46 to the surface of F. nucleatum was demonstrated by immunologic assays. After stimulation of oral epithelial cells with F. nucleatum, gene expression was determined by real-time polymerase chain reaction analysis while protein secretion was monitored by enzyme-linked immunosorbent assays. Heat and protease treatments of bacterial cells reduced CD46 binding. F. nucleatum-bound CD46 mediated the cleavage of C3b in the presence of factor I. Stimulating oral epithelial cells with F. nucleatum at a multiplicity of infection of 50 resulted in a significant upregulation of the gene expression and protein secretion of IL-6, IL-8, and MMP-9 by oral epithelial cells. However, pretreating the epithelial cells with an anti-CD46 polyclonal antibody attenuated the production of IL-6, IL-8, and MMP-9 in response to F. nucleatum. Such an inhibitory effect was not observed with non-specific antibodies. The present study demonstrates that F. nucleatum can bind the complement regulatory protein CD46. The interaction of F. nucleatum with epithelial cell surface CD46 may contribute to increasing the levels of proinflammatory mediators and MMPs in periodontal sites and consequently modulate tissue destruction.

Transmigrated neutrophils in the intestinal lumen engage ICAM 1 to regulate the epithelial barrier and neutrophil recruitment

PubMed Central

Sumagin, Ronen; Robin, Alex Z.; Nusrat, Asma; Parkos, Charles A.

2014-01-01

Neutrophil (PMN) transepithelial migration (TEM) and accumulation in luminal spaces is a hallmark of mucosal inflammation. TEM has been extensively modeled, however the functional consequences and molecular basis of PMN interactions with luminal epithelial ligands are not clear. Here we report that cytokine-induced expression of a PMN ligand, intercellular adhesion molecule-1 (ICAM-1), exclusively on the luminal (apical) membrane of the intestinal epithelium results in accumulation and enhanced motility of transmigrated PMN on the apical epithelial surface. Using complementary in-vitro and in-vivo approaches we demonstrate that ligation of epithelial ICAM-1 by PMN or with specific antibodies results in myosin light chain kinase (MLCK)-dependent increases in epithelial permeability that are associated with enhanced PMN TEM. Effects of ICAM-1 ligation on epithelial permeability and PMN migration in-vivo were blocked after intraluminal addition of peptides derived from the cytoplasmic domain of ICAM-1. These findings provide new evidence for functional interactions between PMN and epithelial cells after migration into the intestinal lumen. While such interactions may aid in clearance of invading microorganisms by promoting PMN recruitment, engagement of ICAM-1 under pathologic conditions would increase accumulation of epithelial-associated PMN, thus contributing to mucosal injury as observed in conditions including ulcerative colitis. PMID:24345805

Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection.

PubMed

Subramanian, T; Zhao, Ling-Jun; Chinnadurai, G

2013-09-01

Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP-E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP-E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection

PubMed Central

Subramanian, T.; Zhao, Ling-jun; Chinnadurai, G.

2013-01-01

Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP-E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP-E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. PMID:23747199

Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katano, Takahito; Ootani, Akifumi; Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501

2013-03-22

Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system withinmore » the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment.« less

Interaction of micron and nano-sized particles with cells of the dura mater.

PubMed

Papageorgiou, Iraklis; Marsh, Rainy; Tipper, Joanne L; Hall, Richard M; Fisher, John; Ingham, Eileen

2014-10-01

Intervertebral total disc replacements (TDR) are used in the treatment of degenerative spinal disc disease. There are, however, concerns that they may be subject to long-term failure due to wear. The adverse effects of TDR wear have the potential to manifest in the dura mater and surrounding tissues. The aim of this study was to investigate the physiological structure of the dura mater, isolate the resident dural epithelial and stromal cells and analyse the capacity of these cells to internalise model polymer particles. The porcine dura mater was a collagen-rich structure encompassing regularly arranged fibroblastic cells within an outermost epithelial cell layer. The isolated dural epithelial cells had endothelial cell characteristics (positive for von Willebrand factor, CD31, E-cadherin and desmoplakin) and barrier functionality whereas the fibroblastic cells were positive for collagen I and III, tenascin and actin. The capacity of the dural cells to take up model particles was dependent on particle size. Nanometer sized particles readily penetrated both types of cells. However, dural fibroblasts engulfed micron-sized particles at a much higher rate than dural epithelial cells. The study suggested that dural epithelial cells may offer some barrier to the penetration of micron-sized particles but not nanometer sized particles. © 2014 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc.

Isolation and culture of corneal cells and their interactions with dissociated trigeminal neurons.

PubMed

Chan, K Y; Haschke, R H

1982-08-01

The three cell types of rabbit cornea (epithelium, stromal fibroblasts and endothelium) were isolated by an improved method using both microdissection and selective enzyme treatment. This technique reproducibly resulted in an almost total recovery of each cell type from a given cornea. When maintained in culture, the three cell types showed different morphologic characteristics, each resembling the in vivo counterpart. The epithelial culture consisted of both attached and floating cells. The attached cells located at the marginal area of a colony were irregular in shape and possessed pseudopodia, while those in the confluent area were polygonal. Floating cells were typically vacuolated, curve-shaped and joined in groups of 2-4 cells as a spherical body enclosing a lucent interior. Comparison of mitotic rates, ultrastructure, keratin levels and other cytologic evidence suggested that the attached cells may correspond to the basal cells and less differentiated wing cells, while the floating cells may be analogous to the more differentiated wing cells and superficial cells. Neurons dissociated from neonatal rabbit trigeminal (Gasserian) ganglia were plated into multiwells partially covered with a given corneal cell type. The percentages of viable and neurite-bearing neurons were evaluated on the first three days. When neurons were grown in contact with each of the corneal cell types, neurites were extended in every case. However, when neurons were not in contact with the corneal cells in the coculture, only epithelial cells permitted neurite outgrowth. The data suggested two types of cellular interactions between corneal cells and sensory neurons, one of which may be the specific release of a neuronotrophic factor by epithelial cells. This culture system represents the first step towards developing an in vitro model for studying various cornea-trigeminal interactions.

Targeting Stromal Androgen Receptor Suppresses Prolactin-Driven Benign Prostatic Hyperplasia (BPH)

PubMed Central

Lai, Kuo-Pao; Huang, Chiung-Kuei; Fang, Lei-Ya; Izumi, Kouji; Lo, Chi-Wen; Wood, Ronald; Kindblom, Jon; Yeh, Shuyuan

2013-01-01

Stromal-epithelial interaction plays a pivotal role to mediate the normal prostate growth, the pathogenesis of benign prostatic hyperplasia (BPH), and prostate cancer development. Until now, the stromal androgen receptor (AR) functions in the BPH development, and the underlying mechanisms remain largely unknown. Here we used a genetic knockout approach to ablate stromal fibromuscular (fibroblasts and smooth muscle cells) AR in a probasin promoter-driven prolactin transgenic mouse model (Pb-PRL tg mice) that could spontaneously develop prostate hyperplasia to partially mimic human BPH development. We found Pb-PRL tg mice lacking stromal fibromuscular AR developed smaller prostates, with more marked changes in the dorsolateral prostate lobes with less proliferation index. Mechanistically, prolactin mediated hyperplastic prostate growth involved epithelial-stromal interaction through epithelial prolactin/prolactin receptor signals to regulate granulocyte macrophage-colony stimulating factor expression to facilitate stromal cell growth via sustaining signal transducer and activator of transcription-3 activity. Importantly, the stromal fibromuscular AR could modulate such epithelial-stromal interacting signals. Targeting stromal fibromuscular AR with the AR degradation enhancer, ASC-J9®, led to the reduction of prostate size, which could be used in future therapy. PMID:23893956

Qualitatively Monitoring Binding and Expression of the Transcription Factors Sp1 and NFI as a Useful Tool to Evaluate the Quality of Primary Cultured Epithelial Stem Cells in Tissue Reconstruction.

PubMed

Le-Bel, Gaëtan; Ghio, Sergio Cortez; Larouche, Danielle; Germain, Lucie; Guérin, Sylvain L

2018-05-27

Electrophoretic mobility shift assays and Western blots are simple, efficient, and rapid methods to study DNA-protein interactions and protein expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem cells through the culture process is essential to produce high quality substitutes. However, the increase in the number of cell passages is associated with a decrease in their ability to proliferate until senescence is reached. This process is likely to be mediated by the altered expression of nuclear-located transcription factors such as Sp1 and NFI, whose expression has been documented to be required for cell adhesion, migration, and differentiation. In some of our recent studies, we observed a correlation between reconstructed tissues exhibiting poor histological and structural characteristics and a low expression of Sp1 in their constituting epithelial cells. Therefore, monitoring both the expression and DNA binding of these transcription factors in human skin and corneal epithelial cells is a useful tool for characterizing the quality of primary cultured epithelial cells.

The Human Polymeric Immunoglobulin Receptor Facilitates Invasion of Epithelial Cells by Streptococcus pneumoniae in a Strain-Specific and Cell Type-Specific Manner

PubMed Central

Brock, Sean C.; McGraw, Patricia A.; Wright, Peter F.; Crowe Jr., James E.

2002-01-01

Streptococcus pneumoniae is a gram-positive bacterial pathogen that causes invasive life-threatening disease worldwide. This organism also commonly colonizes the upper respiratory epithelium in an asymptomatic fashion. To invade, this pathogen must traverse the respiratory epithelial barrier, allowing it to cause disease locally or disseminate hematogenously throughout the body. Previous work has demonstrated that S. pneumoniae choline-binding protein A, a pneumococcal surface protein, interacts specifically with the human polymeric immunoglobulin receptor, which is expressed by cells in the respiratory epithelium. Choline-binding protein A is required for efficient colonization of the nasopharynx in vivo. Additionally, a recent study showed that the R6x laboratory strain of S. pneumoniae invades a human pharyngeal cell line in a human polymeric immunoglobulin receptor-dependent manner. These findings raised the possibility that the interaction between choline-binding protein A and human polymeric immunoglobulin receptor may be a key determinant of S. pneumoniae pathogenesis. However, the strain used in prior invasion studies, R6x, is an unencapsulated, nonpathogenic strain. In the present study we determined the relative ability of strain R6x or pathogenic strains to invade a variety of human polymeric immunoglobulin receptor-expressing epithelial cell lines. The results of this work suggest that human polymeric immunoglobulin receptor-dependent enhanced invasion of epithelial cells by S. pneumoniae is a limited phenomenon that occurs in a strain-specific and cell type-specific manner. PMID:12183558

The Pseudomonas aeruginosa exopolysaccharide Psl facilitates surface adherence and NF-kappaB activation in A549 cells.

PubMed

Byrd, Matthew S; Pang, Bing; Mishra, Meenu; Swords, W Edward; Wozniak, Daniel J

2010-06-29

In order for the opportunistic Gram-negative pathogen Pseudomonas aeruginosa to cause an airway infection, the pathogen interacts with epithelial cells and the overlying mucous layer. We examined the contribution of the biofilm polysaccharide Psl to epithelial cell adherence and the impact of Psl on proinflammatory signaling by flagellin. Psl has been implicated in the initial attachment of P. aeruginosa to biotic and abiotic surfaces, but its direct role in pathogenesis has not been evaluated (L. Ma, K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak, J. Bacteriol. 188:8213-8221, 2006). Using an NF-kappaB luciferase reporter system in the human epithelial cell line A549, we show that both Psl and flagellin are necessary for full activation of NF-kappaB and production of the interleukin 8 (IL-8) chemokine. We demonstrate that Psl does not directly stimulate NF-kappaB activity, but indirectly as a result of increasing contact between bacterial cells and epithelial cells, it facilitates flagellin-mediated proinflammatory signaling. We confirm differential adherence of Psl and/or flagellin mutants by scanning electron microscopy and identify Psl-dependent membrane structures that may participate in adherence. Although we hypothesized that Psl would protect P. aeruginosa from recognition by the epithelial cell line A549, we instead observed a positive role for Psl in flagellin-mediated NF-kappaB activation, likely as a result of increasing contact between bacterial cells and epithelial cells.

The Pseudomonas aeruginosa Exopolysaccharide Psl Facilitates Surface Adherence and NF-κB Activation in A549 Cells

PubMed Central

Byrd, Matthew S.; Pang, Bing; Mishra, Meenu; Swords, W. Edward; Wozniak, Daniel J.

2010-01-01

In order for the opportunistic Gram-negative pathogen Pseudomonas aeruginosa to cause an airway infection, the pathogen interacts with epithelial cells and the overlying mucous layer. We examined the contribution of the biofilm polysaccharide Psl to epithelial cell adherence and the impact of Psl on proinflammatory signaling by flagellin. Psl has been implicated in the initial attachment of P. aeruginosa to biotic and abiotic surfaces, but its direct role in pathogenesis has not been evaluated (L. Ma, K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak, J. Bacteriol. 188:8213–8221, 2006). Using an NF-κB luciferase reporter system in the human epithelial cell line A549, we show that both Psl and flagellin are necessary for full activation of NF-κB and production of the interleukin 8 (IL-8) chemokine. We demonstrate that Psl does not directly stimulate NF-κB activity, but indirectly as a result of increasing contact between bacterial cells and epithelial cells, it facilitates flagellin-mediated proinflammatory signaling. We confirm differential adherence of Psl and/or flagellin mutants by scanning electron microscopy and identify Psl-dependent membrane structures that may participate in adherence. Although we hypothesized that Psl would protect P. aeruginosa from recognition by the epithelial cell line A549, we instead observed a positive role for Psl in flagellin-mediated NF-κB activation, likely as a result of increasing contact between bacterial cells and epithelial cells. PMID:20802825

Vertex Models of Epithelial Morphogenesis

PubMed Central

Fletcher, Alexander G.; Osterfield, Miriam; Baker, Ruth E.; Shvartsman, Stanislav Y.

2014-01-01

The dynamic behavior of epithelial cell sheets plays a central role during numerous developmental processes. Genetic and imaging studies of epithelial morphogenesis in a wide range of organisms have led to increasingly detailed mechanisms of cell sheet dynamics. Computational models offer a useful means by which to investigate and test these mechanisms, and have played a key role in the study of cell-cell interactions. A variety of modeling approaches can be used to simulate the balance of forces within an epithelial sheet. Vertex models are a class of such models that consider cells as individual objects, approximated by two-dimensional polygons representing cellular interfaces, in which each vertex moves in response to forces due to growth, interfacial tension, and pressure within each cell. Vertex models are used to study cellular processes within epithelia, including cell motility, adhesion, mitosis, and delamination. This review summarizes how vertex models have been used to provide insight into developmental processes and highlights current challenges in this area, including progressing these models from two to three dimensions and developing new tools for model validation. PMID:24896108

Characterization of an immortalized human vaginal epithelial cell line.

PubMed

Rajan, N; Pruden, D L; Kaznari, H; Cao, Q; Anderson, B E; Duncan, J L; Schaeffer, A J

2000-02-01

Adherence of type 1 piliated Escherichia coli to vaginal mucosa plays a major role in the pathogenesis of ascending urinary tract infections (UTIs) in women. Progress in understanding the mechanism of adherence to the vaginal surface could be enhanced by the utilization of well-characterized vaginal epithelial cells. The objective of this study was to immortalize vaginal epithelial cells and study their bacterial adherence properties. Primary vaginal cells were obtained from a normal post-menopausal woman, immortalized by infection with E6/E7 genes from human papillomavirus 16 (HPV 16) and cultured in serum free keratinocyte growth factor medium. Positive immunostaining with a pool of antibodies to cytokeratins 1, 5, 10 and 14 (K1, K5, K10 and K14) and to K13 confirmed the epithelial origin of these cells. The immortalized cells showed binding of type 1 piliated E. coli in a pili specific and mannose sensitive manner. This model system should facilitate studies on the interaction of pathogens with vaginal mucosal cells, an essential step in the progression of ascending UTIs in women.

Establishment and characterization of a telomerase immortalized human gingival epithelial cell line.

PubMed

Moffatt-Jauregui, C E; Robinson, B; de Moya, A V; Brockman, R D; Roman, A V; Cash, M N; Culp, D J; Lamont, R J

2013-12-01

Gingival keratinocytes are used in model systems to investigate the interaction between periodontal bacteria and the epithelium in the initial stages of the periodontal disease process. Primary gingival epithelial cells (GECs) have a finite lifespan in culture before they enter senescence and cease to replicate, while epithelial cells immortalized with viral proteins can exhibit chromosomal rearrangements. The aim of this study was to generate a telomerase immortalized human gingival epithelial cell line and compare its in vitro behaviour to that of human GECs. Human primary GECs were immortalized with a bmi1/hTERT combination to prevent cell cycle triggers of senescence and telomere shortening. The resultant cell-line, telomerase immortalized gingival keratinocytes (TIGKs), were compared to GECs for cell morphology, karyotype, growth and cytokeratin expression, and further characterized for replicative lifespan, expression of toll-like receptors and invasion by P. gingivalis. TIGKs showed morphologies, karyotype, proliferation rates and expression of characteristic cytokeratin proteins comparable to GECs. TIGKs underwent 36 passages without signs of senescence and expressed transcripts for toll-like receptors 1-6, 8 and 9. A subpopulation of cells underwent stratification after extended time in culture. The cytokeratin profiles of TIGK monolayers were consistent with basal cells. When allowed to stratify, cytokeratin profiles of TIGKs were consistent with suprabasal cells of the junctional epithelium. Further, TIGKs were comparable to GECs in previously reported levels and kinetics of invasion by wild-type P. gingivalis and an invasion defective ΔserB mutant. Results confirm bmi1/hTERT immortalization of primary GECs generated a robust cell line with similar characteristics to the parental cell type. TIGKs represent a valuable model system for the study of oral bacteria interactions with host gingival cells. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Integrin-Mediated Transforming Growth Factor-β Activation Regulates Homeostasis of the Pulmonary Epithelial-Mesenchymal Trophic Unit

PubMed Central

Araya, Jun; Cambier, Stephanie; Morris, Alanna; Finkbeiner, Walter; Nishimura, Stephen L.

2006-01-01

Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-β is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, αvβ6 and αvβ8, are responsible for almost all of the TGF-β activation in the EMTU. Both αvβ8 and αvβ6 contribute to fetal tracheal epithelial activation of TGF-β, whereas only αvβ8 contributes to fetal tracheal fibroblast activation of TGF-β. Interestingly, fetal tracheal epithelial αvβ8-mediated TGF-β activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in αvβ8-mediated activation of TGF-β. Autocrine αvβ8-mediated TGF-β activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-β within the EMTU. PMID:16877343

Integrin-mediated transforming growth factor-beta activation regulates homeostasis of the pulmonary epithelial-mesenchymal trophic unit.

PubMed

Araya, Jun; Cambier, Stephanie; Morris, Alanna; Finkbeiner, Walter; Nishimura, Stephen L

2006-08-01

Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-beta is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, alpha(v)beta(6) and alpha(v)beta(8), are responsible for almost all of the TGF-beta activation in the EMTU. Both alpha(v)beta(8) and alpha(v)beta(6) contribute to fetal tracheal epithelial activation of TGF-beta, whereas only alpha(v)beta(8) contributes to fetal tracheal fibroblast activation of TGF-beta. Interestingly, fetal tracheal epithelial alpha(v)beta(8)-mediated TGF-beta activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in alpha(v)beta(8)-mediated activation of TGF-beta. Autocrine alpha(v)beta(8)-mediated TGF-beta activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-beta within the EMTU.

Distinct Phospholipase C-β Isozymes Mediate Lysophosphatidic Acid Receptor 1 Effects on Intestinal Epithelial Homeostasis and Wound Closure

PubMed Central

Lee, Sei-Jung; Leoni, Giovanna; Neumann, Philipp-Alexander; Chun, Jerold; Nusrat, Asma

2013-01-01

Maintenance of the epithelial barrier in the intestinal tract is necessary to protect the host from the hostile luminal environment. Phospholipase C-β (PLC-β) has been implicated to control myriad signaling cascades. However, the biological effects of selective PLC-β isozymes are poorly understood. We describe novel findings that lysophosphatidic acid (LPA) regulates PLC-β1 and PLC-β2 via two distinct pathways to enhance intestinal epithelial cell (IEC) proliferation and migration that facilitate wound closure and recovery of the intestinal epithelial barrier. LPA acting on the LPA1 receptor promotes IEC migration by facilitating the interaction of Gαq with PLC-β2. LPA-induced cell proliferation is PLC-β1 dependent and involves translocation of Gαq to the nucleus, where it interacts with PLC-β1 to induce cell cycle progression. An in vivo study using LPA1-deficient mice (Lpar1−/−) shows a decreased number of proliferating IECs and migration along the crypt-luminal axis. Additionally, LPA enhances migration and proliferation of IECs in an LPA1-dependent manner, and Lpar1−/− mice display defective mucosal wound repair that requires cell proliferation and migration. These findings delineate novel LPA1-dependent lipid signaling that facilitates mucosal wound repair via spatial targeting of distinct PLC-βs within the cell. PMID:23478264

Increased Growth of a Newly Established Mouse Epithelial Cell Line Transformed with HPV-16 E7 in Diabetic Mice.

PubMed

He, Lan; Law, Priscilla T Y; Boon, Siaw Shi; Zhang, Chuqing; Ho, Wendy C S; Banks, Lawrence; Wong, C K; Chan, Juliana C N; Chan, Paul K S

2016-01-01

Epidemiological evidence supports that infection with high-risk types of human papillomavirus (HPV) can interact with host and environmental risk factors to contribute to the development of cervical, oropharyngeal, and other anogenital cancers. In this study, we established a mouse epithelial cancer cell line, designated as Chinese University Papillomavirus-1 (CUP-1), from C57BL/KsJ mice through persistent expression of HPV-16 E7 oncogene. After continuous culturing of up to 200 days with over 60 passages, we showed that CUP-1 became an immortalized and transformed epithelial cell line with continuous E7 expression and persistent reduction of retinoblastoma protein (a known target of E7). This model allowed in-vivo study of interaction between HPV and co-factors of tumorigenesis in syngeneic mice. Diabetes has been shown to increase HPV pathogenicity in different pathological context. Herein, with this newly-established cell line, we uncovered that diabetes promoted CUP-1 xenograft growth in syngeneic db/db mice. In sum, we successfully established a HPV-16 E7 transformed mouse epithelial cell line, which allowed subsequent studies of co-factors in multistep HPV carcinogenesis in an immunocompetent host. More importantly, this study is the very first to demonstrate the promoting effect of diabetes on HPV-associated carcinogenesis in vivo, implicating the importance of cancer surveillance in diabetic environment.

Long non-coding RNA ZFAS1 interacts with miR-150-5p to regulate Sp1 expression and ovarian cancer cell malignancy.

PubMed

Xia, Bairong; Hou, Yan; Chen, Hong; Yang, Shanshan; Liu, Tianbo; Lin, Mei; Lou, Ge

2017-03-21

We reported that long non-coding RNA ZFAS1 was upregulated in epithelial ovarian cancer tissues, and was negatively correlated to the overall survival rate of patients with epithelial ovarian cancer in this study. While depletion of ZFAS1 inhibited proliferation, migration, and development of chemoresistance, overexpression of ZFAS1 exhibited an even higher proliferation rate, migration activity, and chemoresistance in epithelial ovarian cancer cell lines. We further found miR-150-5p was a potential target of ZFAS1, which was downregulated in epithelial ovarian cancer tissue. MiR-150-5p subsequently inhibited expression of transcription factor Sp1, as evidence by luciferase assays. Inhibition of miR-150-5p rescued the suppressed proliferation and migration induced by depletion of ZFAS1 in epithelial ovarian cancer cells, at least in part. Taken together, our findings revealed a critical role of ZFAS1/miR-150-5p/Sp1 axis in promoting proliferation rate, migration activity, and development of chemoresistance in epithelial ovarian cancer. And ZFAS1/miR-150-5p may serve as novel markers and therapeutic targets of epithelial ovarian cancer.

Virus interaction with the apical junctional complex.

PubMed

Gonzalez-Mariscal, Lorenza; Garay, Erika; Lechuga, Susana

2009-01-01

In order to infect pathogens must breach the epithelial barriers that separate the organism from the external environment or that cover the internal cavities and ducts of the body. Epithelia seal the passage through the paracellular pathway with the apical junctional complex integrated by tight and adherens junctions. In this review we describe how viruses like coxsackie, swine vesicular disease virus, adenovirus, reovirus, feline calcivirus, herpes viruses 1 and 2, pseudorabies, bovine herpes virus 1, poliovirus and hepatitis C use as cellular receptors integral proteins present at the AJC of epithelial cells. Interaction with these proteins contributes in a significant manner in defining the particular tropism of each virus. Besides these proteins, viruses exhibit a wide range of cellular co-receptors among which proteins present in the basolateral cell surface like integrins are often found. Therefore targeting proteins of the AJC constitutes a strategy that might allow viruses to bypass the physical barrier that blocks their access to receptors expressed on the basolateral surface of epithelial cells.

Cyclic AMP regulates formation of mammary epithelial acini in vitro

PubMed Central

Nedvetsky, Pavel I.; Kwon, Sang-Ho; Debnath, Jayanta; Mostov, Keith E.

2012-01-01

Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three-dimensional culture utilizing MCF10A mammary epithelial cells, acini form due to integrin-dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking contact with the ECM. In this paper, we report that cyclic AMP (cAMP)-dependent protein kinase A (PKA) promotes acinus formation via two mechanisms. First, cAMP accelerates redistribution of α6-integrin to the periphery of the acinus and thus facilitates the polarization of outer acinar cells. Blocking of α6-integrin function by inhibitory antibody prevents cAMP-dependent polarization. Second, cAMP promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed, resulting in perturbed luminal clearance. cAMP-dependent apoptosis is accompanied by a posttranscriptional PKA-dependent increase in the proapoptotic protein Bcl-2 interacting mediator of cell death. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cells in vitro, both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus. PMID:22675028

Ocular surface epithelium induces expression of human mucosal lymphocyte antigen (HML-1) on peripheral blood lymphocytes

PubMed Central

Gomes, J A P; Dua, H S; Rizzo, L V; Nishi, M; Joseph, A; Donoso, L A

2004-01-01

Background/aims: Peripheral blood CD8+ lymphocytes that home to mucosal surfaces express the human mucosal lymphocyte antigen (HML-1). At mucosal surfaces, including the ocular surface, only intraepithelial CD8+ lymphocytes express HML-1. These lymphocytes are retained in the intraepithelial compartment by virtue of the interaction between HML-1 and its natural ligand, E-cadherin, which is expressed on epithelial cells. The purpose of this study was to determine whether ocular surface epithelial cells (ocular mucosa) could induce the expression of human mucosal lymphocyte antigen on peripheral blood lymphocytes. Methods: Human corneal and conjunctival epithelial cells were co-cultured with peripheral blood lymphocytes. Both non-activated and activated lymphocytes were used in the experiments. After 7 days of incubation, lymphocytes were recovered and analysed for the antigens CD8/HML-1, CD4/HML-1, CD3/CD8, CD3/CD4, CD3/CD25, CD8/CD25, and CD4/CD25 by flowcytometry. Results: Significant statistical differences were observed in the CD8/HML-1 expression when conjunctival epithelial cells were co-cultured with non-activated and activated lymphocytes (p = 0.04 for each) and when corneal epithelial cells were co-cultured with non-activated lymphocytes (p = 0.03). Significant statistical difference in CD4/HML-1 expression was observed only when conjunctival epithelial cells were co-cultured with activated lymphocytes (p = 0.02). Conclusion: Ocular surface epithelial cells can induce the expression of human mucosal lymphocyte antigen on CD8+ (and to some extent on CD4+) lymphocytes. This may allow the retention of CD8+ and CD4+ lymphocytes within the epithelial compartment of the conjunctiva and play a part in mucosal homing of lymphocytes. PMID:14736792

Retinal Pigment Epithelial Cells are a Potential Reservoir for Ebola Virus in the Human Eye

PubMed Central

Smith, Justine R.; Todd, Shawn; Ashander, Liam M.; Charitou, Theodosia; Ma, Yuefang; Yeh, Steven; Crozier, Ian; Michael, Michael Z.; Appukuttan, Binoy; Williams, Keryn A.; Lynn, David J.; Marsh, Glenn A.

2017-01-01

Purpose Success of Ebola virus (EBOV) as a human pathogen relates at the molecular level primarily to blockade the host cell type I interferon (IFN) antiviral response. Most individuals who survive Ebola virus disease (EVD) develop a chronic disease syndrome: approximately one-quarter of survivors suffer from uveitis, which has been associated with presence of EBOV within the eye. Clinical observations of post-Ebola uveitis indicate involvement of retinal pigment epithelial cells. Methods We inoculated ARPE-19 human retinal pigment epithelial cells with EBOV, and followed course of infection by immunocytochemistry and measurement of titer in culture supernatant. To interrogate transcriptional responses of infected cells, we combined RNA sequencing with in silico pathway, gene ontology, transcription factor binding site, and network analyses. We measured infection-induced changes of selected transcripts by reverse transcription-quantitative polymerase chain reaction. Results Human retinal pigment epithelial cells were permissive to infection with EBOV, and supported viral replication and release of virus in high titer. Unexpectedly, 28% of 560 upregulated transcripts in EBOV-infected cells were type I IFN responsive, indicating a robust type I IFN response. Following EBOV infection, cells continued to express multiple immunomodulatory molecules linked to ocular immune privilege. Conclusions Human retinal pigment epithelial cells may serve as an intraocular reservoir for EBOV, and the molecular response of infected cells may contribute to the persistence of live EBOV within the human eye. Translational Relevance This bedside-to-bench research links ophthalmic findings in survivors of EVD who suffer from uveitis with interactions between retinal pigment epithelial cells and EBOV. PMID:28721309

S-carboxymethylcysteine inhibits adherence of Streptococcus pneumoniae to human alveolar epithelial cells.

PubMed

Sumitomo, Tomoko; Nakata, Masanobu; Yamaguchi, Masaya; Terao, Yutaka; Kawabata, Shigetada

2012-01-01

Streptococcus pneumoniae is a major pathogen of respiratory infections that utilizes platelet-activating factor receptor (PAFR) for firm adherence to host cells. The mucolytic agent S-carboxymethylcysteine (S-CMC) has been shown to exert inhibitory effects against infection by several respiratory pathogens including S. pneumoniae in vitro and in vivo. Moreover, clinical studies have implicated the benefits of S-CMC in preventing exacerbation of chronic obstructive pulmonary disease, which is considered to be related to respiratory infections. In this study, to assess whether the potency of S-CMC is attributable to inhibition of pneumococcal adherence to host cells, an alveolar epithelial cell line stimulated with interleukin-1α was used as a model of inflamed epithelial cells. Despite upregulation of PAFR by inflammatory activation, treatment with S-CMC efficiently inhibited pneumococcal adherence to host epithelial cells. In order to gain insight into the inhibitory mechanism, the effects of S-CMC on PAFR expression were also investigated. Following treatment with S-CMC, PAFR expression was reduced at both mRNA and post-transcriptional levels. Interestingly, S-CMC was also effective in inhibiting pneumococcal adherence to cells transfected with PAFR small interfering RNAs. These results indicate S-CMC as a probable inhibitor targeting numerous epithelial receptors that interact with S. pneumoniae.

Effects of methyl gallate and gallic acid on the production of inflammatory mediators interleukin-6 and interleukin-8 by oral epithelial cells stimulated with Fusobacterium nucleatum.

PubMed

Kang, Mi-Sun; Jang, Hee-Sook; Oh, Jong-Suk; Yang, Kyu-Ho; Choi, Nam-Ki; Lim, Hoi-Soon; Kim, Seon-Mi

2009-12-01

Interactions between periodontal bacteria and human oral epithelial cells can lead to the activation and expression of a variety of inflammatory mediators in epithelial cells. Fusobacterium nucleatum is a filamentous human pathogen that is strongly associated with periodontal diseases. This study examined the effects of methyl gallate (MG) and gallic acid (GA) on the production of inflammatory mediators, interleukin (IL)-6 and IL-8, by oral epithelial cells stimulated by F. nucleatum. In a real-time reverse transcription-polymerase chain reaction and an enzyme-linked immunosorbent assay, live F. nucleatum induced high levels of gene expression and protein release of IL-6 and IL-8. The effects of MG and GA were examined by treating KB oral epithelial cells with MG and GA and stimulating them with F. nucleatum. MG and GA inhibited significantly the increases in the IL-6 and IL-8 gene and protein levels in a dose-dependent manner. These Compounds also inhibited the growth of F. nucleatum. No visible effects of MG and GA on the adhesion and invasion of KB cells by F. nucleatum were observed. In conclusion, both MG and GA inhibit IL-6 and IL-8 production from F. nucleatum-activated KB cells.

c-Jun N-terminal kinase 1 promotes transforming growth factor-β1-induced epithelial-to-mesenchymal transition via control of linker phosphorylation and transcriptional activity of Smad3.

PubMed

Velden, Jos L J van der; Alcorn, John F; Guala, Amy S; Badura, Elsbeth C H L; Janssen-Heininger, Yvonne M W

2011-04-01

Transforming growth factor (TGF)-β1 is a key mediator of lung remodeling and fibrosis. Epithelial cells are both a source of and can respond to TGF-β1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-β1-induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-β1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-β1-induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablated caused a marked attenuation in JNK1 or TGF-β1-induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor-1, fibronectin-1, high-mobility group A2, CArG box-binding factor-A, and fibroblast-specific protein-1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-β1-induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-β1-induced EMT.

KSA Antigen Ep-CAM Mediates Cell–Cell Adhesion of Pancreatic Epithelial Cells: Morphoregulatory Roles in Pancreatic Islet Development

PubMed Central

Cirulli, V.; Crisa, L.; Beattie, G.M.; Mally, M.I.; Lopez, A.D.; Fannon, A.; Ptasznik, A.; Inverardi, L.; Ricordi, C.; Deerinck, T.; Ellisman, M.; Reisfeld, R.A.; Hayek, A.

1998-01-01

Cell adhesion molecules (CAMs) are important mediators of cell–cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell–cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny. PMID:9508783

Regulation of defensive function on gingival epithelial cells can prevent periodontal disease.

PubMed

Fujita, Tsuyoshi; Yoshimoto, Tetsuya; Kajiya, Mikihito; Ouhara, Kazuhisa; Matsuda, Shinji; Takemura, Tasuku; Akutagawa, Keiichi; Takeda, Katsuhiro; Mizuno, Noriyoshi; Kurihara, Hidemi

2018-05-01

Periodontal disease is a bacterial biofilm-associated inflammatory disease that has been implicated in many systemic diseases. A new preventive method for periodontal disease needs to be developed in order to promote the health of the elderly in a super-aged society. The gingival epithelium plays an important role as a mechanical barrier against bacterial invasion and a part of the innate immune response to infectious inflammation in periodontal tissue. The disorganization of cell-cell interactions and subsequent inflammation contribute to the initiation of periodontal disease. These make us consider that regulation of host defensive functions, epithelial barrier and neutrophil activity, may become novel preventive methods for periodontal inflammation. Based on this concept, we have found that several agents regulate the barrier function of gingival epithelial cells and suppress the accumulation of neutrophils in the gingival epithelium. We herein introduce the actions of irsogladine maleate, azithromycin, amphotericin B, and Houttuynia cordata (dokudami in Japanese), which is commonly used in traditional medicine, on the epithelial barrier and neutrophil migration in gingival epithelial cells in vivo and in vitro , in order to provide support for the clinical application of these agents to the prevention of periodontal inflammation.

Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells.

PubMed

Stein, Daniel C; LeVan, Adriana; Hardy, Britney; Wang, Liang-Chun; Zimmerman, Lindsey; Song, Wenxia

2015-01-01

Neisseria gonorrhoeae (GC) establishes infection at the mucosal surface of the human genital tract, most of which is lined with polarized epithelial cells. GC can cause localized as well as disseminated infections, leading to various complications. GC constantly change their surface structures via phase and antigenic variation, which has been implicated as a means for GC to establish infection at various anatomic locations of male and female genital tracks. However, the exact contribution of each surface molecule to bacterial infectivity remains elusive due to their phase variation. Using a GC derivative that is genetically devoid of all opa genes (MS11∆Opa), this study shows that Opa expression interferes with GC transmigration across polarized human epithelial cells. MS11∆Opa transmigrates across polarized epithelial cells much faster and to a greater extent than MS11Opa+, while adhering at a similar level as MS11Opa+. When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer. Similar to bacteria alone or co-cultured with non-polarized epithelial cells, MS11∆Opa fails to form large microcolonies at the apical surface of polarized epithelial cells. Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites. Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions.

Role of salivary epithelial toll-like receptors 2 and 4 in modulating innate immune responses in chronic periodontitis.

PubMed

Swaminathan, V; Prakasam, S; Puri, V; Srinivasan, M

2013-12-01

Chronic periodontitis is initiated by sequential colonization with a broad array of bacteria and is perpetuated by an immune-inflammatory response to the changing biofilm. Host recognition of microbes is largely mediated by toll-like receptors (TLRs), which interact with conserved pathogen-associated molecular patterns. Based on ligand recognition, TLR-2 and TLR-4 interact with most periodontal pathogens. Extracrevicular bacterial reservoirs, such as the oral epithelial cells, contribute to the persistence of periodontitis. Human saliva is a rich source of oral epithelial cells that express functional TLRs. In this study we investigated the role of salivary epithelial cell (SEC) TLR-2 and TLR-4 in patients with generalized chronic periodontitis. Unstimulated whole saliva (UWS) was collected from patients with generalized chronic periodontitis and from healthy individuals after obtaining informed consent. Epithelial cells isolated from each UWS sample were assessed for TLR-2, TLR-4, peptidoglycan recognition protein (PGRP)-3 and PGRP-4 by quantitative real-time PCR. In addition, the SECs were stimulated in vitro with microbial products for up to 24 h. The culture supernatant was assessed for cytokines by ELISA. Stimulation with TLR-2- or TLR-4-specific ligands induced cytokine secretion with differential kinetics and up-regulated TLR2 and TLR4 mRNAs, respectively, in cultures of SECs from patients with periodontitis. In addition, the SECs from patients with periodontitis exhibited reduced PGRP3 and PGRP4 mRNAs, the TLR-responsive genes with antibacterial properties. SECs derived from the UWS of patients with chronic periodontitis are phenotypically distinct and could represent potential resources for assessing the epithelial responses to periodontal pathogens in the course of disease progression and persistence. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Stromal-Epithelial Interactions and Tamoxifen-Sensitivity: A Bench-to-Bedside Model of Chemoprevention

DTIC Science & Technology

2008-05-01

effects of tamoxifen can be directly attributed to competitive interactions with ER. Tamox- ifen induces apoptosis in cholangiocarcinoma cells and...Pickens A, et al. Apoptosis and tumorigenesis in human cholangiocarcinoma cells. Involvement of Fas/APO-1 (CD95) and calmodulin. Am J Pathol 1999;155:193

Cyclic adenosine monophosphate modulates cell morphology and behavior of a cultured renal epithelial.

PubMed

Amsler, K

1990-07-01

The role of cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) in modulating functions of differentiated renal cells is well established. Its importance in controlling their growth and differentiation is less clear. We have used somatic cell genetic techniques to probe the role of PKA in controlling morphology and behavior of a renal epithelial cell line, LLC-PK1, which acquires many properties characteristic of the renal proximal tubular cell. Mutants of this line altered in PKA activity have been isolated and their behavior compared to that of the parent line. The results indicate that PKA is involved, either directly or indirectly, in maintenance of cell morphology, cell-cell and cell-substratum interactions, density-dependent growth regulation, and expression of one function characteristic of the renal proximal tubular cell, Na-hexose symport. The relevance of these results to the role of PKA in controlling growth and differentiation of renal epithelial cells in vivo is discussed.

A functional role of the extracellular domain of Crumbs in cell architecture and apicobasal polarity.

PubMed

Letizia, Annalisa; Ricardo, Sara; Moussian, Bernard; Martín, Nicolás; Llimargas, Marta

2013-05-15

Regulated cell shape changes in epithelial cells, which contribute to most organs and tissues, are at the basis of morphogenesis. Crumbs (Crb) is an essential apical determinant controlling epithelial apicobasal polarity. Here we provide evidence for a novel role of Crb apical localisation and stabilisation in controlling cell shape through apical domain organisation and adherens junction positioning. We find that Crb apical stabilisation requires the extracellular domain. In vivo results from Drosophila suggest that the extracellular domain assists Crb apical stabilisation by mediating Crb-Crb interactions at opposing cell membranes. We further confirm Crb-Crb extracellular interactions by showing that the extracellular domain of Crb is sufficient to promote cell aggregation in vitro. Furthermore, we report that Crb apical stabilisation mediated by the extracellular domain is also required for maintenance of Crb apicobasal polarity. Our results provide new insights into the mechanisms of apicobasal polarity and the cellular mechanisms of tissue architecture.

Bovine colostrum enhances natural killer cell activity and immune response in a mouse model of influenza infection and mediates intestinal immunity through toll-like receptors 2 and 4.

PubMed

Wong, Eric B; Mallet, Jean-François; Duarte, Jairo; Matar, Chantal; Ritz, Barry W

2014-04-01

Oral administration of bovine colostrum affects intestinal immunity, including an increased percentage of natural killer (NK) cells. However, effects on NK cell cytotoxic activity and resistance to infection as well as a potential mechanism remain unclear. Therefore, we investigated the effects of bovine colostrum (La Belle, Inc, Bellingham, WA) on the NK cytotoxic response to influenza infection and on toll-like receptor (TLR) activity in a primary intestinal epithelial cell culture. We hypothesized that colostrum would increase NK cell activity and that TLR-2 and TLR-4 blocking would reduce interleukin 6 production by epithelial cells in response to contact stimulation with colostrum. Four-month-old female C57BL/6 mice were supplemented with 1 g of colostrum per kilogram of body weight before and after infection with influenza A virus (H1N1). Animals were assessed for weight loss, splenic NK cell activity, and lung virus titers. Colostrum-supplemented mice demonstrated less reduction in body weight after influenza infection, indicating a less severe infection, increased NK cell cytotoxicity, and less virus burden in the lungs compared with controls. Colostrum supplementation enhanced NK cell cytotoxicity and improved the immune response to primary influenza virus infection in mice. To investigate a potential mechanism, a primary culture of small intestine epithelial cells was then stimulated with colostrum. Direct activation of epithelial cells resulted in increased interleukin 6 production, which was inhibited with TLR-2 and TLR-4 blocking antibodies. The interaction between colostrum and immunity may be dependent, in part, on the interaction of colostrum components with innate receptors at the intestinal epithelium, including TLR-2 and TLR-4. Copyright © 2014 Elsevier Inc. All rights reserved.

Human adipose tissue from normal and tumoral breast regulates the behavior of mammary epithelial cells.

PubMed

Pistone Creydt, Virginia; Fletcher, Sabrina Johanna; Giudice, Jimena; Bruzzone, Ariana; Chasseing, Norma Alejandra; Gonzalez, Eduardo Gustavo; Sacca, Paula Alejandra; Calvo, Juan Carlos

2013-02-01

Stromal-epithelial interactions mediate both breast development and breast cancer progression. In the present work, we evaluated the effects of conditioned media (CMs) of human adipose tissue explants from normal (hATN) and tumor (hATT) breast on proliferation, adhesion, migration and metalloproteases activity on tumor (MCF-7 and IBH-7) and non-tumor (MCF-10A) human breast epithelial cell lines. Human adipose tissues were obtained from patients and the conditioned medium from hATN and hATT collected after 24 h of incubation. MCF-10A, MCF-7 and IBH-7 cells were grown and incubated with CMs and proliferation and adhesion, as well as migration ability and metalloprotease activity, of epithelial cells after exposing cell cultures to hATN- or hATT-CMs were quantified. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post hoc tests were performed. Tumor and non-tumor breast epithelial cells significantly increased their proliferation activity after 24 h of treatment with hATT-CMs compared to control-CMs. Furthermore, cellular adhesion of these two tumor cell lines was significantly lower with hATT-CMs than with hATN-CMs. Therefore, hATT-CMs seem to induce significantly lower expression or less activity of the components involved in cellular adhesion than hATN-CMs. In addition, hATT-CMs induced pro-MMP-9 and MMP-9 activity and increased the migration of MCF-7 and IBH-7 cells compared to hATN-CMs. We conclude that the microenvironment of the tumor interacts in a dynamic way with the mutated epithelium. This evidence leads to the possibility to modify the tumor behavior/phenotype through the regulation or modification of its microenvironment. We developed a model in which we obtained CMs from adipose tissue explants completely, either from normal or tumor breast. In this way, we studied the contribution of soluble factors independently of the possible effects of direct cell contact.

The Rap GTPase Activator Drosophila PDZ-GEF Regulates Cell Shape in Epithelial Migration and Morphogenesis▿

PubMed Central

Boettner, Benjamin; Van Aelst, Linda

2007-01-01

Epithelial morphogenesis is characterized by an exquisite control of cell shape and position. Progression through dorsal closure in Drosophila gastrulation depends on the ability of Rap1 GTPase to signal through the adherens junctional multidomain protein Canoe. Here, we provide genetic evidence that epithelial Rap activation and Canoe effector usage are conferred by the Drosophila PDZ-GEF (dPDZ-GEF) exchange factor. We demonstrate that dPDZ-GEF/Rap/Canoe signaling modulates cell shape and apicolateral cell constriction in embryonic and wing disc epithelia. In dPDZ-GEF mutant embryos with strong dorsal closure defects, cells in the lateral ectoderm fail to properly elongate. Postembryonic dPDZ-GEF mutant cells generated in mosaic tissue display a striking extension of lateral cell perimeters in the proximity of junctional complexes, suggesting a loss of normal cell contractility. Furthermore, our data indicate that dPDZ-GEF signaling is linked to myosin II function. Both dPDZ-GEF and cno show strong genetic interactions with the myosin II-encoding gene, and myosin II distribution is severely perturbed in epithelia of both mutants. These findings provide the first insight into the molecular machinery targeted by Rap signaling to modulate epithelial plasticity. We propose that dPDZ-GEF-dependent signaling functions as a rheostat linking Rap activity to the regulation of cell shape in epithelial morphogenesis at different developmental stages. PMID:17846121

Heterocellular interaction enhances recruitment of {alpha} and {beta}-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Talhouk, Rabih S.; Mroue, Rana; Mokalled, Mayssa

2008-11-01

Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation ({beta}-casein expression) was evaluated. Heterocellular interaction is critical for {beta}-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complexmore » components ({alpha}-catenin, {beta}-catenin and ZO-2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although {beta}-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and {beta}-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear {beta}-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of {beta}-catenin in GJ complexes.« less

MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

PubMed

Van Itallie, Christina M; Tietgens, Amber Jean; Aponte, Angel; Gucek, Marjan; Cartagena-Rivera, Alexander X; Chadwick, Richard S; Anderson, James M

2018-02-02

Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells. © 2018. Published by The Company of Biologists Ltd.

Organization of the cytokeratin network in an epithelial cell.

PubMed

Portet, Stéphanie; Arino, Ovide; Vassy, Jany; Schoëvaërt, Damien

2003-08-07

The cytoskeleton is a dynamic three-dimensional structure mainly located in the cytoplasm. It is involved in many cell functions such as mechanical signal transduction and maintenance of cell integrity. Among the three cytoskeletal components, intermediate filaments (the cytokeratin in epithelial cells) are the best candidates for this mechanical role. A model of the establishment of the cytokeratin network of an epithelial cell is proposed to study the dependence of its structural organization on extracellular mechanical environment. To implicitly describe the latter and its effects on the intracellular domain, we use mechanically regulated protein synthesis. Our model is a hybrid of a partial differential equation of parabolic type, governing the evolution of the concentration of cytokeratin, and a set of stochastic differential equations describing the dynamics of filaments. Each filament is described by a stochastic differential equation that reflects both the local interactions with the environment and the non-local interactions via the past history of the filament. A three-dimensional simulation model is derived from this mathematical model. This simulation model is then used to obtain examples of cytokeratin network architectures under given mechanical conditions, and to study the influence of several parameters.

A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis.

PubMed

Carterson, A J; Höner zu Bentrup, K; Ott, C M; Clarke, M S; Pierson, D L; Vanderburg, C R; Buchanan, K L; Nickerson, C A; Schurr, M J

2005-02-01

A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.

Interaction of micron and nano-sized particles with cells of the dura mater

PubMed Central

Papageorgiou, Iraklis; Marsh, Rainy; Tipper, Joanne L; Hall, Richard M; Fisher, John; Ingham, Eileen

2014-01-01

Intervertebral total disc replacements (TDR) are used in the treatment of degenerative spinal disc disease. There are, however, concerns that they may be subject to long-term failure due to wear. The adverse effects of TDR wear have the potential to manifest in the dura mater and surrounding tissues. The aim of this study was to investigate the physiological structure of the dura mater, isolate the resident dural epithelial and stromal cells and analyse the capacity of these cells to internalise model polymer particles. The porcine dura mater was a collagen-rich structure encompassing regularly arranged fibroblastic cells within an outermost epithelial cell layer. The isolated dural epithelial cells had endothelial cell characteristics (positive for von Willebrand factor, CD31, E-cadherin and desmoplakin) and barrier functionality whereas the fibroblastic cells were positive for collagen I and III, tenascin and actin. The capacity of the dural cells to take up model particles was dependent on particle size. Nanometer sized particles readily penetrated both types of cells. However, dural fibroblasts engulfed micron-sized particles at a much higher rate than dural epithelial cells. The study suggested that dural epithelial cells may offer some barrier to the penetration of micron-sized particles but not nanometer sized particles. © 2014 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 1496–1505, 2014. PMID:24604838

CD47 regulates renal tubular epithelial cell self-renewal and proliferation following renal ischemia reperfusion.

PubMed

Rogers, Natasha M; Zhang, Zheng J; Wang, Jiao-Jing; Thomson, Angus W; Isenberg, Jeffrey S

2016-08-01

Defects in renal tubular epithelial cell repair contribute to renal ischemia reperfusion injury, cause acute kidney damage, and promote chronic renal disease. The matricellular protein thrombospondin-1 and its receptor CD47 are involved in experimental renal ischemia reperfusion injury, although the role of this interaction in renal recovery is unknown. We found upregulation of self-renewal genes (transcription factors Oct4, Sox2, Klf4 and cMyc) in the kidney of CD47(-/-) mice after ischemia reperfusion injury. Wild-type animals had minimal self-renewal gene expression, both before and after injury. Suggestive of cell autonomy, CD47(-/-) renal tubular epithelial cells were found to increase expression of the self-renewal genes. This correlated with enhanced proliferative capacity compared with cells from wild-type mice. Exogenous thrombospondin-1 inhibited self-renewal gene expression in renal tubular epithelial cells from wild-type but not CD47(-/-) mice, and this was associated with decreased proliferation. Treatment of renal tubular epithelial cells with a CD47 blocking antibody or CD47-targeting small interfering RNA increased expression of some self-renewal transcription factors and promoted cell proliferation. In a syngeneic kidney transplant model, treatment with a CD47 blocking antibody increased self-renewal transcription factor expression, decreased tissue damage, and improved renal function compared with that in control mice. Thus, thrombospondin-1 via CD47 inhibits renal tubular epithelial cell recovery after ischemia reperfusion injury through inhibition of proliferation/self-renewal. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Cellular Interactions and Biological Responses to Titanium Dioxide Nanoparticles in HepG2 and BEAS-2B Cells: Role of Cell Culture Media

EPA Science Inventory

ABSTRACT We have shown previously that the composition of the biological medium used in vitro can affect the cellular interaction and biological response of titanium dioxide nanoparticles (nano-TiO2) in human lung epithelial cells. However, it is unclear if these effects are co...

An intercellular polyamine transfer via gap junctions regulates proliferation and response to stress in epithelial cells

PubMed Central

Desforges, Bénédicte; Curmi, Patrick A.; Bounedjah, Ouissame; Nakib, Samir; Hamon, Loic; De Bandt, Jean-Pascal; Pastré, David

2013-01-01

In the organism, quiescent epithelial cells have the potential to resume cycling as a result of various stimuli, including wound healing or oxidative stress. Because quiescent cells have a low polyamine level, resuming their growth requires an increase of their intracellular polyamine levels via de novo polyamine synthesis or their uptake from plasma. Another alternative, explored here, is an intercellular exchange with polyamine-rich cycling cells via gap junctions. We show that polyamines promote gap junction communication between proliferating cells by promoting dynamical microtubule plus ends at the cell periphery and thus allow polyamine exchange between cells. In this way, cycling cells favor regrowth in adjacent cells deprived of polyamines. In addition, intercellular interactions mediated by polyamines can coordinate the translational response to oxidative stress through the formation of stress granules. Some putative in vivo consequences of polyamine-mediated intercellular interactions are also discussed regarding cancer invasiveness and tissue regeneration. PMID:23515223

Protein kinase antagonists inhibit invasion of mammalian cells by Fonsecaea pedrosoi.

PubMed

Limongi, Cristiana L; De Souza, Wanderley; Rozental, Sonia

2003-03-01

The phosphorylation process is an important mechanism of cell signalling and regulation. It has been implicated recently in defence strategies against a variety of pathogens that alter host signalling pathways in order to facilitate their invasion and survival within host cells. In this study, the involvement of protein kinases (PKs) has been investigated in attachment and invasion by the pathogenic fungus Fonsecaea pedrosoi within epithelial cells and macrophages. The use of the PK inhibitors staurosporine, genistein and calphostin C prior to infection provided significant information about the role played by PKs in the F. pedrosoi-host cell interaction. All three PK inhibitors could reduce cell invasion by F. pedrosoi significantly. Pre-treatment of macrophages, epithelial cells or conidia with PK inhibitors decreased fungus invasion, and this effect could be overcome by okadaic acid, a phosphatase inhibitor. Immunofluorescence assays showed that tyrosine residues were phosphorylated in the first step of the interaction, while serine residues were phosphorylated in the subsequent step of entry of the parasite into the host cell. These results suggest that both host-cell and conidium PK activities are important in the interaction process, playing a significant role in cell invasion.

Proliferative activity and branching morphogenesis in the human prostate: a closer look at pre- and postnatal prostate growth.

PubMed

Xue, Y; Sonke, G; Schoots, C; Schalken, J; Verhofstad, A; de la Rosette, J; Smedts, F

2001-10-01

To gain further insight into the molecular cell biologic features of prostate development, we investigated the proliferative activity of prostate epithelial and stromal cells and their topographic relationship with neuroendocrine (NE) cell distribution and regional heterogeneity. Consecutive sections from 43 prostates taken during autopsy representing fetuses (12-38 weeks of gestation), infants, prepubertal males and adults were double stained for chromogranin A and MIB-1. MIB-1 labeling index (LI) was calculated in the budding tips, forming acini, major collecting ducts, adjacent and non-adjacent stromal compartments. Furthermore, the topographic relationship between proliferating cells and NE cells was evaluated. In the first half of gestation, cell proliferation as revealed by MIB-1 LI was significantly higher in epithelial structures and stroma than in older fetuses and other age groups. MIB-1 LI was higher in budding tips than in other epithelial regions. MIB-1 LI in stroma adjacent to budding tips was not higher than that adjacent to other epithelial branching segments. Co-expression of chromogranin A and MIB-1 staining was not observed. MIB-1 LI was lower in cells in the direct vicinity of chromogranin A positive NE cells than at a distance from NE cells. Prostate development in the first half of gestation is explosive. Thereafter, the prostate basically is a slow-growing organ. Budding tips are the major growth foci during early prostate development, while stromal growth is evenly distributed throughout the prostate, probably indicating that stromal-epithelial interactions do not manifest in enhanced proliferation at their interface. NE cells may have an inhibitory effect on proliferation of exocrine epithelial cells and are probably only associated with differentiation of prostate exocrine cells in the prostate. Copyright 2001 Wiley-Liss, Inc.

Candida parapsilosis Protects Premature Intestinal Epithelial Cells from Invasion and Damage by Candida albicans

PubMed Central

Gonia, Sara; Archambault, Linda; Shevik, Margaret; Altendahl, Marie; Fellows, Emily; Bliss, Joseph M.; Wheeler, Robert T.; Gale, Cheryl A.

2017-01-01

Candida is a leading cause of late-onset sepsis in premature infants and is thought to invade the host via immature or damaged epithelial barriers. We previously showed that the hyphal form of Candida albicans invades and causes damage to premature intestinal epithelial cells (pIECs), whereas the non-hyphal Candida parapsilosis, also a fungal pathogen of neonates, has less invasion and damage abilities. In this study, we investigated the potential for C. parapsilosis to modulate pathogenic interactions of C. albicans with the premature intestine. While a mixed infection with two fungal pathogens may be expected to result in additive or synergistic damage to pIECs, we instead found that C. parapsilosis was able to protect pIECs from invasion and damage by C. albicans. C. albicans-induced pIEC damage was reduced to a similar extent by multiple different C. parapsilosis strains, but strains differed in their ability to inhibit C. albicans invasion of pIECs, with the inhibitory activity correlating with their adhesiveness for C. albicans and epithelial cells. C. parapsilosis cell-free culture fractions were also able to significantly reduce C. albicans adhesion and damage to pIECs. Furthermore, coadministration of C. parapsilosis cell-free fractions with C. albicans was associated with decreased infection and mortality in zebrafish. These results indicate that C. parapsilosis is able to reduce invasion, damage, and virulence functions of C. albicans. Additionally, the results with cellular and cell-free fractions of yeast cultures suggest that inhibition of pathogenic interactions between C. albicans and host cells by C. parapsilosis occurs via secreted molecules as well as by physical contact with the C. parapsilosis cell surface. We propose that non-invasive commensals can be used to inhibit virulence features of pathogens and deserve further study as a non-pharmacological strategy to protect the fragile epithelial barriers of premature infants. PMID:28382297

Stromal loss of TGFβ drives cancer growth in the epithelium via inflammation | Center for Cancer Research

Cancer.gov

Interactions between epithelial and stromal cells play an important role in cancer development and progression. Epithelial cancers develop when changes occur to tumor suppressor genes in stromal fibroblast cells. For example, loss of tumor suppressor, p53, in stromal fibroblasts leads to p53 inactivation in the epithelium in a prostate cancer model, and disruption of the transforming growth factor-b receptor II (TGF-βRII) in stromal fibroblasts results in intraepithelial dysplasia in prostate cancer and invasive squamous cell carcinoma (SCC) in mouse forestomach.

Conditional immortalization of human thyroid epithelial cells: a tool for analysis of oncogene action.

PubMed Central

Wynford-Thomas, D; Bond, J A; Wyllie, F S; Burns, J S; Williams, E D; Jones, T; Sheer, D; Lemoine, N R

1990-01-01

To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types. Images PMID:1697930

In vivo imaging of epithelial wound healing in the cnidarian Clytia hemisphaerica demonstrates early evolution of purse string and cell crawling closure mechanisms.

PubMed

Kamran, Zach; Zellner, Katie; Kyriazes, Harry; Kraus, Christine M; Reynier, Jean-Baptiste; Malamy, Jocelyn E

2017-12-19

All animals have mechanisms for healing damage to the epithelial sheets that cover the body and line internal cavities. Epithelial wounds heal either by cells crawling over the wound gap, by contraction of a super-cellular actin cable ("purse string") that surrounds the wound, or some combination of the two mechanisms. Both cell crawling and purse string closure of epithelial wounds are widely observed across vertebrates and invertebrates, suggesting early evolution of these mechanisms. Cnidarians evolved ~600 million years ago and are considered a sister group to the Bilateria. They have been much studied for their tremendous regenerative potential, but epithelial wound healing has not been characterized in detail. Conserved elements of wound healing in bilaterians and cnidarians would suggest an evolutionary origin in a common ancestor. Here we test this idea by characterizing epithelial wound healing in live medusae of Clytia hemisphaerica. We identified cell crawling and purse string-mediated mechanisms of healing in Clytia epithelium that appear highly analogous of those seen in higher animals, suggesting that these mechanisms may have emerged in a common ancestor. Interestingly, we found that epithelial wound healing in Clytia is 75 to >600 times faster than in cultured cells or embryos of other animals previously studied, suggesting that Clytia may provide valuable clues about optimized healing efficiency. Finally, in Clytia, we show that damage to the basement membrane in a wound gap causes a rapid shift between the cell crawling and purse string mechanisms for wound closure. This is consistent with work in other systems showing that cells marginal to a wound choose between a super-cellular actin cable or lamellipodia formation to close wounds, and suggests a mechanism underlying this decision. 1. Cell crawling and purse string mechanisms of epithelial wound healing likely evolved before the divergence of Cnidaria from the bilaterian lineage ~ 600mya 2. In Clytia, the choice between cell crawling and purse string mechanisms of wound healing depends on interactions between the epithelial cells and the basement membrane.

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

PubMed

González-Robles, Arturo; Omaña-Molina, Maritza; Salazar-Villatoro, Lizbeth; Flores-Maldonado, Catalina; Lorenzo-Morales, Jacob; Reyes-Batlle, María; Arnalich-Montiel, Francisco; Martínez-Palomo, Adolfo

2017-12-01

Acanthamoeba culbertsoni trophozoites, previously isolated from a human keratitis case with severe intraocular damage, were maintained in axenic culture. Co-incubation of amoebae with MDCK cell monolayers demonstrated an apparent preference of the amoebae to introduce themselves between the cells. The trophozoites appeared to cross the cell monolayer through the tight junctions, which resulted in decreased trans-epithelial resistance (TER) measurements. Unexpectedly, after co-incubation of amoebae with hamster corneas, we observed that the trophozoites were able to cross the different cell layers and reach the corneal stroma after only 12 h of interaction, in contrast to other Acanthamoeba species. These observations suggest that this A. culbertsoni isolate is particularly pathogenic. Further research with diverse methodologies needs to be performed to explain the unique behavior of this Acanthamoeba strain. Copyright © 2017 Elsevier Inc. All rights reserved.

Capsular Sialic Acid of Streptococcus suis Serotype 2 Binds to Swine Influenza Virus and Enhances Bacterial Interactions with Virus-Infected Tracheal Epithelial Cells

PubMed Central

Wang, Yingchao; Gagnon, Carl A.; Savard, Christian; Music, Nedzad; Srednik, Mariela; Segura, Mariela; Lachance, Claude; Bellehumeur, Christian

2013-01-01

Streptococcus suis serotype 2 is an important swine bacterial pathogen, and it is also an emerging zoonotic agent. It is unknown how S. suis virulent strains, which are usually found in low quantities in pig tonsils, manage to cross the first host defense lines to initiate systemic disease. Influenza virus produces a contagious infection in pigs which is frequently complicated by bacterial coinfections, leading to significant economic impacts. In this study, the effect of a preceding swine influenza H1N1 virus (swH1N1) infection of swine tracheal epithelial cells (NTPr) on the ability of S. suis serotype 2 to adhere to, invade, and activate these cells was evaluated. Cells preinfected with swH1N1 showed bacterial adhesion and invasion levels that were increased more than 100-fold compared to those of normal cells. Inhibition studies confirmed that the capsular sialic acid moiety is responsible for the binding to virus-infected cell surfaces. Also, preincubation of S. suis with swH1N1 significantly increased bacterial adhesion to/invasion of epithelial cells, suggesting that S. suis also uses swH1N1 as a vehicle to invade epithelial cells when the two infections occur simultaneously. Influenza virus infection may facilitate the transient passage of S. suis at the respiratory tract to reach the bloodstream and cause bacteremia and septicemia. S. suis may also increase the local inflammation at the respiratory tract during influenza infection, as suggested by an exacerbated expression of proinflammatory mediators in coinfected cells. These results give new insight into the complex interactions between influenza virus and S. suis in a coinfection model. PMID:24082069

Protocadherin-1 binds to SMAD3 and suppresses TGF-β1-induced gene transcription

PubMed Central

Faura Tellez, Grissel; Vandepoele, Karl; Brouwer, Uilke; Koning, Henk; Elderman, Robin M.; Hackett, Tillie-Louise; Willemse, Brigitte W. M.; Holloway, John; Van Roy, Frans; Koppelman, Gerard H.

2015-01-01

Genetic studies have identified Protocadherin-1 (PCDH1) and Mothers against decapentaplegic homolog-3 (SMAD3) as susceptibility genes for asthma. PCDH1 is expressed in bronchial epithelial cells and has been found to interact with SMAD3 in yeast two-hybrid (Y2H) overexpression assays. Here, we test whether PCDH1 and SMAD3 interact at endogenous protein levels in bronchial epithelial cells and evaluate the consequences thereof for transforming growth factor-β1 (TGF-β1)-induced gene transcription. We performed Y2H screens and coimmunoprecipitation (co-IP) experiments of PCDH1 and SMAD3 in HEK293T and 16HBE14o− (16HBE) cell lines. Activity of a SMAD3-driven luciferase reporter gene in response to TGF-β1 was measured in BEAS-2B cells transfected with PCDH1 and in 16HBE cells transfected with PCDH1-small-interfering RNA (siRNA). TGF-β1-induced gene expression was quantified in BEAS-2B clones overexpressing PCDH1 and in human primary bronchial epithelial cells (PBECs) transfected with PCDH1-siRNA. We confirm PCDH1 and SMAD3 interactions by Y2H and by co-IP in HEK293T cells overexpressing both proteins, and at endogenous protein levels in 16HBE cells. TGF-β-induced activation of a SMAD3-driven reporter was reduced by exogenous PCDH1 in BEAS2B cells, whereas it was increased by siRNA-mediated knockdown of endogenous PCDH1 in 16HBE cells. Overexpression of PCDH1 suppressed expression of TGF-β target genes in BEAS-2B cells, whereas knockdown of PCDH1 in human PBECs increased TGF-β-induced gene expression. In conclusion, we demonstrate that PCDH1 binds to SMAD3 and regulates its activation by TGF-β signaling in bronchial epithelial cells. We propose that PCDH1 and SMAD3 act in a single pathway in asthma susceptibility that affects sensitivity of the airway epithelium to TGF-β. PMID:26209277

Cell polarity signaling in the plasticity of cancer cell invasiveness

PubMed Central

Gandalovičová, Aneta; Vomastek, Tomáš; Rosel, Daniel; Brábek, Jan

2016-01-01

Apico-basal polarity is typical of cells present in differentiated epithelium while front-rear polarity develops in motile cells. In cancer development, the transition from epithelial to migratory polarity may be seen as the hallmark of cancer progression to an invasive and metastatic disease. Despite the morphological and functional dissimilarity, both epithelial and migratory polarity are controlled by a common set of polarity complexes Par, Scribble and Crumbs, phosphoinositides, and small Rho GTPases Rac, Rho and Cdc42. In epithelial tissues, their mutual interplay ensures apico-basal and planar cell polarity. Accordingly, altered functions of these polarity determinants lead to disrupted cell-cell adhesions, cytoskeleton rearrangements and overall loss of epithelial homeostasis. Polarity proteins are further engaged in diverse interactions that promote the establishment of front-rear polarity, and they help cancer cells to adopt different invasion modes. Invading cancer cells can employ either the collective, mesenchymal or amoeboid invasion modes or actively switch between them and gain intermediate phenotypes. Elucidation of the role of polarity proteins during these invasion modes and the associated transitions is a necessary step towards understanding the complex problem of metastasis. In this review we summarize the current knowledge of the role of cell polarity signaling in the plasticity of cancer cell invasiveness. PMID:26872368

Geometric constraints during epithelial jamming

NASA Astrophysics Data System (ADS)

Atia, Lior; Bi, Dapeng; Sharma, Yasha; Mitchel, Jennifer A.; Gweon, Bomi; Koehler, Stephan A.; DeCamp, Stephen J.; Lan, Bo; Kim, Jae Hun; Hirsch, Rebecca; Pegoraro, Adrian F.; Lee, Kyu Ha; Starr, Jacqueline R.; Weitz, David A.; Martin, Adam C.; Park, Jin-Ah; Butler, James P.; Fredberg, Jeffrey J.

2018-06-01

As an injury heals, an embryo develops or a carcinoma spreads, epithelial cells systematically change their shape. In each of these processes cell shape is studied extensively whereas variability of shape from cell to cell is regarded most often as biological noise. But where do cell shape and its variability come from? Here we report that cell shape and shape variability are mutually constrained through a relationship that is purely geometrical. That relationship is shown to govern processes as diverse as maturation of the pseudostratified bronchial epithelial layer cultured from non-asthmatic or asthmatic donors, and formation of the ventral furrow in the Drosophila embryo. Across these and other epithelial systems, shape variability collapses to a family of distributions that is common to all. That distribution, in turn, is accounted for by a mechanistic theory of cell-cell interaction, showing that cell shape becomes progressively less elongated and less variable as the layer becomes progressively more jammed. These findings suggest a connection between jamming and geometry that spans living organisms and inert jammed systems, and thus transcends system details. Although molecular events are needed for any complete theory of cell shape and cell packing, observations point to the hypothesis that jamming behaviour at larger scales of organization sets overriding geometric constraints.

Rap1 GTPase is required for mouse lens epithelial maintenance and morphogenesis

PubMed Central

Maddala, Rupalatha; Nagendran, Tharkika; Lang, Richard A.; Morozov, Alexei; Rao, Ponugoti V.

2015-01-01

Rap1, a Ras-like small GTPase, plays a crucial role in cell-matrix adhesive interactions, cell-cell junction formation, cell polarity and migration. The role of Rap1 in vertebrate organ development and tissue architecture, however, remains elusive. We addressed this question in a mouse lens model system using a conditional gene targeting approach. While individual germline deficiency of either Rap1a or Rap1b did not cause overt defects in mouse lens, conditional double deficiency (Rap1 cKO) prior to lens placode formation led to an ocular phenotype including microphthalmia and lens opacification in embryonic mice. The embryonic Rap1 cKO mouse lens exhibited striking defects including loss of E-cadherin- and ZO-1-based cell-cell junctions, disruption of paxillin and β1-integrin-based cell adhesive interactions along with abnormalities in cell shape and apical-basal polarity of epithelium. These epithelial changes were accompanied by increased levels of α-smooth muscle actin, vimentin and N-cadherin, and expression of transcriptional suppressors of E-cadherin (Snai1, Slug and Zeb2), and a mesenchymal metabolic protein (Dihydropyrimidine dehydrogenase). Additionally, while lens differentiation was not overtly affected, increased apoptosis and dysregulated cell cycle progression were noted in epithelium and fibers in Rap1 cKO mice. Collectively these observations uncover a requirement for Rap1 in maintenance of lens epithelial phenotype and morphogenesis. PMID:26212757

Real-Time Sensing of Enteropathogenic E. coli-Induced Effects on Epithelial Host Cell Height, Cell-Substrate Interactions, and Endocytic Processes by Infrared Surface Plasmon Spectroscopy

PubMed Central

Zlotkin-Rivkin, Efrat; Rund, David; Melamed-Book, Naomi; Zahavi, Eitan Erez; Perlson, Eran; Mercone, Silvana; Golosovsky, Michael; Davidov, Dan; Aroeti, Benjamin

2013-01-01

Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell's height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity. PMID:24194932

Inflammatory bowel disease: cause and immunobiology.

PubMed

Baumgart, Daniel C; Carding, Simon R

2007-05-12

Crohn's disease and ulcerative colitis are idiopathic inflammatory bowel disorders. In this paper, we discuss how environmental factors (eg, geography, cigarette smoking, sanitation and hygiene), infectious microbes, ethnic origin, genetic susceptibility, and a dysregulated immune system can result in mucosal inflammation. After describing the symbiotic interaction of the commensal microbiota with the host, oral tolerance, epithelial barrier function, antigen recognition, and immunoregulation by the innate and adaptive immune system, we examine the initiating and perpetuating events of mucosal inflammation. We pay special attention to pattern-recognition receptors, such as toll-like receptors and nucleotide-binding-oligomerisation-domains (NOD), NOD-like receptors and their mutual interaction on epithelial cells and antigen-presenting cells. We also discuss the important role of dendritic cells in directing tolerance and immunity by modulation of subpopulations of effector T cells, regulatory T cells, Th17 cells, natural killer T cells, natural killer cells, and monocyte-macrophages in mucosal inflammation. Implications for novel therapies, which are discussed in detail in the second paper in this Series, are covered briefly.

Use of Methacrylic Acid-Containing Hydrogels to Increase Protein Transport Across the Intestinal Epithelium

NASA Astrophysics Data System (ADS)

Blanchette, James; Lopez, Jennifer; Park, Kinam; Peppas, Nicholas

2002-03-01

Oral protein delivery requires protection from the harsh environment of the stomach, release in the small intestine and passage from the intestinal lumen into the circulation. Hydrogels that swell in response to the pH change when passing from the stomach to the small intestine can accomplish the first two points. The ability to enhance the permeability of intestinal epithelial cells is currently under investigation. Methacrylic acid-containing hydrogels have shown the ability to bind calcium ions that decreases the concentration of free extracellular calcium for these epithelial cells. This change triggers a number of intracellular events including rearrangement of the cytoskeleton leading to increased permeability. Studies done on Caco-2 cells (human colon adenocarcinoma) measuring changes in transepithelial resistance are used to assess the effect of the polymer-cell interactions on the integrity of intestinal epithelial cell monolayers.

MOF Acetylates the Histone Demethylase LSD1 to Suppress Epithelial-to-Mesenchymal Transition.

PubMed

Luo, Huacheng; Shenoy, Anitha K; Li, Xuehui; Jin, Yue; Jin, Lihua; Cai, Qingsong; Tang, Ming; Liu, Yang; Chen, Hao; Reisman, David; Wu, Lizi; Seto, Edward; Qiu, Yi; Dou, Yali; Casero, Robert A; Lu, Jianrong

2016-06-21

The histone demethylase LSD1 facilitates epithelial-to-mesenchymal transition (EMT) and tumor progression by repressing epithelial marker expression. However, little is known about how its function may be modulated. Here, we report that LSD1 is acetylated in epithelial but not mesenchymal cells. Acetylation of LSD1 reduces its association with nucleosomes, thus increasing histone H3K4 methylation at its target genes and activating transcription. The MOF acetyltransferase interacts with LSD1 and is responsible for its acetylation. MOF is preferentially expressed in epithelial cells and is downregulated by EMT-inducing signals. Expression of exogenous MOF impedes LSD1 binding to epithelial gene promoters and histone demethylation, thereby suppressing EMT and tumor invasion. Conversely, MOF depletion enhances EMT and tumor metastasis. In human cancer, high MOF expression correlates with epithelial markers and a favorable prognosis. These findings provide insight into the regulation of LSD1 and EMT and identify MOF as a critical suppressor of EMT and tumor progression. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Two stages of enteropathogenic Escherichia coli intestinal pathogenicity are up and down-regulated by the epithelial cell differentiation.

PubMed

Gabastou, J M; Kernéis, S; Bernet-Camard, M F; Barbat, A; Coconnier, M H; Kaper, J B; Servin, A L

1995-09-01

Pathogens and eucaryotic cells are active partners during the process of pathogenicity. To gain access to enterocytes and to cross the epithelial membrane, many enterovirulent microorganisms interact with the brush border membrane-associated components as receptors. Recent reports provide evidence that intestinal cell differentiation plays a role in microbial pathogenesis. Human enteropathogenic Escherichia coli (EPEC) develop their pathogenicity upon infecting enterocytes. To determine if intestinal epithelial cell differentiation influences EPEC pathogenicity, we examined the infection of human intestinal epithelial cells by JPN 15 (pMAR7) [EAF+ eae+] EPEC strain as a function of the cell differentiation. The human embryonic intestinal INT407 cells, the human colonic T84 cells, the human undifferentiated HT-29 cells (HT-29 Std) and two enterocytic cell lines, HT-29 glc-/+ and Caco-2 cells, were used as cellular models. Cells were infected apically with the EPEC strain and the cell-association and cell-entry were examined by quantitative determination using metabolically radiolabeled bacteria, as well as by light, scanning and transmission electron microscopy. [EAF+ eae+] EPEC bacteria efficiently colonized the cultured human intestinal cells. Diffuse bacterial adhesion occurred to undifferentiated HT-29 Std and INT407 cells, whereas characteristic EPEC cell clusters were observed on fully differentiated enterocytic HT-29 glc-/+ cells and on colonic crypt T84 cells. As shown using the Caco-2 cell line, which spontaneously differentiates in culture, the formation of EPEC clusters increased as a function of the epithelial cell differentiation. In contrast, efficient cell-entry of [EAF+ eae+] EPEC bacteria occurred in recently differentiated Caco-2 cells and decreased when the cells were fully differentiated.(ABSTRACT TRUNCATED AT 250 WORDS)

Human Skin Is the Largest Epithelial Surface for Interaction with Microbes.

PubMed

Gallo, Richard L

2017-06-01

Human skin contains an abundant and diverse population of microbial organisms. Many of these microbes inhabit follicular structures of the skin. Furthermore, numerous studies have shown that the interaction of some members of the skin microbiome with host cells will result in changes in cell function. However, estimates of the potential for the microbiome to influence human health through skin have ignored the inner follicular surface, and therefore vastly underestimated the potential of the skin microbiome to have a systemic effect on the human body. By calculating the surface area of follicular and the interfollicular epithelial surface it is shown that skin provides a vast interface for interactions with the microbiome. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Multiplexed multi-scale imaging: novel roles for the scaffold protein IQGAP1 in epithelial cell development (Conference Presentation)

NASA Astrophysics Data System (ADS)

Schweikhard, Volker

2016-02-01

The precise sub-cellular spatial localization of multi-protein complexes is increasingly recognized as a key mechanism governing the organization of mammalian cells. Consequently, there is a need for novel microscopy techniques capable of investigating such sub-cellular architectures in comprehensive detail. Here, we applied a novel multiplexed STORM super-resolution microscopy technique, in combination with high-throughput immunofluorescence microscopy and live-cell imaging, to investigate the roles of the scaffold protein IQGAP1 in epithelial cells. IQGAP1 is known to orchestrate a wide range of biological processes, including intracellular signaling, cytoskeletal regulation, cell-cell adhesion, and protein trafficking, by forming distinct complexes with a number of known interaction partners, and recruiting these complexes to specific subcellular locations. Our results demonstrate that, in addition to supporting epithelial adherens junctions by associating with specialized cortical actin structures, IQGAP1 plays a second role in which it controls the confinement of a unique, previously undocumented class of membranous compartments to the basal actin cortex. These largely immotile yet highly dynamic structures appear transiently as cells merge into clusters and establish of apical-basolateral (epithelial) polarity, and are identified as an intermediate compartment in the endocytic recycling pathways for cell junction complexes and cell surface receptors. Although these two functions of IQGAP1 occur in parallel and largely independently of each other, they both support the maturation and maintenance of polarized epithelial cell architectures.

Interactions Between Bacteria and the Gut Mucosa: Do Enteric Neurotransmitters Acting on the Mucosal Epithelium Influence Intestinal Colonization or Infection?

PubMed

Green, Benedict T; Brown, David R

2016-01-01

The intestinal epithelium is a critical barrier between the internal and external milieux of the mammalian host. Epithelial interactions between these two host environments have been shown to be modulated by several different, cross-communicating cell types residing in the gut mucosa. These include enteric neurons, whose activity is influenced by bacterial pathogens, and their secreted products. Neurotransmitters appear to influence epithelial associations with bacteria in the intestinal lumen. For example, internalization of Salmonella enterica and Escherichia coli O157:H7 into the Peyer's patch mucosa of the small intestine is altered after the inhibition of neural activity with saxitoxin, a neuronal sodium channel blocker. Catecholamine neurotransmitters, such as dopamine and norepinephrine, also alter bacterial internalization in Peyer's patches. In the large intestine, norepinephrine increases the mucosal adherence of E. coli. These neurotransmitter actions are mediated by well-defined catecholamine receptors situated on the basolateral membranes of epithelial cells rather than through direct interactions with luminal bacteria. Investigations of the involvement of neuroepithelial communication in the regulation of interactions between the intestinal mucosa and luminal bacteria will provide novel insights into the mechanisms underlying bacterial colonization and pathogenesis at mucosal surfaces.

Salmonella enterica serovar Typhimurium adhesion and cytotoxicity during epithelial cell stress is reduced by Lactobacillus rhamnosus GG

PubMed Central

Burkholder, Kristin M; Bhunia, Arun K

2009-01-01

Background Physiological stressors may alter susceptibility of the host intestinal epithelium to infection by enteric pathogens. In the current study, cytotoxic effect, adhesion and invasion of Salmonella enterica serovar Typhimurium (S. Typhimurium) to Caco-2 cells exposed to thermal stress (41°C, 1 h) was investigated. Probiotic bacteria have been shown to reduce interaction of pathogens with the epithelium under non-stress conditions and may have a significant effect on epithelial viability during infection; however, probiotic effect on pathogen interaction with epithelial cells under physiological stress is not known. Therefore, we investigated the influence of Lactobacillus rhamnosus GG and Lactobacillus gasseri on Salmonella adhesion and Salmonella-induced cytotoxicity of Caco-2 cells subjected to thermal stress. Results Thermal stress increased the cytotoxic effect of both S. Typhimurium (P = 0.0001) and nonpathogenic E. coli K12 (P = 0.004) to Caco-2 cells, and resulted in greater susceptibility of cell monolayers to S. Typhimurium adhesion (P = 0.001). Thermal stress had no significant impact on inflammatory cytokines released by Caco-2 cells, although exposure to S. Typhimurium resulted in greater than 80% increase in production of IL-6 and IL-8. Blocking S. Typhimurium with anti-ShdA antibody prior to exposure of Salmonella decreased adhesion (P = 0.01) to non-stressed and thermal-stressed Caco-2 cells. Pre-exposure of Caco-2 cells to L. rhamnosus GG significantly reduced Salmonella-induced cytotoxicity (P = 0.001) and Salmonella adhesion (P = 0.001) to Caco-2 cells during thermal stress, while L. gasseri had no effect. Conclusion Results suggest that thermal stress increases susceptibility of intestinal epithelial Caco-2 cells to Salmonella adhesion, and increases the cytotoxic effect of Salmonella during infection. Use of L. rhamnosus GG as a probiotic may reduce the severity of infection during epithelial cell stress. Mechanisms by which thermal stress increases susceptibility to S. Typhimurium colonization and by which L. rhamnosus GG limits the severity of infection remain to be elucidated. PMID:19589170

Positive and Negative Regulatory Mechanisms for Fine-Tuning Cellularity and Functions of Medullary Thymic Epithelial Cells.

PubMed

Akiyama, Taishin; Tateishi, Ryosuke; Akiyama, Nobuko; Yoshinaga, Riko; Kobayashi, Tetsuya J

2015-01-01

Self-tolerant T cells and regulatory T cells develop in the thymus. A wide variety of cell-cell interactions in the thymus is required for the differentiation, proliferation, and repertoire selection of T cells. Various secreted and cell surface molecules expressed in thymic epithelial cells (TECs) mediate these processes. Moreover, cytokines expressed by cells of hematopoietic origin regulate the cellularity of TECs. Tumor necrosis factor (TNF) family RANK ligand, lymphotoxin, and CD40 ligand, expressed in T cells and innate lymphoid cells (ILCs), promote the differentiation and proliferation of medullary TECs (mTECs) that play critical roles in the induction of immune tolerance. A recent study suggests that interleukin-22 (IL-22) produced by ILCs promotes regeneration of TECs after irradiation. Intriguingly, tumor growth factor-β and osteoprotegerin limit cellularity of mTECs, thereby attenuating regulatory T cell generation. We will review recent insights into the molecular basis for cell-cell interactions regulating differentiation and proliferation of mTECs and also discuss about a perspective on use of mathematical models for understanding this complicated system.

[The cornea: stasis and dynamics].

PubMed

Nishida, Teruo

2008-03-01

The physiological roles of the cornea are to conduct external light into the eye, focus it, together with the lens, onto the retina, and to provide rigidity to the entire eyeball. Good vision thus requires maintenance of the transparency and proper refractive shape of the cornea. Although the cornea appears to be a relatively static structure, dynamic processes operate within and around the cornea at the tissue, cell, and molecular level. In this article, I review the mechanisms responsible for maintenance of corneal homeostasis as well as the development of new modes of treatment for various corneal diseases. I. The static cornea: structure and physiological functions. The cornea is derived from ectoderm, so that it can be considered as transparent skin. It is devoid of blood vessels and manifests the highest sensitivity in the entire body. The surface of the cornea is covered by tear fluid, which serves both as a lubricant and as a conduit for regulatory molecules. The cornea is also supplied with oxygen and various nutrients by the aqueous humor and a loop vascular system in addition to tear fluid. The cornea interacts with its surrounding tissues directly as well as indirectly through tear fluid or aqueous humor, with such interactions playing an important role in the regulation of corneal structure and functions. The resident cells of the cornea-epithelial cells, fibroblasts (keratocytes), and endothelial cells--also engage in mutual interactions through network systems. These interactions as well as those with infiltrated cells and regulation by nerves contribute to the maintenance of the normal structure and functions of the cornea as well as to the repair of corneal injuries. II. The dynamic cornea: maintenance of structure and functions by network systems. Developments in laser and computer technology have allowed observation of the cells and collagen fibers within the cornea. Furthermore, progress in cell and molecular biology has allowed characterization of dynamic network systems-including cell-cell and cell-extracellular matrix interactions as well as cytokines and neural factors-that contribute to the maintenance of corneal transparency and shape. III. Disruption of network systems: persistent corneal epithelial defects and corneal ulcer. Selection of the appropriate treatment for pathologic lesions of the cornea and the accompanying decrease in visual acuity requires localization of the lesion with regard to the epithelium, stroma, or endothelium of the cornea. In certain instances, however, it is not possible to determine the cause of the problem within the cornea. In such cases, the cause of the pathologic lesion and the target for treatment may lie in the surrounding tissues or environment. For example, corneal epithelial wound healing may be delayed, leading to the development of persistent epithelial defects, as a result of disruption of intercellular junctions between epithelial cells, an abnormality of the corneal basement membrane, altered concentrations of various cytokines in tear fluid, a lowered corneal sensation, or allergic reactions in the lid conjunctiva. Loss of corneal epithelial barrier function can further allow inflammatory cytokines present in tear fluid, together with infiltrated cells, to activate keratocytes and elicit excessive degradation of collagen in the stroma, thereby giving rise to corneal ulcer. IV. Development of new drugs for corneal diseases. We have attempted to apply the results of basic scientific research to the development of new drugs for corneal diseases that remain difficult to treat. The process of authorization for new drugs from the Ministry of Health, Labor, and Welfare takes more than two decades, however. The path from the bench to clinical practice is thus a long one. 1. Development of eyedrops for treatment of persistent corneal epithelial defects. We demonstrated the clinical efficacy of fibronectin eyedrops for the treatment of persistent epithelial defects of the cornea. However, the possibility of blood-borne infections has interfered with the development of serum-derived fibronectin as a drug. An automated machine for the preparation of autologous fibronectin eyedrops has therefore recently been developed. Furthermore, in seeking an alternative to fibronectin eyedrops, we are investigating the effects of a peptide corresponding to the second cell-binding domain of fibronectin on corneal epithelial wound healing. Considering that urokinase-type plasminogen activator may be expressed at the site of corneal epithelial defects and facilitates epithelial migration, the potential clinical application of annexin V, which stimulates the secretion of urokinase-type plasminogen activator for the treatment of persistent corneal epithelial defects is also now under investigation in Japan. 2. Development of eyedrops for treatment of neurotrophic keratopathy. Substance P, a neurotransmitter, stimulates corneal epithelial migration in a synergistic manner with insulin-like growth factor (IGF)--1. We have shown that eyedrops containing both the substance P-derived peptide FGLM-amide and the IGF-1--derived peptide SSSR are effective for the treatment of persistent corneal epithelial defects in individuals with diabetic keratopathy or neurotrophic keratopathy, both of which are associated with a reduction in corneal sensation. 3. Development of drugs for corneal ulcer. Treatment of corneal infection with antibiotics does not necessarily halt the process of corneal ulceration, which is characterized by excessive degradation of stromal collagen, or resolve persistent corneal epithelial defects. In addition to eyedrops for the treatment of persistent corneal epithelial defects, we have therefore also been working on the development of new drugs for the treatment of corneal ulcer. To this end, we have established an experimental system in which corneal fibroblasts are cultured in a three-dimensional collagen gel. With this system, we have shown that triptolide and steroids inhibit collagen degradation by corneal fibroblasts. Triptolide or its derivatives are thus potential drugs for the treatment of corneal ulcer and would work by acting directly on corneal fibroblasts rather than by inhibiting the secreted enzymes(matrix metalloproteinases) responsible for collagen degradation.

Paramyxovirus Infection Mimics In Vivo Cellular Dynamics in Three-Demensional Human Bronchio-Epithelial Tissue-Like Assemblies

NASA Technical Reports Server (NTRS)

Deatly, Anne M.; Lin, Yen-Huei; McCarthy, Maureen; Chen, Wei; Miller, Lynn Z.; Quiroz, Jorge; Nowak, Becky M.; Lerch, Robert A.; Udem, Stephen A.; Goodwin, Thomas J.

2012-01-01

Respiratory syncytial virus and parainfluenza virus cause severe respiratory disease, especially in infants, children and the elderly. An in vitro model that accurately mimics infection of the human respiratory epithelium (HRE) would facilitate vaccine development greatly. Monolayer cultures traditionally used to study these viruses do not accurately and precisely differentiate the replication efficiencies of wild type and attenuated viruses. Therefore, we engineered novel three-dimensional (3D) tissue-like assemblies (TLAs) of human broncho-epithelial (HBE) cells to produce a more physiologically relevant in vitro model of the HRE. TLAs resemble HRE structurally and by expression of differentiated epithelial cell markers. Most significantly, wild type viruses exhibited a clear growth advantage over attenuated strains in TLAs unlike monolayer cultures. In addition, the TLAs responded to virus infection by secreting pro-inflammatory mediators similar to the respiratory epithelia of infected children. These characteristics make the TLA model a valuable platform technology to develop and evaluate live, attenuated respiratory virus vaccine candidates for human use. Respiratory virus diseases, the most frequent and least preventable of all infectious diseases, range in severity from the common cold to severe bronchiolitis and pneumonia . Two paramyxoviruses, respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3), are responsible for a majority of the most severe respiratory diseases of infants and young children. RSV causes 70% of all bronchiolitis cases and is a major cause of morbidity and mortality worldwide, especially in infants. PIV3 causes 10-15% of bronchiolitis and pneumonia during infancy, second only to RSV, and 40% of croup in infants To date, licensed vaccines are not available to prevent these respiratory diseases. At present, traditional monkey kidney (Vero and LLC-MK2) and human (HEp-2) tissue culture cells and small animal models (mouse, cotton rat, guinea pig, ferret, and hamster) fail to accurately imitate viral replication and human disease states (8). Lacking an authentic model has impeded the development and evaluation of live, attenuated vaccine candidates. Development of a physiologically relevant in vitro tissue culture model that reproduces characteristics of the HRE, the primary target of RSV and PIV3, would aid in predicting clinical attenuation and safety of vaccine candidates. Successful tissue engineering of a 3D human intestinal model using novel NASA technology inspired the development of a tri-culture 3D model for the HRE. Sequential layering of primary mesenchymal cells (comprised of normal human fibroblasts and endothelial cells) followed by BEAS-2B epithelial cells derived from human bronchi and tracheae were recapitulated on Cultisphere and/or cytodex3 microcarriers in cylindrical vessels that rotate horizontally creating an organized epithelial structure. Horizontal rotation randomizes the gravity vector modeling aspects of microgravity. Mesenchymal and epithelial cells grown under these conditions reproduce the structural organization, multi-cellular complexity, and differentiation state of the HRE. The opportunity to study respiratory viruses in a nasal epithelium model is invaluable because the most promising respiratory virus vaccine candidates are live attenuated viruses for intranasal administration. Here we characterize the interactions of respiratory viruses and epithelial cells grown under modeled microgravity in comparison to gravity-ladened monolayers. 3D HBE TLAs and traditional monolayers (2D) are infected at 35 C, the upper temperature of the upper HRE, to simulate in vivo infection conditions. Growth kinetics of wild type (wt) RSV and PIV3 viruses were compared in 2D and 3D cells to that of strains attenuated in humans or rhesus macaques. This novel 3D HBE model also offers an opportunity to study whether the epithelial cell function, especially in host defenses recapitulated by mimicking the structural organization of the HRE. In vivo, airway epithelial cells play a significant and dynamic role in host defense by blocking paracellular permeability and modulating airway function through cellular interactions or tight junctions. As regulators of the innate immune response, epithelial cells constitutively express cytokines, chemokines, and colony stimulating factors including RANTES, IL-8, IL-6, GM-CSF, and G-CSF for proactive host defense. In response to viral infection, epithelial cells induce potent immuno-modulatory and pro-inflammatory cytokines that recruit phagocytic and inflammatory cells to clear the virus and enhance protection. Although disease pathogenesis is classically attributed to the cytopathic effects of the pathogen, severe disease states associated with RSV and PIV3 are attributed to the inflammatory response, especially in infants. RSV is a potent inducer of cytokines and pro-inflammatory mediators in epithelial cells in vivo. A differentiated human epithelial model independent of the complete functional immune system will help elucidate the role of epithelial cells in respiratory disease. We reported here, virus and host cell interactions in 3D HBE TLAs are similar to that in vivo. Because the epithelial cell organization of the TLAs impacts not only the expression of airway epithelial characteristics, but also cellular communication, the TLAs represent a more physiologically relevant model of the HRE than BEAS-2B or other non-tumour monolayer models of respiratory disease. As a result, wild type respiratory viruses have a clear growth advantage over attenuated viruses in TLAs unlike traditional monolayers. In addition, the TLAs respond to wild type virus infection by secreting pro-inflammatory mediators characteristic of infected HRE. TLAs expressing microbial defense mechanisms provide an excellent model to study the interactions of respiratory pathogens with their host and to identify the innate immunity mediators. Therefore, 3D HBE TLAs offer advantages for the study of respiratory viruses and the development of viral vaccine candidates.

Metastatic prostate cancer-associated P62 inhibits autophagy flux and promotes epithelial to mesenchymal transition by sustaining the level of HDAC6.

PubMed

Jiang, Xianhan; Huang, Yiqiao; Liang, Xue; Jiang, Funeng; He, Yongzhong; Li, Tian; Xu, Guibin; Zhao, Haibo; Yang, Weiqing; Jiang, Ganggang; Su, Zhengming; Jiang, Lingke; Liu, Leyuan

2018-05-01

P62 (also named sequestosome-1, SQSTM1) is involved in autophagy regulation through multiple pathways. It interacts with autophagosomes-associated LC3-II and ubiquitinated protein aggregates to engulf the aggregates in autophagosomes, interacts with HDAC6 to inhibit its deacetylase activity to maintain the levels of acetylated α-tubulin and stabilities of microtubules to enhance autophagosome trafficking, and regulates autophagy initiation and cell survival. We performed immunohistochemistry staining of P62 in prostate tissues from prostate cancer patients and found that levels of P62 in patients with prostate adenocarcinomas (PCA) are significantly higher than those in patients with benign prostate hyperplasia (BPH). High levels of P62 predict high tumor grade and high intensity of metastasis. We created prostate cancer cell lines stably overexpressing P62 and then suppress the expression of P62 in the cell line stably overexpressing P62 with CRISPR technology. Cell proliferation assay with crystal violet, cell migration assay, cell invasion assay, Western blot analysis, and confocal fluorescent microscopy were conducted to test the impact of altered levels of P62 on the growth, migration, invasion, epithelial-to-mesenchymal transition, autophagy flux, HDAC6 activity, and microtubular acetylation of cancer cells. P62 increased the levels of HDAC6 and reduced the acetylation of α-tubulin and the stability of microtubules. Consequently, high levels of P62 caused a promotion of epithelial-to-mesenchymal transition in addition to an impairment of autophagy flux, and further led to an enhancement of proliferation, migration, and invasion of prostate cancer cells. P62 promotes metastasis of PCA by sustaining the level of HDAC6 to inhibit autophagy and promote epithelial-to-mesenchymal transition. © 2018 Wiley Periodicals, Inc.

Cellular migration, transition and interaction during regeneration of the sponge Hymeniacidon heliophila.

PubMed

Coutinho, Cristiano C; Rosa, Ivone de Andrade; Teixeira, John Douglas de Oliveira; Andrade, Leonardo R; Costa, Manoel Luis; Mermelstein, Claudia

2017-01-01

Sponges have a high capacity for regeneration and this process improves biomass production in some species, thus contributing to a solution for the biomass supply problem for biotechnological applications. The aim of this work is to characterize the dynamics of cell behavior during the initial stages of sponge regeneration, using bright-field microscopy, confocal microscopy and SEM. We focused on the first 20 h of regeneration, during which blastema formation and epithelium initialization occur. An innovative sponge organotypic culture of the regenerating internal region is described and investigated by confocal microscopy, cell transplantation and vital staining. Cell-cell interaction and cell density are shown to affect events in morphogenesis such as epithelial/mesenchymal and mesenchymal/epithelial transitions as well as distinct cell movements required for regeneration. Extracellular matrix was organized according to the morphogenetic process observed, with evidence for cell-signaling instructions and remodeling. These data and the method of organotypic culture described here provide support for the development of viable sponge biomass production.

Bone morphogenetic protein and Notch signalling crosstalk in poor-prognosis, mesenchymal-subtype colorectal cancer.

PubMed

Irshad, Shazia; Bansal, Mukesh; Guarnieri, Paolo; Davis, Hayley; Al Haj Zen, Ayman; Baran, Brygida; Pinna, Claudia Maria Assunta; Rahman, Haseeb; Biswas, Sujata; Bardella, Chiara; Jeffery, Rosemary; Wang, Lai Mun; East, James Edward; Tomlinson, Ian; Lewis, Annabelle; Leedham, Simon John

2017-06-01

The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context-dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ-secretase-independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co-localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal-subtype tumours, where it interacts with Notch to induce an epithelial-mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP-Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal-subtype tumours promotes a synergistic BMP-Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal-subtype CRC insensitive to γ-secretase inhibition unless BMP activation is concomitantly addressed. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Know your neighbor: Microbiota and host epithelial cells interact locally to control intestinal function and physiology.

PubMed

Sommer, Felix; Bäckhed, Fredrik

2016-05-01

Interactions between the host and its associated microbiota differ spatially and the local cross talk determines organ function and physiology. Animals and their organs are not uniform but contain several functional and cellular compartments and gradients. In the intestinal tract, different parts of the gut carry out different functions, tissue structure varies accordingly, epithelial cells are differentially distributed and gradients exist for several physicochemical parameters such as nutrients, pH, or oxygen. Consequently, the microbiota composition also differs along the length of the gut, but also between lumen and mucosa of the same intestinal segment, and even along the crypt-villus axis in the epithelium. Thus, host-microbiota interactions are highly site-specific and the local cross talk determines intestinal function and physiology. Here we review recent advances in our understanding of site-specific host-microbiota interactions and discuss their functional relevance for host physiology. © 2016 WILEY Periodicals, Inc.

Cellular interactions of a lipid-based nanocarrier model with human keratinocytes: Unravelling transport mechanisms.

PubMed

Silva, Elisabete; Barreiros, Luísa; Segundo, Marcela A; Costa Lima, Sofia A; Reis, Salette

2017-04-15

Knowledge of delivery system transport through epidermal cell monolayer is vital to improve skin permeation and bioavailability. Recently, nanostructured lipid carriers (NLCs) have gained great attention for transdermal delivery due to their biocompatibility, high drug payload, occlusive properties and skin hydration effect. However, the nanocarriers transport related mechanisms in epidermal epithelial cells are not yet understood. In this research, the internalization and transport pathways of the NLCs across the epidermal epithelial cell monolayer (HaCaT cells) were investigated. The 250nm sized witepsol/miglyol NLCs, prepared by hot homogenization had reduced cytotoxicity and no effect on the integrity of cell membrane in human HaCaT keratinocytes. The internalization was time-, concentration- and energy-dependent, and the uptake of NLCs was a vesicle-mediated process by macropinocytosis and clathrin-mediated pathways. 3% of NLCs were found at the apical membrane side of the HaCaT monolayer through exocytosis mechanism. Additionally, the endoplasmic reticulum, Golgi apparatus and microtubules played crucial roles in the transport of NLCs out of HaCaT cells. NLCs were transported intact across the human keratinocytes monolayer, without disturbing the tight junction's structure. From the transcytosis data only approximately 12% of the internalized NLCs were passed from the apical to the basolateral side. The transcytosis of NLCs throughout the HaCaT cell monolayer towards the basolateral membrane side requires the involvement of the endoplasmic reticulum, Golgi apparatus and microtubules. Our findings may contribute to a systematic understanding of NLCs transport across epidermal epithelial cell monolayers and their optimization for clinical transdermal application. Transdermal drug delivery is a challenging and growing area of clinical application. Lipid nanoparticles such as nanostructured lipid carriers (NLCs) have gained wide interest for transdermal drug delivery. However these nanocarriers' interactions with epidermal epithelial barrier are yet unknown. Unveiling the mechanisms involved in NLCs transport across the epidermal epithelial monolayers will contribute with valuable information to achieve enhanced skin permeability, superior bioavailability and consequently improved therapeutic effect. With our present work we could certainly provide researchers and clinicians guidance for the design of optimized transdermal delivery systems, based on the nanomaterials and biological interactions. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Stable knockdown of Kif5b in MDCK cells leads to epithelial–mesenchymal transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cui, Ju, E-mail: juzi.cui@gmail.com; Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR; Jin, Guoxiang

2015-07-17

Polarization of epithelial cells requires vectorial sorting and transport of polarity proteins to apical or basolateral domains. Kif5b is the mouse homologue of the human ubiquitous Kinesin Heavy Chain (uKHC). To investigate the function of Kif5b in epithelial cells, we examined the phenotypes of Kif5b-deficient MDCK cells. Stable knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate, profound changes in cell morphology, loss of epithelial cell marker, and gain of mesenchymal marker, as well as increased cell migration, invasion, and tumorigenesis abilities. E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells, and their expression levelsmore » were decreased in Kif5b-deficient MDCK cells. Overexpression of E-cadherin and NMMIIA in Kif5b depleted MDCK cells could decrease mesenchymal marker expression and cell migration ability. These results indicate that stable knockdown of Kif5b in MDCK cells can lead to epithelial–mesenchymal transition, which is mediated by defective E-cadherin and NMMIIA expression. - Highlights: • Knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate. • Kif5b deficient MDCK cells underwent epithelial–mesenchymal transition. • E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells. • Decreased E-cadherin and NMMIIA levels mediate EMT in Kif5b deficient MDCK cells. • Overexpression of E-cadherin and NMMIIA reverse the effects of Kif5b knockdown.« less

Analysis of Hippo and TGFβ signaling in polarizing epithelial cells and mouse embryos.

PubMed

Narimatsu, Masahiro; Labibi, Batool; Wrana, Jeffrey L; Attisano, Liliana

2016-01-01

The Hippo signaling pathway is involved in numerous biological events ranging from early development to organogenesis and when disrupted, impacts various human diseases including cancer. The Hippo pathway also interacts with and controls the activity of other signaling pathways such as the TGFβ/Smad pathway, in which Hippo pathway activity influences the subcellular localization of Smad transcription factors. Here, we describe techniques for examining crosstalk between Hippo and TGFβ signaling in polarizing mammary epithelial cells. In addition, we provide detailed methods for analyzing the subcellular localization of the Hippo pathway effectors, Taz and Yap using both in vitro cultured epithelial cells and in vivo in pregastrulation mouse embryos. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

PubMed Central

Ulm, Ashley; Mayhew, Christopher N.; Debley, Jason; Khurana Hershey, Gurjit K.; Ji, Hong

2016-01-01

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research. PMID:27022951

Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri

PubMed Central

2012-01-01

Background Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. Results We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. Conclusions We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material. PMID:22429795

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells.

PubMed

Ulm, Ashley; Mayhew, Christopher N; Debley, Jason; Khurana Hershey, Gurjit K; Ji, Hong

2016-03-10

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

Haemophilus haemolyticus Interaction with Host Cells Is Different to Nontypeable Haemophilus influenzae and Prevents NTHi Association with Epithelial Cells

PubMed Central

Pickering, Janessa L.; Prosser, Amy; Corscadden, Karli J.; de Gier, Camilla; Richmond, Peter C.; Zhang, Guicheng; Thornton, Ruth B.; Kirkham, Lea-Ann S.

2016-01-01

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that resides in the upper respiratory tract and contributes to a significant burden of respiratory related diseases in children and adults. Haemophilus haemolyticus is a respiratory tract commensal that can be misidentified as NTHi due to high levels of genetic relatedness. There are reports of invasive disease from H. haemolyticus, which further blurs the species boundary with NTHi. To investigate differences in pathogenicity between these species, we optimized an in vitro epithelial cell model to compare the interaction of 10 H. haemolyticus strains with 4 NTHi and 4 H. influenzae-like haemophili. There was inter- and intra-species variability but overall, H. haemolyticus had reduced capacity to attach to and invade nasopharyngeal and bronchoalveolar epithelial cell lines (D562 and A549) within 3 h when compared with NTHi. H. haemolyticus was cytotoxic to both cell lines at 24 h, whereas NTHi was not. Nasopharyngeal epithelium challenged with some H. haemolyticus strains released high levels of inflammatory mediators IL-6 and IL-8, whereas NTHi did not elicit an inflammatory response despite higher levels of cell association and invasion. Furthermore, peripheral blood mononuclear cells stimulated with H. haemolyticus or NTHi released similar and high levels of IL-6, IL-8, IL-10, IL-1β, and TNFα when compared with unstimulated cells but only NTHi elicited an IFNγ response. Due to the relatedness of H. haemolyticus and NTHi, we hypothesized that H. haemolyticus may compete with NTHi for colonization of the respiratory tract. We observed that in vitro pre-treatment of epithelial cells with H. haemolyticus significantly reduced NTHi attachment, suggesting interference or competition between the two species is possible and warrants further investigation. In conclusion, H. haemolyticus interacts differently with host cells compared to NTHi, with different immunostimulatory and cytotoxic properties. This study provides an in vitro model for further investigation into the pathogenesis of Haemophilus species and the foundation for exploring whether H. haemolyticus can be used to prevent NTHi disease. PMID:27242968

Cellular senescence and autophagy in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF).

PubMed

Kuwano, Kazuyoshi; Araya, Jun; Hara, Hiromichi; Minagawa, Shunsuke; Takasaka, Naoki; Ito, Saburo; Kobayashi, Kenji; Nakayama, Katsutoshi

2016-11-01

Aging is associated with impairments in homeostasis. Although aging and senescence are not equivalent, the number of senescent cells increases with aging. Cellular senescence plays important roles in tissue repair or remodeling, as well as embryonic development. Autophagy is a process of lysosomal self-degradation that maintains a homeostatic balance between the synthesis, degradation, and recycling of cellular proteins. Autophagy diminishes with aging; additionally, accelerated aging can be attributed to reduced autophagy. Cellular senescence has been widely implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD), a disease of accelerated lung aging, presumably by impairing cell repopulation and by aberrant cytokine secretion in the senescence-associated secretory phenotype. The possible participation of autophagy in the pathogenic sequence of COPD has been extensively explored. Although it has been reported that increased autophagy may induce epithelial cell death, an insufficient reserve of autophagy can induce cellular senescence in bronchial epithelial cells of COPD. Furthermore, advanced age is one of the most important risk factors for the development of idiopathic pulmonary fibrosis (IPF). Telomere shortening is found in blood leukocytes and alveolar epithelial cells from patients with IPF. Accelerated senescence of epithelial cells plays a role in IPF pathogenesis by perpetuating abnormal epithelial-mesenchymal interactions. Insufficient autophagy may be an underlying mechanism of accelerated epithelial cell senescence and myofibroblast differentiation in IPF. Herein, we review the molecular mechanisms of cellular senescence and autophagy and summarize the role of cellular senescence and autophagy in both COPD and IPF. Copyright © 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

HIV Impairs Lung Epithelial Integrity and Enters the Epithelium to Promote Chronic Lung Inflammation.

PubMed

Brune, Kieran A; Ferreira, Fernanda; Mandke, Pooja; Chau, Eric; Aggarwal, Neil R; D'Alessio, Franco R; Lambert, Allison A; Kirk, Gregory; Blankson, Joel; Drummond, M Bradley; Tsibris, Athe M; Sidhaye, Venkataramana K

2016-01-01

Several clinical studies show that individuals with HIV are at an increased risk for worsened lung function and for the development of COPD, although the mechanism underlying this increased susceptibility is poorly understood. The airway epithelium, situated at the interface between the external environment and the lung parenchyma, acts as a physical and immunological barrier that secretes mucins and cytokines in response to noxious stimuli which can contribute to the pathobiology of chronic obstructive pulmonary disease (COPD). We sought to determine the effects of HIV on the lung epithelium. We grew primary normal human bronchial epithelial (NHBE) cells and primary lung epithelial cells isolated from bronchial brushings of patients to confluence and allowed them to differentiate at an air- liquid interface (ALI) to assess the effects of HIV on the lung epithelium. We assessed changes in monolayer permeability as well as the expression of E-cadherin and inflammatory modulators to determine the effect of HIV on the lung epithelium. We measured E-cadherin protein abundance in patients with HIV compared to normal controls. Cell associated HIV RNA and DNA were quantified and the p24 viral antigen was measured in culture supernatant. Surprisingly, X4, not R5, tropic virus decreased expression of E-cadherin and increased monolayer permeability. While there was some transcriptional regulation of E-cadherin, there was significant increase in lysosome-mediated protein degradation in cells exposed to X4 tropic HIV. Interaction with CXCR4 and viral fusion with the epithelial cell were required to induce the epithelial changes. X4 tropic virus was able to enter the airway epithelial cells but not replicate in these cells, while R5 tropic viruses did not enter the epithelial cells. Significantly, X4 tropic HIV induced the expression of intercellular adhesion molecule-1 (ICAM-1) and activated extracellular signal-regulated kinase (ERK). We demonstrate that HIV can enter airway epithelial cells and alter their function by impairing cell-cell adhesion and increasing the expression of inflammatory mediators. These observed changes may contribute local inflammation, which can lead to lung function decline and increased susceptibility to COPD in HIV patients.

Endothelial induced EMT in breast epithelial cells with stem cell properties.

PubMed

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J R; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A; Petersen, Ole William; Magnusson, Magnus K; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Endothelial Induced EMT in Breast Epithelial Cells with Stem Cell Properties

PubMed Central

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J. R.; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A.; Petersen, Ole William; Magnusson, Magnus K.; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44high/CD24low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer. PMID:21915264

EphA2 and ephrin-A5 are not a receptor-ligand pair in the ocular lens.

PubMed

Cheng, Catherine; Fowler, Velia M; Gong, Xiaohua

2017-09-01

Eph-ephrin bidirectional signaling is essential for eye lens transparency in humans and mice. Our previous studies in mouse lenses demonstrate that ephrin-A5 is mainly expressed in the anterior epithelium, where it is required for maintaining the anterior epithelial monolayer. In contrast, EphA2 is localized in equatorial epithelial and fiber cells where it is essential for equatorial epithelial and fiber cell organization and hexagonal cell shape. Immunostaining of lens epithelial and fiber cells reveals that EphA2 and ephrin-A5 are also co-expressed in anterior fiber cell tips, equatorial epithelial cells and newly formed lens fibers, although they are not precisely colocalized. Due to this complex expression pattern and the promiscuous interactions between Eph receptors and ephrin ligands, as well as their complex bidirectional signaling pathways, cataracts in ephrin-A5(-/-) or EphA2(-/-) lenses may arise from loss of function or abnormal signaling mechanisms. To test whether abnormal signaling mechanisms may play a role in cataractogenesis in ephrin-A5(-/-) or EphA2(-/-) lenses, we generated EphA2 and ephrin-A5 double knockout (DKO) mice. We compared the phenotypes of EphA2(-/-) and ephrin-A5(-/-) lenses to that of DKO lenses. DKO lenses displayed an additive lens phenotype that was not significantly different from the two single KO lens phenotypes. Similar to ephrin-A5(-/-) lenses, DKO lenses had abnormal anterior epithelial cells leading to a large mass of epithelial cells that invade into the underlying fiber cell layer, directly resulting in anterior cataracts in ephrin-A5(-/-) and DKO lenses. Yet, similar to EphA2(-/-) lenses, DKO lenses also had abnormal packing of equatorial epithelial cells with disorganized meridional rows, lack of a lens fulcrum and disrupted fiber cells. The DKO lens phenotype rules out abnormal signaling by EphA2 in ephrin-A5(-/-) lenses or by ephrin-A5 in EphA2(-/-) lenses as possible cataract mechanisms. Thus, these results indicate that EphA2 and ephrin-A5 do not form a lens receptor-ligand pair, and that EphA2 and ephrin-A5 have other binding partners in the lens to help align differentiating equatorial epithelial cells or maintain the anterior epithelium, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

SU-E-J-107: Supervised Learning Model of Aligned Collagen for Human Breast Carcinoma Prognosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bredfeldt, J; Liu, Y; Conklin, M

Purpose: Our goal is to develop and apply a set of optical and computational tools to enable large-scale investigations of the interaction between collagen and tumor cells. Methods: We have built a novel imaging system for automating the capture of whole-slide second harmonic generation (SHG) images of collagen in registry with bright field (BF) images of hematoxylin and eosin stained tissue. To analyze our images, we have integrated a suite of supervised learning tools that semi-automatically model and score collagen interactions with tumor cells via a variety of metrics, a method we call Electronic Tumor Associated Collagen Signatures (eTACS). Thismore » group of tools first segments regions of epithelial cells and collagen fibers from BF and SHG images respectively. We then associate fibers with groups of epithelial cells and finally compute features based on the angle of interaction and density of the collagen surrounding the epithelial cell clusters. These features are then processed with a support vector machine to separate cancer patients into high and low risk groups. Results: We validated our model by showing that eTACS produces classifications that have statistically significant correlation with manual classifications. In addition, our system generated classification scores that accurately predicted breast cancer patient survival in a cohort of 196 patients. Feature rank analysis revealed that TACS positive fibers are more well aligned with each other, generally lower density, and terminate within or near groups of epithelial cells. Conclusion: We are working to apply our model to predict survival in larger cohorts of breast cancer patients with a diversity of breast cancer types, predict response to treatments such as COX2 inhibitors, and to study collagen architecture changes in other cancer types. In the future, our system may be used to provide metastatic potential information to cancer patients to augment existing clinical assays.« less

Ionizing radiation induces heritable disruption of epithelial cell interactions

NASA Technical Reports Server (NTRS)

Park, Catherine C.; Henshall-Powell, Rhonda L.; Erickson, Anna C.; Talhouk, Rabih; Parvin, Bahram; Bissell, Mina J.; Barcellos-Hoff, Mary Helen; Chatterjee, A. (Principal Investigator)

2003-01-01

Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, beta-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell-cell communication, aberrant cell-extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization.

Tannic acid attenuates TGF-β1-induced epithelial-to-mesenchymal transition by effectively intervening TGF-β signaling in lung epithelial cells.

PubMed

Pattarayan, Dhamotharan; Sivanantham, Ayyanar; Krishnaswami, Venkateshwaran; Loganathan, Lakshmanan; Palanichamy, Rajaguru; Natesan, Subramanian; Muthusamy, Karthikeyan; Rajasekaran, Subbiah

2018-03-01

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and an irreversible lung disorder characterized by the accumulation of fibroblasts and myofibroblasts in the extracellular matrix. The transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) is thought to be one of the possible sources for a substantial increase in the number of fibroblasts/myofibroblasts in IPF lungs. Tannic acid (TA), a natural dietary polyphenolic compound has been shown to possess diverse pharmacological effects. However, whether TA can inhibit TGF-β1-mediated EMT in lung epithelial cells remains enigmatic. Both the human adenocarcinomic alveolar epithelial (A549) and normal bronchial epithelial (BEAS-2B) cells were treated with TGF-β1 with or without TA. Results showed that TA addition, markedly inhibited TGF-β1-induced EMT as assessed by reduced expression of N-cadherin, type-1-collagen, fibronectin, and vimentin. Furthermore, TA inhibited TGF-β1-induced cell proliferation through inducing cell cycle arrest at G0/G1 phase. TGF-β1-induced increase in the phosphorylation of Smad (Smad2 and 3), Akt as well as that of mitogen activated protein kinase (ERK1/2, JNK1/2, and p38) mediators was effectively inhibited by TA. On the other hand, TA reduced the TGF-β1-induced increase in TGF-β receptors expression. Using molecular docking approach, FTIR, HPLC and Western blot analyses, we further identified the direct binding of TA to TGF-β1. Finally, we conclude that TA might directly interact with TGF-β1, thereby repressing TGF-β signaling and subsequent EMT process in lung epithelial cells. Further animal studies are needed to clarify its potential therapeutic benefit in pulmonary fibrosis. © 2017 Wiley Periodicals, Inc.

Regulation of Bacteria-Induced Intercellular Adhesion Molecule-1 by CCAAT/Enhancer Binding Proteins

PubMed Central

Manzel, Lori J.; Chin, Cecilia L.; Behlke, Mark A.; Look, Dwight C.

2009-01-01

Direct interaction between bacteria and epithelial cells may initiate or amplify the airway response through induction of epithelial defense gene expression by nuclear factor-κB (NF-κB). However, multiple signaling pathways modify NF-κB effects to modulate gene expression. In this study, the effects of CCAAT/enhancer binding protein (C/EBP) family members on induction of the leukocyte adhesion glycoprotein intercellular adhesion molecule-1 (ICAM-1) was examined in primary cultures of human tracheobronchial epithelial cells incubated with nontypeable Haemophilus influenzae. Increased ICAM-1 gene transcription in response to H. influenzae required gene sequences located at −200 to −135 in the 5′-flanking region that contain a C/EBP-binding sequence immediately upstream of the NF-κB enhancer site. Constitutive C/EBPβ was found to have an important role in epithelial cell ICAM-1 regulation, while the adjacent NF-κB sequence binds the RelA/p65 and NF-κB1/p50 members of the NF-κB family to induce ICAM-1 expression in response to H. influenzae. The expression of C/EBP proteins is not regulated by p38 mitogen-activated protein kinase activation, but p38 affects gene transcription by increasing the binding of TATA-binding protein to TATA-box–containing gene sequences. Epithelial cell ICAM-1 expression in response to H. influenzae was decreased by expressing dominant-negative protein or RNA interference against C/EBPβ, confirming its role in ICAM-1 regulation. Although airway epithelial cells express multiple constitutive and inducible C/EBP family members that bind C/EBP sequences, the results indicate that C/EBPβ plays a central role in modulation of NF-κB–dependent defense gene expression in human airway epithelial cells after exposure to H. influenzae. PMID:18703796

Growth control in colon epithelial cells: gadolinium enhances calcium-mediated growth regulation.

PubMed

Attili, Durga; Jenkins, Brian; Aslam, Muhammad Nadeem; Dame, Michael K; Varani, James

2012-12-01

Gadolinium, a member of the lanthanoid family of transition metals, interacts with calcium-binding sites on proteins and other biological molecules. The overall goal of the present investigation was to determine if gadolinium could enhance calcium-induced epithelial cell growth inhibition in the colon. Gadolinium at concentrations as low as 1-5 μM combined with calcium inhibits proliferation of human colonic epithelial cells more effectively than calcium alone. Gadolinium had no detectable effect on calcium-induced differentiation in the same cells based on change in cell morphology, induction of E-cadherin synthesis, and translocation of E-cadherin from the cytosol to the cell surface. When the colon epithelial cells were treated with gadolinium and then exposed to increased calcium concentrations, movement of extracellular calcium into the cell was suppressed. In contrast, gadolinium treatment had no effect on ionomycin-induced release of stored intracellular calcium into the cytoplasm. Whether these in vitro observations can be translated into an approach for reducing abnormal proliferation in the colonic mucosa (including polyp formation) is not known. These results do, however, provide an explanation for our recent findings that a multi-mineral supplement containing all of the naturally occurring lanthanoid metals including gadolinium are more effective than calcium alone in preventing colon polyp formation in mice on a high-fat diet.

Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells

PubMed Central

Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

2015-01-01

The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting. PMID:26068810

Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells

NASA Astrophysics Data System (ADS)

Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

2015-06-01

The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting.

Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells.

PubMed

Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

2015-06-12

The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting.

Chemo-mechanical modeling of tumor growth in elastic epithelial tissue

NASA Astrophysics Data System (ADS)

Bratsun, Dmitry A.; Zakharov, Andrey P.; Pismen, Len

2016-08-01

We propose a multiscale chemo-mechanical model of the cancer tumor development in the epithelial tissue. The epithelium is represented by an elastic 2D array of polygonal cells with its own gene regulation dynamics. The model allows the simulation of the evolution of multiple cells interacting via the chemical signaling or mechanically induced strain. The algorithm includes the division and intercalation of cells as well as the transformation of normal cells into a cancerous state triggered by a local failure of the spatial synchronization of the cellular rhythms driven by transcription/translation processes. Both deterministic and stochastic descriptions of the system are given for chemical signaling. The transformation of cells means the modification of their respective parameters responsible for chemo-mechanical interactions. The simulations reproduce a distinct behavior of invasive and localized carcinoma. Generally, the model is designed in such a way that it can be readily modified to take account of any newly understood gene regulation processes and feedback mechanisms affecting chemo-mechanical properties of cells.

Kif3a Controls Murine Nephron Number Via GLI3 Repressor, Cell Survival, and Gene Expression in a Lineage-Specific Manner

PubMed Central

Chi, Lijun; Galtseva, Alevtina; Chen, Lin; Mo, Rong; Hui, Chi-chung; Rosenblum, Norman D.

2013-01-01

The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3Δ699/+) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms. PMID:23762375

Loss of Proliferation and Antigen Presentation Activity following Internalization of Polydispersed Carbon Nanotubes by Primary Lung Epithelial Cells

PubMed Central

Kumari, Mandavi; Sachar, Sumedha; Saxena, Rajiv K.

2012-01-01

Interactions between poly-dispersed acid functionalized single walled carbon nanotubes (AF-SWCNTs) and primary lung epithelial (PLE) cells were studied. Peritoneal macrophages (PMs, known phagocytic cells) were used as positive controls in this study. Recovery of live cells from cultures of PLE cells and PMs was significantly reduced in the presence of AF-SWCNTs, in a time and dose dependent manner. Both PLE cells as well as PMs could take up fluorescence tagged AF-SWCNTs in a time dependent manner and this uptake was significantly blocked by cytochalasin D, an agent that blocks the activity of acto-myosin fibers and therefore the phagocytic activity of cells. Confocal microscopic studies confirmed that AF-SWCNTs were internalized by both PLE cells and PMs. Intra-trachially instilled AF-SWCNTs could also be taken up by lung epithelial cells as well as alveolar macrophages. Freshly isolated PLE cells had significant cell division activity and cell cycling studies indicated that treatment with AF-SWCNTs resulted in a marked reduction in S-phase of the cell cycle. In a previously standardized system to study BCG antigen presentation by PLE cells and PMs to sensitized T helper cells, AF-SWCNTs could significantly lower the antigen presentation ability of both cell types. These results show that mouse primary lung epithelial cells can efficiently internalize AF-SWCNTs and the uptake of nanotubes interfered with biological functions of PLE cells including their ability to present BCG antigens to sensitized T helper cells. PMID:22384094

Neutrophils and the Inflammatory Tissue Microenvironment in the Mucosa

PubMed Central

Campbell, Eric L.; Kao, Daniel J.; Colgan, Sean P.

2016-01-01

The interaction of neutrophils (PMNs) and epithelial cells are requisite lines of communication during mucosal inflammatory responses. Consequences of such interactions often determine endpoint organ function, and for this reason, much interest has developed around defining the constituents of the tissue microenvironment of inflammatory lesions. Physiologic in vitro and in vivo models have aided in discovery of components that define the basic inflammatory machinery that mold the inflammatory tissue microenvironment. Here, we will review the recent literature related to the contribution of PMNs to molding of the tissue microenvironment, with an emphasis on the gastrointestinal (GI) tract. We focus on endogenous pathways for promoting tissue homeostasis and the molecular determinants of neutrophil-epithelial cell interactions during ongoing inflammation. These recent studies highlight the dynamic nature of these pathways and lend insight into the complexity of treating mucosal inflammation. PMID:27558331

Cell density and N-cadherin interactions regulate cell proliferation in the sensory epithelia of the inner ear.

PubMed

Warchol, Mark E

2002-04-01

Sensory hair cells in the inner ears of nonmammalian vertebrates can regenerate after injury. In many species, replacement hair cells are produced by the proliferation of epithelial supporting cells. Thus, the ability of supporting cells to undergo renewed proliferation is a key determinant of regenerative ability. The present study used cultures of isolated inner ear sensory epithelia to identify cellular signals that regulate supporting cell proliferation. Small pieces of sensory epithelia from the chicken utricle were cultured in glass microwells. Under those conditions, cell proliferation was inversely related to local cell density. The signaling molecules N-cadherin, beta-catenin, and focal adhesion kinase were immunolocalized in the cultured epithelial cells, and high levels of phosphotyrosine immunoreactivity were present at cell-cell junctions and focal contacts of proliferating cells. Binding of microbeads coated with a function-blocking antibody to N-cadherin inhibited ongoing proliferation. The growth of epithelial cells was also affected by the density of extracellular matrix molecules. The results suggest that cell density, cell-cell contact, and the composition of the extracellular matrix may be critical influences on the regulation of sensory regeneration in the inner ear.

Productive Infection of Human Embryonic Stem Cell-Derived NKX2.1+ Respiratory Progenitors with Human Rhinovirus.

PubMed

Jenny, Robert A; Hirst, Claire; Lim, Sue Mei; Goulburn, Adam L; Micallef, Suzanne J; Labonne, Tanya; Kicic, Anthony; Ling, Kak-Ming; Stick, Stephen M; Ng, Elizabeth S; Trounson, Alan; Giudice, Antonietta; Elefanty, Andrew G; Stanley, Edouard G

2015-06-01

Airway epithelial cells generated from pluripotent stem cells (PSCs) represent a resource for research into a variety of human respiratory conditions, including those resulting from infection with common human pathogens. Using an NKX2.1-GFP reporter human embryonic stem cell line, we developed a serum-free protocol for the generation of NKX2.1(+) endoderm that, when transplanted into immunodeficient mice, matured into respiratory cell types identified by expression of CC10, MUC5AC, and surfactant proteins. Gene profiling experiments indicated that day 10 NKX2.1(+) endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Nevertheless, NKX2.1(+) endoderm supported the infection and replication of the common respiratory pathogen human rhinovirus HRV1b. Moreover, NKX2.1(+) endoderm upregulated expression of IL-6, IL-8, and IL-1B in response to infection, a characteristic of human airway epithelial cells. Our experiments provide proof of principle for the use of PSC-derived respiratory epithelial cells in the study of cell-virus interactions. This report provides proof-of-principle experiments demonstrating, for the first time, that human respiratory progenitor cells derived from stem cells in the laboratory can be productively infected with human rhinovirus, the predominant cause of the common cold. ©AlphaMed Press.

Breast Cancer Research at NASA

NASA Technical Reports Server (NTRS)

1998-01-01

Epithelial and fibroblast cell coculture: Long-term growth human mammary epithelial cells (HMEC) admixed in coculture with fibroblast from the same initial breast tissue grown as 3-dimenstional constructions in the presence of attachment beads in the NASA Bioreactor. A: A typical constrct about 2.0 mm in diameter without beads on the surface. The center of these constrcts is hollow, and beads are organized about the irner surface. Although the coculture provides smaller constructs than the monoculture, the metabolic of the organized cells is about the same. B, C, D: Closer views of cells showing that the shape of cells and cell-to-cell interactions apprear different in the coculture than in the monoculture constructs. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Robert Richmond, NASA/Marshall Space Flight Center (MSFC).

Microgravity

NASA Image and Video Library

1998-10-10

Epithelial and fibroblast cell coculture: Long-term growth human mammary epithelial cells (HMEC) admixed in coculture with fibroblast from the same initial breast tissue grown as 3-dimenstional constructions in the presence of attachment beads in the NASA Bioreactor. A: A typical constrct about 2.0 mm in diameter without beads on the surface. The center of these constrcts is hollow, and beads are organized about the irner surface. Although the coculture provides smaller constructs than the monoculture, the metabolic of the organized cells is about the same. B, C, D: Closer views of cells showing that the shape of cells and cell-to-cell interactions apprear different in the coculture than in the monoculture constructs. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Robert Richmond, NASA/Marshall Space Flight Center (MSFC).

Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells

PubMed Central

Karuri, Nancy W.; Liliensiek, Sara; Teixeira, Ana I.; Abrams, George; Campbell, Sean; Nealey, Paul F.; Murphy, Christopher J.

2006-01-01

Summary The basement membrane possesses a rich 3-dimensional nanoscale topography that provides a physical stimulus, which may modulate cell-substratum adhesion. We have investigated the strength of cell-substratum adhesion on nanoscale topographic features of a similar scale to that of the native basement membrane. SV40 human corneal epithelial cells were challenged by well-defined fluid shear, and cell detachment was monitored. We created silicon substrata with uniform grooves and ridges having pitch dimensions of 400-4000 nm using X-ray lithography. F-actin labeling of cells that had been incubated for 24 hours revealed that the percentage of aligned and elongated cells on the patterned surfaces was the same regardless of pitch dimension. In contrast, at the highest fluid shear, a biphasic trend in cell adhesion was observed with cells being most adherent to the smaller features. The 400 nm pitch had the highest percentage of adherent cells at the end of the adhesion assay. The effect of substratum topography was lost for the largest features evaluated, the 4000 nm pitch. Qualitative and quantitative analyses of the cells during and after flow indicated that the aligned and elongated cells on the 400 nm pitch were more tightly adhered compared to aligned cells on the larger patterns. Selected experiments with primary cultured human corneal epithelial cells produced similar results to the SV40 human corneal epithelial cells. These findings have relevance to interpretation of cell-biomaterial interactions in tissue engineering and prosthetic design. PMID:15226393

Up-Regulation and Profibrotic Role of Osteopontin in Human Idiopathic Pulmonary Fibrosis

PubMed Central

Pardo, Annie; Gibson, Kevin; Cisneros, José; Richards, Thomas J; Yang, Yinke; Becerril, Carina; Yousem, Samueal; Herrera, Iliana; Ruiz, Victor; Selman, Moisés; Kaminski, Naftali

2005-01-01

Background Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disorder characterized by fibroproliferation and excessive accumulation of extracellular matrix in the lung. Methods and Findings Using oligonucleotide arrays, we identified osteopontin as one of the genes that significantly distinguishes IPF from normal lungs. Osteopontin was localized to alveolar epithelial cells in IPF lungs and was also significantly elevated in bronchoalveolar lavage from IPF patients. To study the fibrosis-relevant effects of osteopontin we stimulated primary human lung fibroblasts and alveolar epithelial cells (A549) with recombinant osteopontin. Osteopontin induced a significant increase of migration and proliferation in both fibroblasts and epithelial cells. Epithelial growth was inhibited by the pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) and antibody to CD44, while fibroproliferation was inhibited by GRGDS and antibody to αvβ3 integrin. Fibroblast and epithelial cell migration were inhibited by GRGDS, anti-CD44, and anti-αvβ3. In fibroblasts, osteopontin up-regulated tissue inhibitor of metalloprotease-1 and type I collagen, and down-regulated matrix metalloprotease-1 (MMP-1) expression, while in A549 cells it caused up-regulation of MMP-7. In human IPF lungs, osteopontin colocalized with MMP-7 in alveolar epithelial cells, and application of weakest link statistical models to microarray data suggested a significant interaction between osteopontin and MMP-7. Conclusions Our results provide a potential mechanism by which osteopontin secreted from the alveolar epithelium may exert a profibrotic effect in IPF lungs and highlight osteopontin as a potential target for therapeutic intervention in this incurable disease. PMID:16128620

KRAS Mutation and Epithelial-Macrophage Interplay in Pancreatic Neoplastic Transformation.

PubMed

Bishehsari, Faraz; Zhang, Lijuan; Barlass, Usman; Preite, Nailliw; Turturro, Sanja; Najor, Matthew S; Shetuni, Brandon B; Zayas, Janet P; Mahdavinia, Mahboobeh; Abukhdeir, Abde M; Keshavarzian, Ali

2018-05-14

Pancreatic ductal adenocarcinoma (PDA) is characterized by epithelial mutations in KRAS and prominent tumor-associated inflammation, including macrophage infiltration. But knowledge of early interactions between neoplastic epithelium and macrophages in PDA carcinogenesis is limited. Using a pancreatic organoid model, we found that the expression of mutant KRAS in organoids increased i) ductal to acinar gene expression ratios, ii) epithelial cells proliferation, and iii) colony formation capacity in vitro, and endowed pancreatic cells with the ability to generate neoplastic tumors in vivo. KRAS mutations induced a pro-tumorigenic phenotype in macrophages. Altered macrophages decreased epithelial Pigment Epithelial Derived Factor (PEDF) expression and induced a cancerous phenotype. We validated our findings using annotated patient samples from The Cancer Genome Atlas (TCGA) as well as in our human PDA specimens. Epithelium-macrophage cross talk occurs early in pancreatic carcinogenesis where KRAS directly induces cancer-related phenotypes in epithelium, and also promotes a pro-tumorigenic phenotype in macrophages, in turn augmenting neoplastic growth. This article is protected by copyright. All rights reserved. © 2018 UICC.

Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction.

PubMed

Zhao, Gui Hua; Liu, Ye; Cheng, Yun Tang; Zhao, Qing Song; Qiu, Xiao; Xu, Chao; Xiao, Ting; Zhu, Song; Liu, Gong Zhen; Yin, Kun

2018-06-26

Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat's small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5-2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.

Homophilic and heterophilic polycystin 1 interactions regulate E-cadherin recruitment and junction assembly in MDCK cells

PubMed Central

Streets, Andrew J.; Wagner, Bart E.; Harris, Peter C.; Ward, Christopher J.; Ong, Albert C. M.

2009-01-01

Summary Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited human renal disease and is caused by mutations in two genes, PKD1 (85%) and PKD2 (15%). Cyst epithelial cells are characterised by a complex cellular phenotype including changes in proliferation, apoptosis, basement membrane composition and apicobasal polarity. Since polycystin 1 (PC1), the PKD1 protein, has been located in the basolateral membrane of kidney epithelial cells, we hypothesised that it might have a key role in mediating or stabilising cell-cell interactions. In non-ciliated L929 cells, stable or transient surface expression of the PC1 extracellular domain was sufficient to confer an adhesive phenotype and stimulate junction formation. In MDCK cells, we found that PC1 was recruited to the lateral membranes coincident with E-cadherin within 30 minutes after a `calcium switch'. Recruitment of both proteins was significantly delayed when cells were treated with a PC1 blocking antibody raised to the PKD domains. Finally, PC1 and E-cadherin could be coimmunoprecipitated together from MDCK cells. We conclude that PC1 has a key role in initiating junction formation via initial homophilic interactions and facilitates junction assembly and the establishment of apicobasal polarity by E-cadherin recruitment. PMID:19351715

Numb regulates cell–cell adhesion and polarity in response to tyrosine kinase signalling

PubMed Central

Wang, Zezhou; Sandiford, Shelley; Wu, Chenggang; Li, Shawn Shun-Cheng

2009-01-01

Epithelial-mesenchymal transition (EMT), which can be caused by aberrant tyrosine kinase signalling, marks epithelial tumour progression and metastasis, yet the underlying molecular mechanism is not fully understood. Here, we report that Numb interacts with E-cadherin (E-cad) through its phosphotyrosine-binding domain (PTB) and thereby regulates the localization of E-cad to the lateral domain of epithelial cell–cell junction. Moreover, Numb engages the polarity complex Par3–aPKC–Par6 by binding to Par3 in polarized Madin-Darby canine kidney cells. Intriguingly, after Src activation or hepatocyte growth factor (HGF) treatment, Numb decouples from E-cad and Par3 and associates preferably with aPKC–Par6. Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF-induced EMT. Knockdown of Numb by small hairpin RNA caused a basolateral-to-apicolateral translocation of E-cad and β-catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF-induced cell scattering, a decrease in cell–cell adhesion, and an increase in cell migration. Our work identifies Numb as an important regulator of epithelial polarity and cell–cell adhesion and a sensor of HGF signalling or Src activity during EMT. PMID:19609305

Influence of adhesion and bacteriocin production by Lactobacillus salivarius on the intestinal epithelial cell transcriptional response.

PubMed

O'Callaghan, John; Buttó, Ludovica F; MacSharry, John; Nally, Kenneth; O'Toole, Paul W

2012-08-01

Lactobacillus salivarius strain UCC118 is a human intestinal isolate that has been extensively studied for its potential probiotic effects in human and animal models. The objective of this study was to determine the effect of L. salivarius UCC118 on gene expression responses in the Caco-2 cell line to improve understanding of how the strain might modulate intestinal epithelial cell phenotypes. Exposure of Caco-2 cells to UCC118 led to the induction of several human genes (TNFAIP3, NFKBIA, and BIRC3) that are negative regulators of inflammatory signaling pathways. Induction of chemokines (CCL20, CXCL-1, and CXCL-2) with antimicrobial functions was also observed. Disruption of the UCC118 sortase gene srtA causes reduced bacterial adhesion to epithelial cells. Transcription of three mucin genes was reduced significantly when Caco-2 cells were stimulated with the ΔsrtA derivative of UCC118 compared to cells stimulated with the wild type, but there was no significant change in the transcription levels of the anti-inflammatory genes. UCC118 genes that were significantly upregulated upon exposure to Caco-2 cells were identified by bacterial genome microarray and consisted primarily of two groups of genes connected with purine metabolism and the operon for synthesis of the Abp118 bacteriocin. Following incubation with Caco-2 cells, the bacteriocin synthesis genes were transcribed at higher levels in the wild type than in the ΔsrtA derivative. These data indicate that L. salivarius UCC118 influences epithelial cells both through modulation of the inflammatory response and by modulation of intestinal cell mucin production. Sortase-anchored cell surface proteins of L. salivarius UCC118 have a central role in promoting the interaction between the bacterium and epithelial cells.

Transcriptional Networks in Epithelial-Mesenchymal Transition

PubMed Central

Venkov, Christo; Plieth, David; Ni, Terri; Karmaker, Amitava; Bian, Aihua; George, Alfred L.; Neilson, Eric G.

2011-01-01

Backround Epithelial-mesenchymal transition (EMT) changes polarized epithelial cells into migratory phenotypes associated with loss of cell-cell adhesion molecules and cytoskeletal rearrangements. This form of plasticity is seen in mesodermal development, fibroblast formation, and cancer metastasis. Methods and Findings Here we identify prominent transcriptional networks active during three time points of this transitional process, as epithelial cells become fibroblasts. DNA microarray in cultured epithelia undergoing EMT, validated in vivo, were used to detect various patterns of gene expression. In particular, the promoter sequences of differentially expressed genes and their transcription factors were analyzed to identify potential binding sites and partners. The four most frequent cis-regulatory elements (CREs) in up-regulated genes were SRY, FTS-1, Evi-1, and GC-Box, and RNA inhibition of the four transcription factors, Atf2, Klf10, Sox11, and SP1, most frequently binding these CREs, establish their importance in the initiation and propagation of EMT. Oligonucleotides that block the most frequent CREs restrain EMT at early and intermediate stages through apoptosis of the cells. Conclusions Our results identify new transcriptional interactions with high frequency CREs that modulate the stability of cellular plasticity, and may serve as targets for modulating these transitional states in fibroblasts. PMID:21980432

Epithelial control of gut-associated lymphoid tissue formation through p38α-dependent restraint of NF-κB signaling

PubMed Central

Caballero-Franco, Celia; Guma, Monica; Choo, Min-Kyung; Sano, Yasuyo; Enzler, Thomas; Karin, Michael; Mizoguchi, Atsushi; Park, Jin Mo

2015-01-01

The protein kinase p38α mediates cellular responses to environmental and endogenous cues that direct tissue homeostasis and immune responses. Studies of mice lacking p38α in several different cell types have demonstrated that p38α signaling is essential to maintaining the proliferation-differentiation balance in developing and steady-state tissues. The mechanisms underlying these roles involve cell-autonomous control of signaling and gene expression by p38α. Here we show that p38α regulates gut-associated lymphoid tissue (GALT) formation in a non-cell-autonomous manner. From an investigation of mice with intestinal epithelial cell-specific deletion of the p38α gene, we find that p38α serves to limit NF-κB signaling and thereby attenuate GALT-promoting chemokine expression in the intestinal epithelium. Loss of this regulation results in GALT hyperplasia and, in some animals, mucosa-associated B cell lymphoma. These anomalies occur independently of luminal microbial stimuli and are likely driven by direct epithelial-lymphoid interactions. Our study illustrates a novel p38α-dependent mechanism preventing excessive generation of epithelial-derived signals that drive lymphoid tissue overgrowth and malignancy. PMID:26792803

Epimorphin expression in interstitial pneumonia

PubMed Central

Terasaki, Yasuhiro; Fukuda, Yuh; Suga, Moritaka; Ikeguchi, Naoki; Takeya, Motohiro

2005-01-01

Epimorphin modulates epithelial morphogenesis in embryonic mouse organs. We previously suggested that epimorphin contributes to repair of bleomycin-induced pulmonary fibrosis in mice via epithelium-mesenchyme interactions. To clarify the role of epimorphin in human lungs, we evaluated epimorphin expression and localization in normal lungs, lungs with nonspecific interstitial pneumonia (NSIP), and lungs with usual interstitial pneumonia (UIP); we also studied the effect of recombinant epimorphin on cultured human alveolar epithelial cells in vitro. Northern and Western blotting analyses revealed that epimorphin expression in NSIP samples were significantly higher than those in control lungs and lungs with UIP. Immunohistochemistry showed strong epimorphin expression in mesenchymal cells of early fibrotic lesions and localization of epimorphin protein on mesenchymal cells and extracellular matrix of early fibrotic lesions in the nonspecific interstitial pneumonia group. Double-labeled fluorescent images revealed expression of matrix metalloproteinase 2 in re-epithelialized cells overlying epimorphin-positive early fibrotic lesions. Immunohistochemistry and metalloproteinase activity assay demonstrated augmented expression of metalloproteinase induced by recombinant epimorphin in human alveolar epithelial cells. These findings suggest that epimorphin contributes to repair of pulmonary fibrosis in nonspecific interstitial pneumonia, perhaps partly by inducing expression of matrix metalloproteinase 2, which is an important proteolytic factor in lung remodeling. PMID:15651999

Serratia marcescens is injurious to intestinal epithelial cells

PubMed Central

Ochieng, John B; Boisen, Nadia; Lindsay, Brianna; Santiago, Araceli; Ouma, Collins; Ombok, Maurice; Fields, Barry; Stine, O Colin; Nataro, James P

2014-01-01

Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens. PMID:25426769

Serratia marcescens is injurious to intestinal epithelial cells.

PubMed

Ochieng, John B; Boisen, Nadia; Lindsay, Brianna; Santiago, Araceli; Ouma, Collins; Ombok, Maurice; Fields, Barry; Stine, O Colin; Nataro, James P

2014-01-01

Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens.

Early Molecular Events in Murine Gastric Epithelial Cells Mediated by Helicobacter pylori CagA.

PubMed

Banerjee, Aditi; Basu, Malini; Blanchard, Thomas G; Chintalacharuvu, Subba R; Guang, Wei; Lillehoj, Erik P; Czinn, Steven J

2016-10-01

Murine models of Helicobacter pylori infection are used to study host-pathogen interactions, but lack of severe gastritis in this model has limited its usefulness in studying pathogenesis. We compared the murine gastric epithelial cell line GSM06 to the human gastric epithelial AGS cell line to determine whether similar events occur when cultured with H. pylori. The lysates of cells infected with H. pylori isolates or an isogenic cagA-deficient mutant were assessed for translocation and phosphorylation of CagA and for activation of stress pathway kinases by immunoblot. Phosphorylated CagA was detected in both cell lines within 60 minutes. Phospho-ERK 1/2 was present within several minutes and distinctly present in GSM06 cells at 60 minutes. Similar results were obtained for phospho-JNK, although the 54 kDa phosphoprotein signal was dominant in AGS, whereas the lower molecular weight band was dominant in GSM06 cells. These results demonstrate that early events in H. pylori pathogenesis occur within mouse epithelial cells similar to human cells and therefore support the use of the mouse model for the study of acute CagA-associated host cell responses. These results also indicate that reduced disease in H. pylori-infected mice may be due to lack of the Cag PAI, or by differences in the mouse response downstream of the initial activation events. © 2016 John Wiley & Sons Ltd.

Do myoepithelial cells hold the key for breast tumorprogression?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Polyak, Kornelia; Hu, Min

2005-11-18

Mammary myoepithelial cells have been the foster child of breast cancer biology and have been largely ignored since they were considered to be less important for tumorigenesis than luminal epithelial cells from which most of breast carcinomas are thought to arise. In recent years as our knowledge in stem cell biology and the cellular microenvironment has been increasing myoepithelial cells are slowly starting to gain more attention. Emerging data raise the hypothesis if myoepithelial cells play a key role in breast tumor progression by regulating the in situ to invasive carcinoma transition and if myoepithelial cells are part of themore » mammary stem cell niche. Paracrine interactions between myoepithelial and luminal epithelial cells are known to be important for cell cycle arrest, establishing epithelial cell polarity, and inhibiting migration and invasion. Based on these functions normal mammary myoepithelial cells have been called ''natural tumor suppressors''. However, during tumor progression myoepithelial cells seem to loose these properties and eventually they themselves diminish as tumors become invasive. Better understanding of myoepithelial cell function and their role in tumor progression may lead to their exploitation for cancer therapeutic and preventative measures.« less

Polarity Proteins as Regulators of Cell Junction Complexes: Implications for Breast Cancer

PubMed Central

Bazzoun, Dana; Lelièvre, Sophie; Talhouk, Rabih

2013-01-01

The epithelium of multicellular organisms possesses a well-defined architecture, referred to as polarity that coordinates the regulation of essential cell features. Polarity proteins are intimately linked to the protein complexes that make the tight, adherens and gap junctions; they contribute to the proper localization and assembly of these cell-cell junctions within cells and consequently to functional tissue organization. The establishment of cell-cell junctions and polarity are both implicated in the regulation of epithelial modifications in normal and cancer situations. Uncovering the mechanisms through which cell-cell junctions and epithelial polarization are established and how their interaction with the microenvironment direct cell and tissue organization has opened new venues for the development of cancer therapies. In this review, we focus on the breast epithelium to highlight how polarity and cell-cell junction proteins interact together in normal and cancerous contexts to regulate major cellular mechanisms such as migration. The impact of these proteins on epigenetic mechanisms responsible for resetting cells towards oncogenesis is discussed in light of increasing evidence that tissue polarity modulates chromatin function. Finally, we give an overview of recent breast cancer therapies that target proteins involved in cell-cell junctions. PMID:23458609

Lactobacillus delbrueckii subsp lactis CIDCA 133 modulates response of human epithelial and dendritic cells infected with Bacillus cereus.

PubMed

Rolny, I S; Tiscornia, I; Racedo, S M; Pérez, P F; Bollati-Fogolín, M

2016-11-30

It is known that probiotic microorganisms are able to modulate pathogen virulence. This ability is strain dependent and involves multiple interactions between microorganisms and relevant host's cell populations. In the present work we focus on the effect of a potentially probiotic lactobacillus strain (Lactobacillus delbrueckii subsp. lactis CIDCA 133) in an in vitro model of Bacillus cereus infection. Our results showed that infection of intestinal epithelial HT-29 cells by B. cereus induces nuclear factor kappa B (NF-κB) pathway. Noteworthy, the presence of strain L. delbrueckii subsp.lactis CIDCA 133 increases stimulation. However, B. cereus-induced interleukin (IL)-8 production by epithelial cells is partially abrogated by L. delbrueckii subsp. lactis CIDCA 133. These findings suggest that signalling pathways other than that of NF-κB are involved. In a co-culture system (HT-29 and monocyte-derived dendritic cells), B. cereus was able to translocate from the epithelial (upper) to the dendritic cell compartment (lower). This translocation was partially abrogated by the presence of lactobacilli in the upper compartment. In addition, infection of epithelial cells in the co-culture model, led to an increase in the expression of CD86 by dendritic cells. This effect could not be modified in the presence of lactobacilli. Interestingly, infection of enterocytes with B. cereus triggers production of proinflammatory cytokines by dendritic cells (IL-8, IL-6 and tumour necrosis factor alpha (TNF-α)). The production of TNF-α (a protective cytokine in B. cereus infections) by dendritic cells was increased in the presence of lactobacilli. The present work demonstrates for the first time the effect of L. delbrueckii subsp. lactis CIDCA 133, a potentially probiotic strain, in an in vitro model of B. cereus infection. The presence of the probiotic strain modulates cell response both in infected epithelial and dendritic cells thus suggesting a possible beneficial effect of selected lactobacilli strains on the course of B. cereus infection.

EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling.

PubMed

Kaplan, Nihal; Ventrella, Rosa; Peng, Han; Pal-Ghosh, Sonali; Arvanitis, Constadina; Rappoport, Joshua Z; Mitchell, Brian J; Stepp, Mary Ann; Lavker, Robert M; Getsios, Spiro

2018-01-01

Progenitor cells of the limbal epithelium reside in a discrete area peripheral to the more differentiated corneal epithelium and maintain tissue homeostasis. What regulates the limbal-corneal epithelial boundary is a major unanswered question. Ephrin-A1 ligand is enriched in the limbal epithelium, whereas EphA2 receptor is concentrated in the corneal epithelium. This reciprocal pattern led us to assess the role of ephrin-A1 and EphA2 in limbal-corneal epithelial boundary organization. EphA2-expressing corneal epithelial cells engineered to express ephrin-A1 were used to study boundary formation in vitro in a manner that mimicked the relative abundance of these juxtamembrane signaling proteins in the limbal and corneal epithelium in vivo. Interaction of these two distinct cell populations following initial seeding into discrete culture compartments was assessed by live cell imaging. Immunofluoresence and immunoblotting was used to evaluate the contribution of downstream growth factor signaling and cell-cell adhesion systems to boundary formation at sites of heterotypic contact between ephrin-A1 and EphA2 expressing cells. Ephrin-A1-expressing cells impeded and reversed the migration of EphA2-expressing corneal epithelial cells upon heterotypic contact formation leading to coordinated migration of the two cell populations in the direction of an ephrin-A1-expressing leading front. Genetic silencing and pharmacologic inhibitor studies demonstrated that the ability of ephrin-A1 to direct migration of EphA2-expressing cells depended on an a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and epidermal growth factor receptor (EGFR) signaling pathway that limited E-cadherin-mediated adhesion at heterotypic boundaries. Ephrin-A1/EphA2 signaling complexes play a key role in limbal-corneal epithelial compartmentalization and the response of these tissues to injury.

Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J.

2010-01-01

Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that showed signs of damage by viruses and virus titers (see figure) that indicated large increases in the populations of viruses during the days following inoculation.

IL1{beta}-mediated Stromal COX-2 signaling mediates proliferation and invasiveness of colonic epithelial cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Yingting, E-mail: yitizhu@yahoo.com; Tissue Tech Inc, Miami, FL 33173; Zhu, Min

2012-11-15

COX-2 is a major inflammatory mediator implicated in colorectal inflammation and cancer. However, the exact origin and role of COX-2 on colorectal inflammation and carcinogenesis are still not well defined. Recently, we reported that COX-2 and iNOS signalings interact in colonic CCD18Co fibroblasts. In this article, we investigated whether activation of COX-2 signaling by IL1{beta} in primary colonic fibroblasts obtained from normal and cancer patients play a critical role in regulation of proliferation and invasiveness of human colonic epithelial cancer cells. Our results demonstrated that COX-2 level was significantly higher in cancer associated fibroblasts than that in normal fibroblasts withmore » or without stimulation of IL-1{beta}, a powerful stimulator of COX-2. Using in vitro assays for estimating proliferative and invasive potential, we discovered that the proliferation and invasiveness of the epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts than with normal fibroblasts, with or without stimulation of IL1{beta}. Further analysis indicated that the major COX-2 product, prostaglandin E{sub 2}, directly enhanced proliferation and invasiveness of the epithelial cancer cells in the absence of fibroblasts. Moreover, a selective COX-2 inhibitor, NS-398, blocked the proliferative and invasive effect of both normal and cancer associate fibroblasts on the epithelial cancer cells, with or without stimulation of IL-1{beta}. Those results indicate that activation of COX-2 signaling in the fibroblasts plays a major role in promoting proliferation and invasiveness of the epithelial cancer cells. In this process, PKC is involved in the activation of COX-2 signaling induced by IL-1{beta} in the fibroblasts.« less

Anti-proliferative activities of finasteride in benign prostate epithelial cells require stromal fibroblasts and c-Jun gene.

PubMed

Wang, Kai; Jin, Song; Fan, Dongdong; Wang, Mingshuai; Xing, Nianzeng; Niu, Yinong

2017-01-01

This study aimed to identify the role of mouse fibroblast-mediated c-Jun and IGF-1 signaling in the therapeutic effect of finasteride on benign prostatic epithelial cells. BPH-1 cells, alone or with fibroblasts (c-Jun+/+ or c-Jun-/-), were implanted subcutaneously in male nude mice who were then treated with finasteride. The degrees of cell proliferation, apoptosis, and sizes of the xenografts were determined. BPH-1 cells were grown alone or co-cultured with mouse fibroblasts in the presence of finasteride and the level of IGF-1 secreted into the medium by the fibroblasts was determined. The proliferation-associated signaling pathway in BPH-1 cells was also evaluated. Fibroblasts and c-Jun promoted xenograft growth, stimulated Ki-67 expression, and inhibited BPH-1 apoptosis. Finasteride did not induce the shrinkage of xenografts in the combined-grafted groups despite repressing Ki-67 expression and inducing cell apoptosis. The addition of c-Jun-/- fibroblasts did not promote xenograft growth. In the absence of c-Jun and fibroblasts, finasteride did not alter xenograft growth, Ki-67 expression, or cell apoptosis. The in vitro results demonstrated that when BPH-1 cells were grown in monoculture, treatment with finasteride did not induce cell death and stimulated the expression of pro-proliferative signaling molecules, while in the presence of fibroblasts containing c-Jun, finasteride treatment repressed epithelial cell proliferation, the level of IGF-1 in the medium, and the activation of downstream pro-proliferative signaling pathways. Taken together, our results suggest that fibroblasts, c-Jun, and IGF-1 play key roles in mediating stromal-epithelial interactions that are required for the therapeutic effects of finasteride in benign prostate epithelial cells.

Anti-proliferative activities of finasteride in benign prostate epithelial cells require stromal fibroblasts and c-Jun gene

PubMed Central

Fan, Dongdong; Wang, Mingshuai; Xing, Nianzeng; Niu, Yinong

2017-01-01

This study aimed to identify the role of mouse fibroblast-mediated c-Jun and IGF-1 signaling in the therapeutic effect of finasteride on benign prostatic epithelial cells. BPH-1 cells, alone or with fibroblasts (c-Jun+/+ or c-Jun-/-), were implanted subcutaneously in male nude mice who were then treated with finasteride. The degrees of cell proliferation, apoptosis, and sizes of the xenografts were determined. BPH-1 cells were grown alone or co-cultured with mouse fibroblasts in the presence of finasteride and the level of IGF-1 secreted into the medium by the fibroblasts was determined. The proliferation-associated signaling pathway in BPH-1 cells was also evaluated. Fibroblasts and c-Jun promoted xenograft growth, stimulated Ki-67 expression, and inhibited BPH-1 apoptosis. Finasteride did not induce the shrinkage of xenografts in the combined-grafted groups despite repressing Ki-67 expression and inducing cell apoptosis. The addition of c-Jun-/- fibroblasts did not promote xenograft growth. In the absence of c-Jun and fibroblasts, finasteride did not alter xenograft growth, Ki-67 expression, or cell apoptosis. The in vitro results demonstrated that when BPH-1 cells were grown in monoculture, treatment with finasteride did not induce cell death and stimulated the expression of pro-proliferative signaling molecules, while in the presence of fibroblasts containing c-Jun, finasteride treatment repressed epithelial cell proliferation, the level of IGF-1 in the medium, and the activation of downstream pro-proliferative signaling pathways. Taken together, our results suggest that fibroblasts, c-Jun, and IGF-1 play key roles in mediating stromal-epithelial interactions that are required for the therapeutic effects of finasteride in benign prostate epithelial cells. PMID:28196103

Bacteria-bacteria interactions within the microbiota of the ancestral metazoan Hydra contribute to fungal resistance.

PubMed

Fraune, Sebastian; Anton-Erxleben, Friederike; Augustin, René; Franzenburg, Sören; Knop, Mirjam; Schröder, Katja; Willoweit-Ohl, Doris; Bosch, Thomas C G

2015-07-01

Epithelial surfaces of most animals are colonized by diverse microbial communities. Although it is generally agreed that commensal bacteria can serve beneficial functions, the processes involved are poorly understood. Here we report that in the basal metazoan Hydra, ectodermal epithelial cells are covered with a multilayered glycocalyx that provides a habitat for a distinctive microbial community. Removing this epithelial microbiota results in lethal infection by the filamentous fungus Fusarium sp. Restoring the complex microbiota in gnotobiotic polyps prevents pathogen infection. Although mono-associations with distinct members of the microbiota fail to provide full protection, additive and synergistic interactions of commensal bacteria are contributing to full fungal resistance. Our results highlight the importance of resident microbiota diversity as a protective factor against pathogen infections. Besides revealing insights into the in vivo function of commensal microbes in Hydra, our findings indicate that interactions among commensal bacteria are essential to inhibit pathogen infection.

Bacteria–bacteria interactions within the microbiota of the ancestral metazoan Hydra contribute to fungal resistance

PubMed Central

Fraune, Sebastian; Anton-Erxleben, Friederike; Augustin, René; Franzenburg, Sören; Knop, Mirjam; Schröder, Katja; Willoweit-Ohl, Doris; Bosch, Thomas CG

2015-01-01

Epithelial surfaces of most animals are colonized by diverse microbial communities. Although it is generally agreed that commensal bacteria can serve beneficial functions, the processes involved are poorly understood. Here we report that in the basal metazoan Hydra, ectodermal epithelial cells are covered with a multilayered glycocalyx that provides a habitat for a distinctive microbial community. Removing this epithelial microbiota results in lethal infection by the filamentous fungus Fusarium sp. Restoring the complex microbiota in gnotobiotic polyps prevents pathogen infection. Although mono-associations with distinct members of the microbiota fail to provide full protection, additive and synergistic interactions of commensal bacteria are contributing to full fungal resistance. Our results highlight the importance of resident microbiota diversity as a protective factor against pathogen infections. Besides revealing insights into the in vivo function of commensal microbes in Hydra, our findings indicate that interactions among commensal bacteria are essential to inhibit pathogen infection. PMID:25514534

Iron-Induced Virulence of Salmonella enterica Serovar Typhimurium at the Intestinal Epithelial Interface Can Be Suppressed by Carvacrol

PubMed Central

Kortman, Guus A. M.; Roelofs, Rian W. H. M.; Swinkels, Dorine W.; de Jonge, Marien I.; Burt, Sara A.

2014-01-01

Oral iron therapy can increase the abundance of bacterial pathogens, e.g., Salmonella spp., in the large intestine of African children. Carvacrol is a natural compound with antimicrobial activity against various intestinal bacterial pathogens, among which is the highly prevalent Salmonella enterica serovar Typhimurium. This study aimed to explore a presumed interaction between carvacrol and bacterial iron handling and to assess the potential of carvacrol in preventing the increase of bacterial pathogenicity during high iron availability. S. Typhimurium was cultured with increasing concentrations of iron and carvacrol to study the effects of these combined interventions on growth, adhesion to intestinal epithelial cells, and iron uptake/influx in both bacterial and epithelial cells. In addition, the ability of carvacrol to remove iron from the high-affinity ligand transferrin and an Fe-dye complex was examined. Carvacrol retarded growth of S. Typhimurium at all iron conditions. Furthermore, iron-induced epithelial adhesion was effectively reduced by carvacrol at high iron concentrations. The reduction of growth and virulence by carvacrol was not paralleled by a change in iron uptake or influx into S. Typhimurium. In contrast, bioavailability of iron for epithelial cells was moderately decreased under these conditions. Further, carvacrol was shown to lack the properties of an iron binding molecule; however, it was able to weaken iron-ligand interactions by which it may possibly interfere with bacterial virulence. In conclusion, our in vitro data suggest that carvacrol has the potential to serve as a novel dietary supplement to prevent pathogenic overgrowth and colonization in the large intestine during oral iron therapy. PMID:24379194

The ancillary protein 1 of Streptococcus pyogenes FCT-1 pili mediates cell adhesion and biofilm formation through heterophilic as well as homophilic interactions

PubMed Central

Becherelli, Marco; Manetti, Andrea G O; Buccato, Scilla; Viciani, Elisa; Ciucchi, Laura; Mollica, Giulia; Grandi, Guido; Margarit, Imma

2012-01-01

Summary Gram-positive pili are known to play a role in bacterial adhesion to epithelial cells and in the formation of biofilm microbial communities. In the present study we undertook the functional characterization of the pilus ancillary protein 1 (AP1_M6) from Streptococcus pyogenes isolates expressing the FCT-1 pilus variant, known to be strong biofilm formers. Cell binding and biofilm formation assays using S. pyogenes in-frame deletion mutants, Lactococcus expressing heterologous FCT-1 pili and purified recombinant AP1_M6, indicated that this pilin is a strong cell adhesin that is also involved in bacterial biofilm formation. Moreover, we show that AP1_M6 establishes homophilic interactions that mediate inter-bacterial contact, possibly promoting bacterial colonization of target epithelial cells in the form of three-dimensional microcolonies. Finally, AP1_M6 knockout mutants were less virulent in mice, indicating that this protein is also implicated in GAS systemic infection. PMID:22320452

Active Tension Network model reveals an exotic mechanical state realized in epithelial tissues

NASA Astrophysics Data System (ADS)

Noll, Nicholas; Mani, Madhav; Heemskerk, Idse; Streicha, Sebastian; Shraiman, Boris

Mechanical interactions play a crucial role in epithelial morphogenesis, yet understanding the complex mechanisms through which stress and deformation affect cell behavior remains an open problem. Here we formulate and analyze the Active Tension Network (ATN) model, which assumes that mechanical balance of cells is dominated by cortical tension and introduces tension dependent active remodeling of the cortex. We find that ATNs exhibit unusual mechanical properties: i) ATN behaves as a fluid at short times, but at long times it supports external tension, like a solid; ii) its mechanical equilibrium state has extensive degeneracy associated with a discrete conformal - ''isogonal'' - deformation of cells. ATN model predicts a constraint on equilibrium cell geometry, which we demonstrate to hold in certain epithelial tissues. We further show that isogonal modes are observed in a fruit fly embryo, accounting for the striking variability of apical area of ventral cells and helping understand the early phase of gastrulation. Living matter realizes new and exotic mechanical states, understanding which helps understand biological phenomena.

Human intraepithelial lymphocytes.

PubMed

Mayassi, Toufic; Jabri, Bana

2018-04-20

The location of intraepithelial lymphocytes (IEL) between epithelial cells, their effector memory, cytolytic and inflammatory phenotype positions them to kill infected epithelial cells and protect the intestine against pathogens. Human TCRαβ + CD8αβ + IEL have the dual capacity to recognize modified self via natural killer (NK) receptors (autoreactivity) as well as foreign antigen via the T cell receptor (TCR), which is accomplished in mouse by two cell subsets, the naturally occurring TCRαβ + CD8αα + and adaptively induced TCRαβ + CD8αβ + IEL subsets, respectively. The private/oligoclonal nature of the TCR repertoire of both human and mouse IEL suggests local environmental factors dictate the specificity of IEL responses. The line between sensing of foreign antigens and autoreactivity is blurred for IEL in celiac disease, where recognition of stress ligands by induced activating NK receptors in conjunction with inflammatory signals such as IL-15 can result in low-affinity TCR/non-cognate antigen and NK receptor/stress ligand interactions triggering destruction of intestinal epithelial cells.

USP17 is upregulated in osteosarcoma and promotes cell proliferation, metastasis, and epithelial-mesenchymal transition through stabilizing SMAD4.

PubMed

Song, Chenyang; Liu, Wenge; Li, Jiandong

2017-07-01

USP17 is upregulated in several cancers, indicating that USP17 might play essential functions in tumor development. However, the function of USP17 in osteosarcoma is still unknown. Our work aimed to investigate the function of USP17 in osteosarcoma. We found that the expression of USP17 was upregulated in osteosarcoma tissues and cell lines, including MG-63 and U2OS. Several functional experiments, such as colony formation analysis, Cell Counting Kit-8 assay, wound healing analysis, and transwell assay, showed that USP17 promoted cell proliferation, migration, and invasion. Moreover, we found that USP17 facilitated migration and invasion through promoting epithelial-mesenchymal transition. SMAD4 has been found to regulate epithelial-mesenchymal transition, co-immunopurification, and glutathione S-transferase pull-down analysis demonstrated that USP17 interacted with SMAD4. Furthermore, USP17 stabilized SMAD4 through its deubiquitinase activity. In conclusion, this study shows that USP17 enhances osteosarcoma cell proliferation and invasion through stabilizing SMAD4.

Disruption of Sorting Nexin 5 Causes Respiratory Failure Associated with Undifferentiated Alveolar Epithelial Type I Cells in Mice

PubMed Central

Im, Sun-Kyoung; Jeong, HyoBin; Jeong, Hyun-Woo; Kim, Kyong-Tai; Hwang, Daehee; Ikegami, Machiko; Kong, Young-Yun

2013-01-01

Sorting nexin 5 (Snx5) has been posited to regulate the degradation of epidermal growth factor receptor and the retrograde trafficking of cation-independent mannose 6-phosphate receptor/insulin-like growth factor II receptor. Snx5 has also been suggested to interact with Mind bomb-1, an E3 ubiquitin ligase that regulates the activation of Notch signaling. However, the in vivo functions of Snx5 are largely unknown. Here, we report that disruption of the Snx5 gene in mice (Snx5-/- mice) resulted in partial perinatal lethality; 40% of Snx5-/- mice died shortly after birth due to cyanosis, reduced air space in the lungs, and respiratory failure. Histological analysis revealed that Snx5-/- mice exhibited thickened alveolar walls associated with undifferentiated alveolar epithelial type I cells. In contrast, alveolar epithelial type II cells were intact, exhibiting normal surfactant synthesis and secretion. Although the expression levels of surfactant proteins and saturated phosphatidylcholine in the lungs of Snx5-/- mice were comparable to those of Snx5+/+ mice, the expression levels of T1α, Aqp5, and Rage, markers for distal alveolar epithelial type I cells, were significantly decreased in Snx5 -/- mice. These results demonstrate that Snx5 is necessary for the differentiation of alveolar epithelial type I cells, which may underlie the adaptation to air breathing at birth. PMID:23526992

Galectin-3 Inhibits Galectin-8/Parkin-Mediated Ubiquitination of Group A Streptococcus.

PubMed

Cheng, Yi-Lin; Wu, Yan-Wei; Kuo, Chih-Feng; Lu, Shiou-Ling; Liu, Fu-Tong; Anderson, Robert; Lin, Chiou-Feng; Liu, Yi-Ling; Wang, Wan-Yu; Chen, Ying-Da; Zheng, Po-Xing; Wu, Jiunn-Jong; Lin, Yee-Shin

2017-07-25

Group A streptococcus (GAS) is an important human pathogen that causes a wide variety of cutaneous and systemic infections. Although originally thought to be an extracellular bacterium, numerous studies have demonstrated that GAS can trigger internalization into nonimmune cells to escape from immune surveillance or antibiotic-mediated killing. Epithelial cells possess a defense mechanism involving autophagy-mediated targeting and killing of GAS within lysosome-fused autophagosomes. In endothelial cells, in contrast, we previously showed that autophagy is not sufficient for GAS killing. In the present study, we showed higher galectin-3 (Gal-3) expression and lower Gal-8 expression in endothelial cells than in epithelial cells. The recruitment of Gal-3 to GAS is higher and the recruitment of Gal-8 to GAS is lower in endothelial cells than in epithelial cells. We further showed that Gal-3 promotes GAS replication and diminishes the recruitment of Gal-8 and ubiquitin, the latter of which is a critical protein for autophagy sequestration. After knockdown of Gal-3 in endothelial cells, the colocalization of Gal-8, parkin, and ubiquitin-decorated GAS is significantly increased, as is the interaction of Gal-8 and parkin, an E3 ligase. Furthermore, inhibition of Gal-8 in epithelial cells attenuates recruitment of parkin; both Gal-8 and parkin contribute to ubiquitin recruitment and GAS elimination. Animal studies confirmed that Gal-3-knockout mice develop less-severe skin damage and that GAS replication can be detected only in the air pouch and not in organs and endothelial cells. These results demonstrate that Gal-3 inhibits ubiquitin recruitment by blocking Gal-8 and parkin recruitment, resulting in GAS replication in endothelial cells. IMPORTANCE In epithelial cells, GAS can be efficiently killed within the lysosome-fused autophaosome compartment. However, we previously showed that, in spite of LC-3 recruitment, the autophagic machinery is not sufficient for GAS killing in endothelial cells. In this report, we provide the first evidence that Gal-3, highly expressed in endothelial cells, blocks the tagging of ubiquitin to GAS by inhibiting recruitment of Gal-8 and parkin, leading to an enhancement of GAS replication. We also provide the first demonstration that Gal-8 can interact with parkin, the critical E3 ligase, for resistance to intracellular bacteria by facilitating the decoration of bacteria with ubiquitin chains. Our findings reveal that differential levels of Gal-3 and Gal-8 expression and recruitment to GAS between epithelial cells and endothelial cells may contribute to the different outcomes of GAS elimination or survival and growth of GAS in these two types of cells. Copyright © 2017 Cheng et al.

EGFR-Dependent Regulation of Matrix-Independent Epithelial Cell Survival

DTIC Science & Technology

2006-04-01

ultraviolet (UV) irradiation ( Mudgil et al., 2003), decreased adhesive interactions between tumor cells and adjacent epithelia Much remains to be...historical perspective on integrin signal transduction. Nat. Cell Biol. 4, E83-E90. Mudgil , A. V., Segal, N., Andriani, F., Wang, Y., Fusenig, N. E. and

THE EFFECTS OF COMBINATORIAL EXPOSURE OF PRO-INFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINES ON AIRWAY EPITHELIAL CELL RELEASE OF CHEMOTACTIC MEDIATORS

EPA Science Inventory

Asthma is a chronic inflammatory disorder of the airways affecting nearly 15 million individuals nationally. Within the inflamed asthmatic airway there exist complex interactions between many cells and the cytokines they release, in particular mast cells, eosinophils, T-lymphocy...

EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

PubMed Central

Kaplan, Nihal; Ventrella, Rosa; Peng, Han; Pal-Ghosh, Sonali; Arvanitis, Constadina; Rappoport, Joshua Z.; Mitchell, Brian J.; Stepp, Mary Ann; Lavker, Robert M.

2018-01-01

Purpose Progenitor cells of the limbal epithelium reside in a discrete area peripheral to the more differentiated corneal epithelium and maintain tissue homeostasis. What regulates the limbal–corneal epithelial boundary is a major unanswered question. Ephrin-A1 ligand is enriched in the limbal epithelium, whereas EphA2 receptor is concentrated in the corneal epithelium. This reciprocal pattern led us to assess the role of ephrin-A1 and EphA2 in limbal–corneal epithelial boundary organization. Methods EphA2-expressing corneal epithelial cells engineered to express ephrin-A1 were used to study boundary formation in vitro in a manner that mimicked the relative abundance of these juxtamembrane signaling proteins in the limbal and corneal epithelium in vivo. Interaction of these two distinct cell populations following initial seeding into discrete culture compartments was assessed by live cell imaging. Immunofluoresence and immunoblotting was used to evaluate the contribution of downstream growth factor signaling and cell–cell adhesion systems to boundary formation at sites of heterotypic contact between ephrin-A1 and EphA2 expressing cells. Results Ephrin-A1–expressing cells impeded and reversed the migration of EphA2-expressing corneal epithelial cells upon heterotypic contact formation leading to coordinated migration of the two cell populations in the direction of an ephrin-A1–expressing leading front. Genetic silencing and pharmacologic inhibitor studies demonstrated that the ability of ephrin-A1 to direct migration of EphA2-expressing cells depended on an a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and epidermal growth factor receptor (EGFR) signaling pathway that limited E-cadherin–mediated adhesion at heterotypic boundaries. Conclusions Ephrin-A1/EphA2 signaling complexes play a key role in limbal–corneal epithelial compartmentalization and the response of these tissues to injury. PMID:29351356

Mycoplasma pulmonis Inhibits Electrogenic Ion Transport across Murine Tracheal Epithelial Cell Monolayers

PubMed Central

Lambert, Linda C.; Trummell, Hoa Q.; Singh, Ashvani; Cassell, Gail H.; Bridges, Robert J.

1998-01-01

Murine chronic respiratory disease is characterized by persistent colonization of tracheal and bronchial epithelial cell surfaces by Mycoplasma pulmonis, submucosal and intraluminal immune and inflammatory cells, and altered airway activity. To determine the direct effect of M. pulmonis upon transepithelial ion transport in the absence of immune and inflammatory cell responses, primary mouse tracheal epithelial cell monolayers (MTEs) were apically infected and assayed in Ussing chambers. M. pulmonis-infected MTEs, but not those infected with a nonmurine mycoplasma, demonstrated reductions in amiloride-sensitive Na+ absorption, cyclic AMP, and cholinergic-stimulated Cl− secretion and transepithelial resistance. These effects were shown to require interaction of viable organisms with the apical surface of the monolayer and to be dependent upon organism number and duration of infection. Altered transport due to M. pulmonis was not merely a result of epithelial cell death as evidenced by the following: (i) active transport of Na+ and Cl−, albeit at reduced rates; (ii) normal cell morphology, including intact tight junctions, as demonstrated by electron microscopy; (iii) maintenance of a mean transepithelial resistance of 440 Ω/cm2; and (iv) lack of leakage of fluid from the basolateral to the apical surface of the monolayer. Alteration in epithelial ion transport in vitro is consistent with impaired pulmonary clearance and altered airway function in M. pulmonis-infected animals. Furthermore, the ability of M. pulmonis to alter transport without killing the host cell may explain its successful parasitism and long-term persistence in the host. Further study of the MTE-M. pulmonis model should elucidate the molecular mechanisms which mediate this reduction in transepithelial ion transport. PMID:9423868

Lung Epithelial Healing: A Modified Seed and Soil Concept

PubMed Central

Brechbuhl, Heather M.; Smith, Mary Kathryn; Smith, Russell W.; Ghosh, Moumita

2012-01-01

Airway epithelial healing is defined as restoration of health or soundness; to cure. Our research indicates that two types of progenitor cells participate in this process: the tissue-specific stem cell (TSC) and the facultative basal progenitor (FBP). The TSC restores the epithelium to its normal structure and function. Thus, the TSC regenerates the epithelium. In contrast, the FBP-derived epithelium is characterized by regions of cellular hyperplasia and hypoplasia. Since the FBP-derived epithelium deviates from normal, we term the FBP-mediated process repair. Our work indicates that the TSC responds to signals from other epithelial cells, including the FBP. These signals instruct the TSC to proliferate or to select one of several differentiation pathways. We interpret these data in the context of Stephen Padget’s “seed and soil” paradigm. Therein, Padget explained that metastasis of a tumor, the seed, to a specific site, the soil, was determined by the growth and differentiation requirements of the tumor cell. By extending the seed and soil paradigm to airway epithelial healing, we suggest that proliferation and differentiation of the TSC, the seed, is determined by its interactions with other cell types, the soil. Based on this concept, we provide a set of suggestions for development of cell-based therapies that are directed toward chronic airways disease. PMID:22550238

A novel role of EMMPRIN/CD147 in transformation of quiescent fibroblasts to cancer-associated fibroblasts by breast cancer cells

PubMed Central

Xu, Jing; Lu, Yang; Qiu, Songbo; Chen, Zhi-Nan; Fan, Zhen

2013-01-01

We tested the novel hypothesis that EMMPRIN/CD147, a transmembrane glycoprotein overexpressed in breast cancer cells, has a previously unknown role in transforming fibroblasts to cancer-associated fibroblasts, and that cancer-associated fibroblasts in turn induce epithelial-to-mesenchymal transition of breast cancer cells. Co-culture of fibroblasts with breast cancer cells or treatment of fibroblasts with breast cancer cell conditioned culture medium or recombinant EMMPRIN/CD147 induced expression of α-SMA in the fibroblasts in an EMMPRIN/CD147-dependent manner and promoted epithelial-to-mesenchymal transition of breast cancer cells and enhanced cell migration potential. These findings support a novel role of EMMPRIN/CD147 in regulating the interaction between cancer and stroma. PMID:23474495

Regulation of Leukocyte Infiltration into Ovarian Cancer by Tumour-Stroma Interactions; A Microarray View of Cancer Microenvironment

DTIC Science & Technology

2005-03-01

and EpCAM-linked magnetic beads to separate the cells. Success is assessed on flow cytometry using 2G3, Laminin, FAPa and CK7 markers. On the array...that are >90% enriched for CK7 in the epithelial component, and >80% FAPa for the non-epithelial component. At this moment, however, we have not got

The Nuclear and Adherent Junction Complex Component Protein Ubinuclein Negatively Regulates the Productive Cycle of Epstein-Barr Virus in Epithelial Cells▿

PubMed Central

Gruffat, Henri; Lupo, Julien; Morand, Patrice; Boyer, Véronique; Manet, Evelyne

2011-01-01

The Epstein-Barr Virus (EBV) productive cycle is initiated by the expression of the viral trans-activator EB1 (also called Zebra, Zta, or BZLF1), which belongs to the basic leucine zipper transcription factor family. We have previously identified the cellular NACos (nuclear and adherent junction complex components) protein ubinuclein (Ubn-1) as a partner for EB1, but the function of this complex has never been studied. Here, we have evaluated the consequences of this interaction on the EBV productive cycle and find that Ubn-1 overexpression represses the EBV productive cycle whereas Ubn-1 downregulation by short hairpin RNA (shRNA) increases virus production. By a chromatin immunoprecipitation (ChIP) assay, we show that Ubn-1 blocks EB1-DNA interaction. We also show that in epithelial cells, relocalization and sequestration of Ubn-1 to the tight junctions of nondividing cells allow increased activation of the productive cycle. We propose a model in which Ubn-1 is a modulator of the EBV productive cycle: in proliferating epithelial cells, Ubn-1 is nuclear and inhibits activation of the productive cycle, whereas in differentiated cells, Ubn-1 is sequestrated to tight junctions, thereby allowing EB1 to fully function in the nucleus. PMID:21084479

Extracellular vesicles released by mesenchymal-like prostate carcinoma cells modulate EMT state of recipient epithelial-like carcinoma cells through regulation of AR signaling.

PubMed

El-Sayed, Ihsan Y; Daher, Ahmad; Destouches, Damien; Firlej, Virginie; Kostallari, Enis; Maillé, Pascale; Huet, Eric; Haidar-Ahmad, Nathaline; Jenster, Guido; de la Taille, Alexandre; Abou Merhi, Raghida; Terry, Stéphane; Vacherot, Francis

2017-12-01

Extracellular vesicles released from cancer cells may play an important role in cancer progression by shuttling oncogenic information into recipient cells. However, our knowledge is still fragmentary and there remain numerous questions regarding the mechanisms at play and the functional consequences of these interactions. We have recently established a mesenchymal-like prostate cancer cell line (22Rv1/CR-1; Mes-PCa). In this study, we assessed the effects of the extracellular vesicles released by these cells on recipient androgen-dependent epithelial VCaP prostate cancer cells. Mes-PCa derived vesicles were found to promote mesenchymal features in the recipient epithelial-like prostate cancer cells. This transformation was accompanied by a modulation of androgen receptor signaling and activation of TGFβ signaling pathway. Moreover, recipient cells acquiring mesenchymal traits displayed enhanced migratory and invasive features as well as increased resistance to the androgen receptor antagonist, enzalutamide. Our results suggest a previously unappreciated role for Mes-PCa secreted vesicles in cancer promotion by transferring cell-mediated signals and promoting phenotypic changes in recipient prostate cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

The zebrafish galectins Drgal1-L2 and Drgal3-L1 bind in vitro to the infectious hematopoietic necrosis virus (IHNV) glycoprotein and reduce viral adhesion to fish epithelial cells*

PubMed Central

Feng, Chiguang; González-Montalbán, Núria; Ravindran, Chinnarajan; Jackson, Shawn; de las Heras-Sánchez, Ana; Giomarelli, Barbara; Ahmed, Hafiz; Haslam, Stuart M.; Wu, Gang; Dell, Anne; Ammayappan, Arun; Vakharia, Vikram N.; Vasta, Gerardo R.

2015-01-01

The infectious hematopoietic necrosis virus (IHNV; Rhabdoviridae, Novirhabdovirus) infects teleost fish, such as salmon and trout, and is responsible for significant losses in the aquaculture industry and in wild fish populations. Although IHNV enters the host through the skin at the base of the fins, the viral adhesion and entry mechanisms are not fully understood. In recent years, evidence has accumulated in support of the key roles played by protein-carbohydrate interactions between host lectins secreted to the extracellular space and virion envelope glycoproteins in modulating viral adhesion and infectivity. In this study, we assessed in vitro the potential role(s) of zebrafish (Danio rerio) proto type galectin-1 (Drgal1-L2) and a chimera galectin-3 (Drgal3-L1) in IHNV adhesion to epithelial cells. Our results suggest that the extracellular Drgal1-L2 and Drgal3-L1 interact directly and in a carbohydrate-dependent manner with the IHNV glycosylated envelope and glycans on the epithelial cell surface, significantly reducing viral adhesion. PMID:26429411

Modeling the Epithelial Morphogenesis of Germ Band Retraction in Three Dimensions

NASA Astrophysics Data System (ADS)

McCleery, W. Tyler; Veldhuis, Jim; Brodland, G. Wayne; Crews, Sarah M.; Hutson, M. Shane

2015-03-01

Embryogenesis of higher-order organisms is driven by an intricate coordination of cellular mechanics. Mechanical analysis of certain developmental events, e.g., dorsal closure in the fruit fly D. melanogaster, has been sufficiently described using two-dimensional models. Here, we present a three-dimensional modeling technique to investigate germ band retraction (GBR) - a whole-embryo, irreducibly 3D morphogenetic event. At the start of GBR, the epithelial tissue known as the germ band is initially wrapped around the posterior end of an ellipsoidal fly embryo. This tissue then retracts as an adjacent epithelial tissue, the amnioserosa, simultaneously contracts. We hypothesize that proper GBR requires maintenance of cell-cell connectivity in the amnioserosa, as well as both cell and tissue topology on the embryo's ellipsoidal surface. The exact interfacial tensions are less important. We test the dynamic interactions between these two tissues on a 3D ellipsoidal last. To speed simulation run times and focus on the relevant tissues, epithelial cells are defined as polygons constrained to lie on the surface of the ellipsoidal last. These cells have adjustable parameters such as edge tensions and cell pressures. Tissue movements are simulated by balancing these dynamic cell-level forces with viscous resistance and allowing cells to exchange neighbors. This modeling approach helps elucidate the multicellular stress fields in normal and aberrant development, providing deeper insight into the mechanical interdependence of developing tissues.

Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mytych, Jennifer, E-mail: jennifermytych@gmail.com; Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa; Wos, Izabela

Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we showmore » that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells. - Highlights: • Activated and senescent THP-1 monocytes induced cyto- and genotoxicity in HeLa cells. • Altered monocytes provoked oxidative and nitrosative stress-induced DNA damage. • DNA damage activated DDR pathways and lead to premature senescence and apoptosis. • Klotho reduced ROS/RNS-mediated toxicity through insulin/IGF-IR pathway inhibition. • Klotho protects HeLa cells from cyto- and genotoxicity induced by altered monocytes.« less

Growth Control in Colon Epithelial Cells: Gadolinium Enhances Calcium-Mediated Growth Regulation

PubMed Central

Attili, Durga; Jenkins, Brian; Aslam, Muhammad Nadeem; Dame, Michael K.

2013-01-01

Gadolinium, a member of the lanthanoid family of transition metals, interacts with calcium-binding sites on proteins and other biological molecules. The overall goal of the present investigation was to determine if gadolinium could enhance calcium-induced epithelial cell growth inhibition in the colon. Gadolinium at concentrations as low as 1–5 µM combined with calcium inhibits proliferation of human colonic epithelial cells more effectively than calcium alone. Gadolinium had no detectable effect on calcium-induced differentiation in the same cells based on change in cell morphology, induction of E-cadherin synthesis, and translocation of E-cadherin from the cytosol to the cell surface. When the colon epithelial cells were treated with gadolinium and then exposed to increased calcium concentrations, movement of extracellular calcium into the cell was suppressed. In contrast, gadolinium treatment had no effect on ionomycin-induced release of stored intracellular calcium into the cytoplasm. Whether these in vitro observations can be translated into an approach for reducing abnormal proliferation in the colonic mucosa (including polyp formation) is not known. These results do, however, provide an explanation for our recent findings that a multi-mineral supplement containing all of the naturally occurring lanthanoid metals including gadolinium are more effective than calcium alone in preventing colon polyp formation in mice on a high-fat diet. PMID:23008064

Membrane-shed vesicles from the parasite Trichomonas vaginalis: characterization and their association with cell interaction.

PubMed

Nievas, Yesica R; Coceres, Veronica M; Midlej, Victor; de Souza, Wanderley; Benchimol, Marlene; Pereira-Neves, Antonio; Vashisht, Ajay A; Wohlschlegel, James A; Johnson, Patricia J; de Miguel, Natalia

2018-06-01

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor-ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

PubMed Central

Liévin-Le Moal, Vanessa

2013-01-01

SUMMARY Hosts are protected from attack by potentially harmful enteric microorganisms, viruses, and parasites by the polarized fully differentiated epithelial cells that make up the epithelium, providing a physical and functional barrier. Enterovirulent bacteria interact with the epithelial polarized cells lining the intestinal barrier, and some invade the cells. A better understanding of the cross talk between enterovirulent bacteria and the polarized intestinal cells has resulted in the identification of essential enterovirulent bacterial structures and virulence gene products playing pivotal roles in pathogenesis. Cultured animal cell lines and cultured human nonintestinal, undifferentiated epithelial cells have been extensively used for understanding the mechanisms by which some human enterovirulent bacteria induce intestinal disorders. Human colon carcinoma cell lines which are able to express in culture the functional and structural characteristics of mature enterocytes and goblet cells have been established, mimicking structurally and functionally an intestinal epithelial barrier. Moreover, Caco-2-derived M-like cells have been established, mimicking the bacterial capture property of M cells of Peyer's patches. This review intends to analyze the cellular and molecular mechanisms of pathogenesis of human enterovirulent bacteria observed in infected cultured human colon carcinoma enterocyte-like HT-29 subpopulations, enterocyte-like Caco-2 and clone cells, the colonic T84 cell line, HT-29 mucus-secreting cell subpopulations, and Caco-2-derived M-like cells, including cell association, cell entry, intracellular lifestyle, structural lesions at the brush border, functional lesions in enterocytes and goblet cells, functional and structural lesions at the junctional domain, and host cellular defense responses. PMID:24006470

Targeted overexpression of EZH2 in the mammary gland disrupts ductal morphogenesis and causes epithelial hyperplasia.

PubMed

Li, Xin; Gonzalez, Maria E; Toy, Katherine; Filzen, Tracey; Merajver, Sofia D; Kleer, Celina G

2009-09-01

The Polycomb group protein enhancer of zeste homolog 2 (EZH2), which has roles during development of numerous tissues, is a critical regulator of cell type identity. Overexpression of EZH2 has been detected in invasive breast carcinoma tissue samples and is observed in human breast tissue samples of morphologically normal lobules up to 12 years before the development of breast cancer. The function of EZH2 during preneoplastic progression in the mammary gland is unknown. To investigate the role of EZH2 in the mammary gland, we targeted the expression of EZH2 to mammary epithelial cells using the mouse mammary tumor virus long terminal repeat. EZH2 overexpression resulted in aberrant terminal end bud architecture. By the age of 4 months, 100% of female mouse mammary tumor virus-EZH2 virgin mice developed intraductal epithelial hyperplasia resembling the human counterpart accompanied by premature differentiation of ductal epithelial cells and up-regulation of the luminal marker GATA-3. In addition, remodeling of the mammary gland after parturition was impaired and EZH2 overexpression caused delayed involution. Mechanistically, we found that EZH2 physically interacts with beta-catenin, inducing beta-catenin nuclear accumulation in mammary epithelial cells and activating Wnt/beta-catenin signaling. The biological significance of these data to human hyperplasias is demonstrated by EZH2 up-regulation and colocalization with beta-catenin in human intraductal epithelial hyperplasia, the earliest histologically identifiable precursor of breast carcinoma.

Targeted Overexpression of EZH2 in the Mammary Gland Disrupts Ductal Morphogenesis and Causes Epithelial Hyperplasia

PubMed Central

Li, Xin; Gonzalez, Maria E.; Toy, Katherine; Filzen, Tracey; Merajver, Sofia D.; Kleer, Celina G.

2009-01-01

The Polycomb group protein enhancer of zeste homolog 2 (EZH2), which has roles during development of numerous tissues, is a critical regulator of cell type identity. Overexpression of EZH2 has been detected in invasive breast carcinoma tissue samples and is observed in human breast tissue samples of morphologically normal lobules up to 12 years before the development of breast cancer. The function of EZH2 during preneoplastic progression in the mammary gland is unknown. To investigate the role of EZH2 in the mammary gland, we targeted the expression of EZH2 to mammary epithelial cells using the mouse mammary tumor virus long terminal repeat. EZH2 overexpression resulted in aberrant terminal end bud architecture. By the age of 4 months, 100% of female mouse mammary tumor virus-EZH2 virgin mice developed intraductal epithelial hyperplasia resembling the human counterpart accompanied by premature differentiation of ductal epithelial cells and up-regulation of the luminal marker GATA-3. In addition, remodeling of the mammary gland after parturition was impaired and EZH2 overexpression caused delayed involution. Mechanistically, we found that EZH2 physically interacts with β-catenin, inducing β-catenin nuclear accumulation in mammary epithelial cells and activating Wnt/β-catenin signaling. The biological significance of these data to human hyperplasias is demonstrated by EZH2 up-regulation and colocalization with β-catenin in human intraductal epithelial hyperplasia, the earliest histologically identifiable precursor of breast carcinoma. PMID:19661437

Comparative analysis of the interaction of HSPs in dendritic cells, macrophages, RGM-1 cells infected by Helicobacter pylori

PubMed Central

Yao, Yongliang; Wu, Jianhong; Gu, Tao; Cheng, Yang; Li, Guangxin

2016-01-01

Helicobacter pylori may cause chronic gastritis, even gastric cancer, however, antigen-presenting cells (APCs) are most important immune cells involved in the induction and expression of the underlying inflammatory responses to resist H. pylori. To study the interaction of HSPs in dendritic cells (DCs), macrophages and RGM-1 cells infected with H. pylori, HSP-27, HSP-60, HSP-70 and HSP-90 proteins were analyzed in the mucosa tissue or serum of gastritis patients caused by H. pylori, and in cell supernatant of DCs, macrophages, RGM-1 infected by H. pylori, or in above host cells. We found that HSP-27, HSP-60, HSP-70 and HSP-90 decreased in gastric epithelial cells, but increased significantly in DCs, macrophages. Meanwhile, inflammation associated proteins iNOS-2 and COX-2 were participated in the expression of HSPs in the process of host cells defensing against H. pylori infection. These findings contribute to understand the functions of HSP-27, HSP-60, HSP-70 and HSP-90 in H. pylori infection APCs and gastric epithelial cells indicating that HSPs would be diagnostic markers for H. pylori infection. PMID:27830002

Follicular dermal papilla structures by organization of epithelial and mesenchymal cells in interfacial polyelectrolyte complex fibers.

PubMed

Lim, Tze Chiun; Leong, Meng Fatt; Lu, Hongfang; Du, Chan; Gao, Shujun; Wan, Andrew C A; Ying, Jackie Y

2013-09-01

The hair follicle is a regenerating organ that produces a new hair shaft during each growth cycle. Development and cycling of the hair follicle is governed by interactions between the epithelial and mesenchymal components. Therefore, development of an engineered 3D hair follicle would be useful for studying these interactions to identify strategies for treatment of hair loss. We have developed a technique suitable for assembly of different cell types in close proximity in fibrous hydrogel scaffolds with resolutions of ∼50 μm. By assembly of dermal papilla (DP) and keratinocytes, structures similar to the native hair bulb arrangement are formed. Gene expression of these constructs showed up-regulation of molecules involved in epithelial-mesenchymal interactions of the hair follicle. Implantation of the follicular structures in SCID mice led to the formation of hair follicle-like structures, thus demonstrating their hair inductive ability. The transparency of the fiber matrix and the small dimensions of the follicular structures allowed the direct quantitation of DP cell proliferation by confocal microscopy, clearly illustrating the promoting or inhibitory effects of hair growth regulating agents. Collectively, our results suggested a promising application of these 3D engineered follicular structures for in vitro screening and testing of drugs for hair growth therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Microfluidic lung airway-on-a-chip with arrayable suspended gels for studying epithelial and smooth muscle cell interactions.

PubMed

Humayun, Mouhita; Chow, Chung-Wai; Young, Edmond W K

2018-05-01

Chronic lung diseases (CLDs) are regulated by complex interactions between many different cell types residing in lung airway tissues. Specifically, interactions between airway epithelial cells (ECs) and airway smooth muscle cells (SMCs) have been shown in part to play major roles in the pathogenesis of CLDs, but the underlying molecular mechanisms are not well understood. To advance our understanding of lung pathophysiology and accelerate drug development processes, new innovative in vitro tissue models are needed that can reconstitute the complex in vivo microenvironment of human lung tissues. Organ-on-a-chip technologies have recently made significant strides in recapitulating physiological properties of in vivo lung tissue microenvironments. However, novel advancements are still needed to enable the study of airway SMC-EC communication with matrix interactions, and to provide higher throughput capabilities and manufacturability. We have developed a thermoplastic-based microfluidic lung airway-on-a-chip model that mimics the lung airway tissue microenvironment, and in particular, the interactions between SMCs, ECs, and supporting extracellular matrix (ECM). The microdevice is fabricated from acrylic using micromilling and solvent bonding techniques, and consists of three vertically stacked microfluidic compartments with a bottom media reservoir for SMC culture, a middle thin hydrogel layer, and an upper microchamber for achieving air-liquid interface (ALI) culture of the epithelium. A unique aspect of the design lies in the suspended hydrogel with upper and lower interfaces for EC and SMC culture, respectively. A mixture of type I collagen and Matrigel was found to promote EC adhesion and monolayer formation, and SMC adhesion and alignment. Optimal culturing protocols were established that enabled EC-SMC coculture for more than 31 days. Epithelial monolayers displayed common morphological markers including ZO-1 tight junctions and F-actin cell cortices, while SMCs exhibited enhanced cell alignment and expression of α-SMA. The thermoplastic device construction facilitates mass manufacturing, allows EC-SMC coculture systems to be arrayed for increased throughput, and can be disassembled to allow extraction of the suspended gel for downstream analyses. This airway-on-a-chip device has potential to significantly advance our understanding of SMC-EC-matrix interactions, and their roles in the development of CLDs.

The tight junction protein ZO-1 and an interacting transcription factor regulate ErbB-2 expression

PubMed Central

Balda, Maria S.; Matter, Karl

2000-01-01

Epithelial tight junctions regulate paracellular diffusion and restrict the intermixing of apical and basolateral plasma membrane components. We now identify a Y-box transcription factor, ZONAB (ZO-1-associated nucleic acid-binding protein), that binds to the SH3 domain of ZO-1, a submembrane protein of tight junctions. ZONAB localizes to the nucleus and at tight junctions, and binds to sequences of specific promoters containing an inverted CCAAT box. In reporter assays, ZONAB and ZO-1 functionally interact in the regulation of the ErbB-2 promoter in a cell density-dependent manner. In stably transfected overexpressing cells, ZO-1 and ZONAB control expression of endogenous ErbB-2 and function in the regulation of paracellular permeability. These data indicate that tight junctions directly participate in the control of gene expression and suggest that they function in the regulation of epithelial cell differentiation. PMID:10790369

An adapted in vitro assay to assess Campylobacter jejuni interaction with intestinal epithelial cells: Taking into stimulation with TNFα.

PubMed

Rodrigues, Ramila Cristiane; Pocheron, Anne-Lise; Cappelier, Jean-Michel; Tresse, Odile; Haddad, Nabila

2018-06-01

Campylobacter jejuni is the most prevalent foodborne bacterial infection agent. This pathogen seems also involved in inflammatory bowel diseases in which pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), play a major role. C. jejuni pathogenicity has been extensively studied using in vitro cell culture methods, and more precisely "healthy" cells. In fact, no information is available regarding the behavior of C. jejuni in contact with TNFα-stimulated cells. Therefore, this research was designed to investigate the effect of TNFα on C. jejuni interaction with human intestinal epithelial cells (HT29 and HT29-MTX). To ensure IL-8 production induced by TNFα, human rtTNFα was added to HT29 and HT29-MTX before adhesion and invasion assays. About 10 8 CFU bacteria of C. jejuni strains cells were added to measure their adherence and invasion abilities using TNFα-stimulated cells versus non stimulated cells. Exposure to TNFα results in IL-8 overproduction by intestinal epithelial cells. In addition, the effect of TNFα pre-treatment on C. jejuni adhesion and internalization into eukaryotic cells is strain-dependent. Indeed, the adhesion/invasion process is affected in <50% of the strains tested when TNFα is added to the intestinal cells. Interestingly, TNFα affects more strains in their ability to adhere to and invade the mucus-secreting HT29-MTX cells. Among the 10 strains tested, the aero-tolerant C. jejuni Bf strain is one of the most virulent. These results suggest that the TNFα signalling pathway could participate in the internalization of C. jejuni in human intestinal cells and can help in understanding the pathogenicity of this microorganism in contact with TNFα-stimulated cells. Copyright © 2018. Published by Elsevier B.V.

Development and Use of the Lens Epithelial Explant System to Study Lens Differentiation and Cataractogenesis

PubMed Central

West-Mays, Judith A.; Pino, Guiseppe; Lovicu, Frank J.

2010-01-01

Over the last two decades much progress has been made in identifying and characterizing many of the molecules involved in understanding normal lens biology and its pathology. Much of this has been made possible through the establishment and use of the lens epithelial explant system. This simplistic tissue culture model, comprised of a sheet of lens epithelium on its native substratum, has been used effectively to study many cellular processes, including lens epithelial cell proliferation, fiber cell differentiation, cell apoptosis as well as epithelial to mesenchymal transformation of cells. In doing so, a number of key growth factors and cytokines, including members of the FGF, Wnt and TGFβ family have been shown to play essential roles in many of these cellular events. This has led to further studies exploring the signaling pathways downstream of these molecules in the lens, paving the way for the development of a number of in situ models (primarily transgenic mouse lines) to further explore in more detail the nature of these molecular and cellular interactions. To reciprocate, the lens epithelial explant system is increasingly being used to further characterize the nature of many complex phenotypes and pathologies observed in these in situ models, allowing us to selectively isolate and examine the direct impact of an individual molecule on a specific cellular response in lens cells. There is no question that the lens epithelial explant system has served as a powerful tool to further our understanding of lens biology and pathology, and there is no doubt that it will continue to serve in such a capacity, as new developments are realized and putative treatments for aberrant lens cell behaviour are to be trialed. PMID:20006728

Host control of human papillomavirus infection and disease.

PubMed

Doorbar, John

2018-02-01

Most human papillomaviruses cause inapparent infections, subtly affecting epithelial homeostasis, to ensure genome persistence in the epithelial basal layer. As with conspicuous papillomas, these self-limiting lesions shed viral particles to ensure population level maintenance and depend on a balance between viral gene expression, immune cell stimulation and immune surveillance for persistence. The complex immune evasion strategies, characteristic of high-risk HPV types, also allow the deregulated viral gene expression that underlies neoplasia. Neoplasia occurs at particular epithelial sites where vulnerable cells such as the reserve or cuboidal cells of the cervical transformation zone are found. Beta papillomavirus infection can also predispose an individual with immune deficiencies to the development of cancers. The host control of HPV infections thus involves local interactions between keratinocytes and the adaptive immune response. Effective immune detection and surveillance limits overt disease, leading to HPV persistence as productive microlesions or in a true latent state. Copyright © 2017. Published by Elsevier Ltd.

A novel role for integrin-linked kinase in epithelial sheet morphogenesis.

PubMed

Vespa, Alisa; D'Souza, Sudhir J A; Dagnino, Lina

2005-09-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.

Lactobacillus plantarum Enhanced IL-22 Production in Natural Killer (NK) Cells That Protect the Integrity of Intestinal Epithelial Cell Barrier Damaged by Enterotoxigenic Escherichia coli.

PubMed

Qiu, Yueqin; Jiang, Zongyong; Hu, Shenglan; Wang, Li; Ma, Xianyong; Yang, Xuefen

2017-11-13

Interleukin (IL)-22-producing Natural Killer (NK) cells protect the gut epithelial cell barrier from pathogens. A strain of probiotics, Lactobacillus plantarum (L. plantarum, LP), was previously found by our laboratory to significantly improve the mucosal barrier integrity and function of the small intestine in pigs. However, it was unclear whether LP benefited the intestinal mucosal barrier via interactions with the intestinal NK cells. The present study, therefore, was focused on the therapeutic effect of NK cells that were stimulated by LP on attenuating enterotoxigenic Escherichia coli (ETEC)-induced the damage to the integrity of the epithelial cell barrier. The results showed that LP can efficiently increase protein levels of the natural cytotoxicity receptor (NCR) family, and the expression levels of IL-22 mRNA and protein in NK cells. Transfer of NK cells stimulated by LP conferred protection against ETEC K88-induced intestinal epithelial barrier damage in NCM460 cells. We found that NK cells stimulated by LP could partially offset the reduction in NCM460 cell monolayers transepithelial electrical resistance (TEER) caused by ETEC K88, and increase ZO-1 and occludin mRNA and protein expressions by ETEC K88-infected NCM460 cells. Furthermore, adding NK cells that were stimulated by LP to ETEC K88-infected NCM460cells, IL-22R1, p-Stat3, and p-Tyk2 expression by NCM460 cells was increased. Mechanistic experiment showed that NK cells stimulated by LP lost the function of maintaining TEER of NCM460 cells challenged with ETEC K88, when polyclonal anti-IL-22 antibody was used to block IL-22 production. Collectively, our results suggested that LP stimulation of NK could enhance IL-22 production, which might be able to provide defense against ETEC-induced damage to the integrity of intestinal epithelial barrier.

Differential tissue growth and cell adhesion alone drive early tooth morphogenesis: An ex vivo and in silico study

PubMed Central

Savriama, Yoland; Jernvall, Jukka

2018-01-01

From gastrulation to late organogenesis animal development involves many genetic and bio-mechanical interactions between epithelial and mesenchymal tissues. Ectodermal organs, such as hairs, feathers and teeth are well studied examples of organs whose development is based on epithelial-mesenchymal interactions. These develop from a similar primordium through an epithelial folding and its interaction with the mesenchyme. Despite extensive knowledge on the molecular pathways involved, little is known about the role of bio-mechanical processes in the morphogenesis of these organs. We propose a simple computational model for the biomechanics of one such organ, the tooth, and contrast its predictions against cell-tracking experiments, mechanical relaxation experiments and the observed tooth shape changes over developmental time. We found that two biomechanical processes, differential tissue growth and differential cell adhesion, were enough, in the model, for the development of the 3D morphology of the early tooth germ. This was largely determined by the length and direction of growth of the cervical loops, lateral folds of the enamel epithelium. The formation of these cervical loops was found to require accelerated epithelial growth relative to other tissues and their direction of growth depended on specific differential adhesion between the three tooth tissues. These two processes and geometrical constraints in early tooth bud also explained the shape asymmetry between the lateral cervical loops and those forming in the anterior and posterior of the tooth. By performing mechanical perturbations ex vivo and in silico we inferred the distribution and direction of tensile stresses in the mesenchyme that restricted cervical loop lateral growth and forced them to grow downwards. Overall our study suggests detailed quantitative explanations for how bio-mechanical processes lead to specific morphological 3D changes over developmental time. PMID:29481561

A novel mechanism by which hepatocyte growth factor blocks tubular epithelial to mesenchymal transition.

PubMed

Yang, Junwei; Dai, Chunsun; Liu, Youhua

2005-01-01

Hepatocyte growth factor (HGF) is a potent antifibrotic cytokine that blocks tubular epithelial to mesenchymal transition (EMT) induced by TGF-beta1. However, the underlying mechanism remains largely unknown. This study investigated the signaling events that lead to HGF blockade of the TGF-beta1-initiated EMT. Incubation of human kidney epithelial cells HKC with HGF only marginally affected the expression of TGF-beta1 and its type I and type II receptors, suggesting that disruption of TGF-beta1 signaling likely plays a critical role in mediating HGF inhibition of TGF-beta1 action. However, HGF neither affected TGF-beta1-induced Smad-2 phosphorylation and its subsequent nuclear translocation nor influenced the expression of inhibitory Smad-6 and -7 in tubular epithelial cells. HGF specifically induced the expression of Smad transcriptional co-repressor SnoN but not Ski and TG-interacting factor at both mRNA and protein levels in HKC cells. SnoN physically interacted with activated Smad-2 by forming transcriptionally inactive complex and overrode the profibrotic action of TGF-beta1. In vivo, HGF did not affect Smad-2 activation and its nuclear accumulation in tubular epithelium, but it restored SnoN protein abundance in the fibrotic kidney in obstructive nephropathy. Hence, HGF blocks EMT by antagonizing TGF-beta1's action via upregulating Smad transcriptional co-repressor SnoN expression. These findings not only identify a novel mode of interaction between the signals activated by HGF receptor tyrosine kinase and TGF-beta receptor serine/threonine kinases but also illustrate the feasibility of confining Smad activity as an effective strategy for blocking renal fibrosis.

Generation of organotypic raft cultures from primary human keratinocytes.

PubMed

Anacker, Daniel; Moody, Cary

2012-02-22

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)(1). The life cycle of HPV is tightly linked to the differentiation of squamous epithelium(2). Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production(3,4,5). In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras(6) and modified by Kopan et al.(7), the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies(8). Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses(9). Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-β1 in Salivary Gland Epithelial and Mesenchymal Differentiation

PubMed Central

Janebodin, Kajohnkiart; Buranaphatthana, Worakanya; Ieronimakis, Nicholas; Hays, Aislinn L.; Reyes, Morayma

2013-01-01

Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-β1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-β1 on salivary gland cell differentiation. TGF-β1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-βR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-β signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-β1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors. PMID:23841093

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paquette, Stéphane G.; Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario; Banner, David

Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directlymore » triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction. - Highlights: • We investigated H1N1pdm/sH1N1 infection in primary epithelial cells. • H1N1pdm directly initiated a robust inflammatory gene signature, sH1N1 did not. • H1N1pdm viral RNA triggered a stronger response than sH1N1. • H1N1pdm induces greater response due to direct virus–cell interaction. • These results have potential to impact vaccine and therapeutic development.« less

Stromal-epithelial interactions modulate the effect of ovarian steroids on goat uterine epithelial cell interleukin-18 release.

PubMed

Qi, X F; Nan, Z C; Jin, Y P; Qu, Y Y; Zhao, X J; Wang, A H

2012-05-01

A primary role of epithelial-stromal interactions in mediating steroid hormone action in the uterus has been established. The present study was undertaken to determine the mode of ovarian steroid action in regulating IL-18 release by goat endometrial epithelial cells (EECs) in the presence and absence of endometrial stromal cells (ESCs). Primary and telomerase-immortalized goat EECs grown alone or cocultured with ESCs were treated with two ovarian steroids, 17β-estradiol (E(2)) and progesterone (P(4)). The IL-18 mRNA and protein expression in EECs were studied by reverse transcript (RT) PCR, ELISA, and Western blot assay. The E(2) and/or P(4) treatment of EECs led to a significant increase in both IL-18 mRNA and protein expression either in the primary or in the immortalized EECs compared with that in EECs without the steroid treatment. However, in the presence of ESCs, IL-18 expression by EECs treated with steroids was significantly decreased compared with cells untreated with E(2) and/or P(4). In addition, significantly high abundance of IL-18 mRNA and protein expression by primary and telomerase-immortalized goat EECs was observed in the presence of ESCs compared with those cells without ESCs. These findings suggest that steroids are important for the control of IL-18 expression in goat EECs. Underlying ESCs are needed to mediate the inhibitory effects of steroids on the IL-18 secretory activity of goat EECs in vitro. The IL-18 abundance expressed by goat EECs in vitro are enhanced by underlying ESCs without the treatment of E(2) and/or P(4). Copyright © 2012 Elsevier Inc. All rights reserved.

Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments.

PubMed

Miller, Daniel P; Hutcherson, Justin A; Wang, Yan; Nowakowska, Zuzanna M; Potempa, Jan; Yoder-Himes, Deborah R; Scott, David A; Whiteley, Marvin; Lamont, Richard J

2017-01-01

Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis , and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism.

The signaling adapter Gab1 regulates cell polarity by acting as a PAR protein scaffold

PubMed Central

Yang, Ziqiang; Xue, Bin; Umitsu, Masataka; Ikura, Mitsuhiko; Muthuswamy, Senthil K.; Neel, Benjamin G.

2012-01-01

Summary Cell polarity plays a key role in development and is disrupted in tumors, yet the molecules and mechanisms that regulate polarity remain poorly defined. We found that the scaffolding adaptor GAB1 interacts with two polarity proteins, PAR1 and PAR3. GAB1 binds PAR1 and enhances its kinase activity. GAB1 brings PAR1 and PAR3 into a transient complex, stimulating PAR3 phosphorylation by PAR1. GAB1 and PAR6 bind the PAR3 PDZ1 domain and thereby compete for PAR3 binding. Consequently, GAB1 depletion causes PAR3 hypo-phosphorylation and increases PAR3/PAR6 complex formation, resulting in accelerated and enhanced tight junction formation, increased trans-epithelial resistance and lateral domain shortening. Conversely, GAB1 over-expression, in a PAR1/PAR3-dependent manner, disrupts epithelial apical-basal polarity, promotes multi-lumen cyst formation, and enhances growth factor-induced epithelial cell scattering. Our results identify GAB1 as a novel negative regulator of epithelial cell polarity that functions as a scaffold for modulating PAR protein complexes on the lateral membrane. PMID:22883624

Interface between breast cancer cells and the tumor microenvironment using platelet-rich plasma to promote tumor angiogenesis - influence of platelets and fibrin bundles on the behavior of breast tumor cells

PubMed Central

Andrade, Sheila Siqueira; Sumikawa, Joana Tomomi; Castro, Eloísa Dognani; Batista, Fabricio Pereira; Paredes-Gamero, Edgar; Oliveira, Lilian Carolina; Guerra, Izabel Monastério; Peres, Giovani Bravin; Cavalheiro, Renan Pelluzzi; Juliano, Luiz; Nazário, Afonso Pinto; Facina, Gil; Tsai, Siu Mui; Oliva, Maria Luiza Vilela; Girão, Manoel João Batista Castello

2017-01-01

Cancer progression is associated with an evolving tissue interface of direct epithelial-tumor microenvironment interactions. In biopsies of human breast tumors, extensive alterations in molecular pathways are correlated with cancer staging on both sides of the tumor-stroma interface. These interactions provide a pivotal paracrine signaling to induce malignant phenotype transition, the epithelial-mesenchymal transition (EMT). We explored how the direct contact between platelets-fibrin bundles primes metastasis using platelet-rich plasma (PRP) as a source of growth factors and mimics the provisional fibrin matrix between actively growing breast cancer cells and the tumor stroma. We have demonstrated PRP functions, modulating cell proliferation that is tumor-subtype and cancer cell-type-specific. Epithelial and stromal primary cells were prepared from breast cancer biopsies from 21 women with different cancer subtypes. Cells supplemented with PRP were immunoblotted with anti-phospho and total Src-Tyr-416, FAK-Try-925, E-cadherin, N-cadherin, TGF-β, Smad2, and Snail monoclonal antibodies. Breast tumor cells from luminal B and HER2 subtypes showed the most malignant profiles and the expression of thrombin and other classes of proteases at levels that were detectable through FRET peptide libraries. The angiogenesis process was investigated in the interface obtained between platelet-fibrin-breast tumor cells co-cultured with HUVEC cells. Luminal B and HER2 cells showed robust endothelial cell capillary-like tubes ex vivo. The studied interface contributes to the attachment of endothelial cells, provides a source of growth factors, and is a solid substrate. Thus, replacement of FBS supplementation with PRP supplementation represents an efficient and simple approach for mimicking the real multifactorial tumor microenvironment. PMID:28187434

Interface between breast cancer cells and the tumor microenvironment using platelet-rich plasma to promote tumor angiogenesis - influence of platelets and fibrin bundles on the behavior of breast tumor cells.

PubMed

Andrade, Sheila Siqueira; Sumikawa, Joana Tomomi; Castro, Eloísa Dognani; Batista, Fabricio Pereira; Paredes-Gamero, Edgar; Oliveira, Lilian Carolina; Guerra, Izabel Monastério; Peres, Giovani Bravin; Cavalheiro, Renan Pelluzzi; Juliano, Luiz; Nazário, Afonso Pinto; Facina, Gil; Tsai, Siu Mui; Oliva, Maria Luiza Vilela; Girão, Manoel João Batista Castello

2017-03-07

Cancer progression is associated with an evolving tissue interface of direct epithelial-tumor microenvironment interactions. In biopsies of human breast tumors, extensive alterations in molecular pathways are correlated with cancer staging on both sides of the tumor-stroma interface. These interactions provide a pivotal paracrine signaling to induce malignant phenotype transition, the epithelial-mesenchymal transition (EMT). We explored how the direct contact between platelets-fibrin bundles primes metastasis using platelet-rich plasma (PRP) as a source of growth factors and mimics the provisional fibrin matrix between actively growing breast cancer cells and the tumor stroma. We have demonstrated PRP functions, modulating cell proliferation that is tumor-subtype and cancer cell-type-specific. Epithelial and stromal primary cells were prepared from breast cancer biopsies from 21 women with different cancer subtypes. Cells supplemented with PRP were immunoblotted with anti-phospho and total Src-Tyr-416, FAK-Try-925, E-cadherin, N-cadherin, TGF-β, Smad2, and Snail monoclonal antibodies. Breast tumor cells from luminal B and HER2 subtypes showed the most malignant profiles and the expression of thrombin and other classes of proteases at levels that were detectable through FRET peptide libraries. The angiogenesis process was investigated in the interface obtained between platelet-fibrin-breast tumor cells co-cultured with HUVEC cells. Luminal B and HER2 cells showed robust endothelial cell capillary-like tubes ex vivo. The studied interface contributes to the attachment of endothelial cells, provides a source of growth factors, and is a solid substrate. Thus, replacement of FBS supplementation with PRP supplementation represents an efficient and simple approach for mimicking the real multifactorial tumor microenvironment.

A surface-charge study on cellular-uptake behavior of F3-peptide-conjugated iron oxide nanoparticles.

PubMed

Zhang, Yu; Yang, Mo; Park, Ji-Ho; Singelyn, Jennifer; Ma, Huiqing; Sailor, Michael J; Ruoslahti, Erkki; Ozkan, Mihrimah; Ozkan, Cengiz

2009-09-01

Surface-charge measurements of mammalian cells in terms of Zeta potential are demonstrated as a useful biological characteristic in identifying cellular interactions with specific nanomaterials. A theoretical model of the changes in Zeta potential of cells after incubation with nanoparticles is established to predict the possible patterns of Zeta-potential change to reveal the binding and internalization effects. The experimental results show a distinct pattern of Zeta-potential change that allows the discrimination of human normal breast epithelial cells (MCF-10A) from human cancer breast epithelial cells (MCF-7) when the cells are incubated with dextran coated iron oxide nanoparticles that contain tumor-homing F3 peptides, where the tumor-homing F3 peptide specifically bound to nucleolin receptors that are overexpressed in cancer breast cells.

Cytotoxicity of protein corona-graphene oxide nanoribbons on human epithelial cells

NASA Astrophysics Data System (ADS)

Mbeh, Doris A.; Akhavan, Omid; Javanbakht, Taraneh; Mahmoudi, Morteza; Yahia, L.'Hocine

2014-11-01

Graphene oxide nanoribbons (GONRs) were synthesized using an oxidative unzipping of multi-walled carbon nanotubes. The interactions of the GONRs with various concentrations of fetal bovine serum or human plasma serum indicated that the GONRs were functionalized substantially by the albumin originated from the two different protein sources. Then, concentration-dependent cytotoxicity of the protein-functionalized GONRs on human epithelial cells was studied. Although the GONRs with concentrations ≤50 μg/mL did not exhibit significant cytotoxicity on the cells (with the cell viability >85%), the concentration of 100 μg/mL exhibited significant cytotoxicity including prevention of cell proliferation and induction of cell apoptosis. These results can provide more in-depth understanding about cytotoxic effects of graphene nanostructures which can be functionalized by the proteins of media.

Genome Wide Analysis of Differentially Expressed Genes in HK-2 Cells, a Line of Human Kidney Epithelial Cells in Response to Oxalate

PubMed Central

Koul, Sweaty; Khandrika, Lakshmipathi; Meacham, Randall B.; Koul, Hari K.

2012-01-01

Nephrolithiasis is a multi-factorial disease which, in the majority of cases, involves the renal deposition of calcium oxalate. Oxalate is a metabolic end product excreted primarily by the kidney. Previous studies have shown that elevated levels of oxalate are detrimental to the renal epithelial cells; however, oxalate renal epithelial cell interactions are not completely understood. In this study, we utilized an unbiased approach of gene expression profiling using Affymetrix HG_U133_plus2 gene chips to understand the global gene expression changes in human renal epithelial cells [HK-2] after exposure to oxalate. We analyzed the expression of 47,000 transcripts and variants, including 38,500 well characterized human genes, in the HK2 cells after 4 hours and 24 hours of oxalate exposure. Gene expression was compared among replicates as per the Affymetrix statistical program. Gene expression among various groups was compared using various analytical tools, and differentially expressed genes were classified according to the Gene Ontology Functional Category. The results from this study show that oxalate exposure induces significant expression changes in many genes. We show for the first time that oxalate exposure induces as well as shuts off genes differentially. We found 750 up-regulated and 2276 down-regulated genes which have not been reported before. Our results also show that renal cells exposed to oxalate results in the regulation of genes that are associated with specific molecular function, biological processes, and other cellular components. In addition we have identified a set of 20 genes that is differentially regulated by oxalate irrespective of duration of exposure and may be useful in monitoring oxalate nephrotoxicity. Taken together our studies profile global gene expression changes and provide a unique insight into oxalate renal cell interactions and oxalate nephrotoxicity. PMID:23028475

G protein-coupled receptor kinase 2 positively regulates epithelial cell migration

PubMed Central

Penela, Petronila; Ribas, Catalina; Aymerich, Ivette; Eijkelkamp, Niels; Barreiro, Olga; Heijnen, Cobi J; Kavelaars, Annemieke; Sánchez-Madrid, Francisco; Mayor, Federico

2008-01-01

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration. PMID:18369319

Epstein-Barr virus and nasopharyngeal carcinoma

PubMed Central

Young, Lawrence S.; Dawson, Christopher W.

2014-01-01

Since its discovery 50 years ago, Epstein-Barr virus (EBV) has been linked to the development of cancers originating from both lymphoid and epithelial cells. Approximately 95% of the world's population sustains an asymptomatic, life-long infection with EBV. The virus persists in the memory B-cell pool of normal healthy individuals, and any disruption of this interaction results in virus-associated B-cell tumors. The association of EBV with epithelial cell tumors, specifically nasopharyngeal carcinoma (NPC) and EBV-positive gastric carcinoma (EBV-GC), is less clear and is currently thought to be caused by the aberrant establishment of virus latency in epithelial cells that display premalignant genetic changes. Although the precise role of EBV in the carcinogenic process is currently poorly understood, the presence of the virus in all tumor cells provides opportunities for developing novel therapeutic and diagnostic approaches. The study of EBV and its role in carcinomas continues to provide insight into the carcinogenic process that is relevant to a broader understanding of tumor pathogenesis and to the development of targeted cancer therapies. PMID:25418193

Juglone alleviates pneumolysin-induced human alveolar epithelial cell injury via inhibiting the hemolytic activity of pneumolysin.

PubMed

Song, Meng; Lu, Gejin; Li, Meng; Deng, Xuming; Wang, Jianfeng

2017-08-01

Streptococcus pneumoniae (the pneumococcus) is an opportunistic pathogen responsible for several human diseases, including acute otitis media, pneumonia, sepsis and bacterial meningitis, and possesses numerous virulence factors associated with pneumococcal infection and pathogenesis. With the capacity to form pores in cholesterol-rich membranes, pneumolysin (PLY) is a key virulence factor of S. pneumoniae and causes severe tissue damage during pneumococcal infection. Juglone (JG), a natural 1,4-naphthoquinone widely found in the roots, leaves, woods and fruits of Juglandaceae walnut trees, inhibits PLY-induced hemolysis via inhibition of the oligomerization of PLY and exhibits minimal anti-S. pneumoniae activity. In addition, when human alveolar epithelial (A549) cells were co-cultured with PLY and JG, PLY-mediated cell injury was significantly alleviated. These results indicate that JG directly interacts with PLY to reduce the cytotoxicity of the toxin in human alveolar epithelial cells. Hence, JG is an effective inhibitor of PLY and protects lung cells from PLY-mediated cell injury. This study also provides the basis for the development of anti-virulence drugs for the treatment of S. pneumoniae infections.

PERP, a host tetraspanning membrane protein, is required for S almonella‐induced inflammation

PubMed Central

Hallstrom, Kelly N.; Srikanth, C. V.; Agbor, Terence A.; Dumont, Christopher M.; Peters, Kristen N.; Paraoan, Luminita; Casanova, James E.; Boll, Erik J.

2015-01-01

Summary S almonella enterica  Typhimurium induces intestinal inflammation through the activity of type III secreted effector (T3SE) proteins. Our prior results indicate that the secretion of the T3SE SipA and the ability of SipA to induce epithelial cell responses that lead to induction of polymorphonuclear transepithelial migration are not coupled to its direct delivery into epithelial cells from S almonella. We therefore tested the hypothesis that SipA interacts with a membrane protein located at the apical surface of intestinal epithelial cells. Employing a split ubiquitin yeast‐two‐hybrid screen, we identified the tetraspanning membrane protein, p53 effector related to PMP‐22 (PERP), as a SipA binding partner. SipA and PERP appear to have intersecting activities as we found PERP to be involved in proinflammatory pathways shown to be regulated by SipA. In sum, our studies reveal a critical role for PERP in the pathogenesis of S. Typhimurium, and for the first time demonstrate that SipA, a T3SE protein, can engage a host protein at the epithelial surface. PMID:25486861

Conditionally immortal ovarian cell lines for investigating the influence of ovarian stroma on the estrogen sensitivity and tumorigenicity of ovarian surface epithelial cells.

PubMed

Jiang, Feng; Saunders, Beatriz O; Haller, Edward; Livingston, Sandra; Nicosia, Santo V; Bai, Wenlong

2003-01-01

The tendency of the ovarian surface epithelium (OSE) to undergo metaplastic and morphogenetic changes during the life cycle, at variance with the adjacent peritoneal mesothelial cells, suggests that its biology may be regulated by underlying ovarian stromal cues. However, little is known about the role that the ovarian stroma plays in the pathobiology of the OSE, largely because of the lack of a suitable in vitro model. Here, we describe the establishment and characterization of conditionally immortalized ovarian stromal and surface epithelial cell lines from H-2K(b)-tsA58 transgenic mice that carry the thermolabile mutant of SV-40 large T antigen under the control of an interferon-gamma (IFN-gamma)-inducible promoter. These cells express functional T antigens, grow continuously under permissive conditions at 33 degrees C in the presence of IFN-gamma, and stop dividing when the activity and expression of the tumor antigen is suppressed by restrictive conditions without IFN-gamma at 39 degrees C. Morphological, immunohistochemical, and ultrastructural analyses show that conditionally immortal OSE cells form cobblestone-like monolayers, express cytokeratin and vimentin, contain several microvilli, and develop tight junctions, whereas stromal cells are spindle-like, express vimentin but not cytokeratin, and contain rare microvilli, thus exhibiting epithelial and stromal phenotypes, respectively. At variance with the reported behavior of rat epithelial cells, conditionally immortal mouse epithelial cells are not spontaneously transformed after continuous culture in vitro. More importantly, conditioned media from stromal cells cultured under permissive conditions increase the specific activity of the endogenous estrogen receptor in BG-1 human ovarian epithelial cancer cells and promote these cells' anchorage-independent growth, suggesting the paracrine influence of a stromal factor. In addition, stromal cells cultured under restrictive conditions retain this growth-stimulatory activity, which, therefore, appears to be independent of T antigen expression. These established cell lines should provide a useful in vitro model system for studying the role of cellular interactions in OSE cell growth and tumorigenesis.

Citrus-derived oils inhibit Satphylococcus aureus growth and alter its interaction with bovine mammary cells

USDA-ARS?s Scientific Manuscript database

This experiment examined the effects of cold-pressed, terpeneless citrus oil (CDO) on growth of Staphylococcus aureus, which a major cause of contagious bovine mastitis, and invasion of epithelial cells as modeled with bovine mammary cells (MAC-T). The broth dilution method (Muthaiyan et al., 2012)...

The Epithelial Cell Adhesion Molecule EpCAM Is Required for Epithelial Morphogenesis and Integrity during Zebrafish Epiboly and Skin Development

PubMed Central

Slanchev, Krasimir; Carney, Thomas J.; Stemmler, Marc P.; Koschorz, Birgit; Amsterdam, Adam; Schwarz, Heinz; Hammerschmidt, Matthias

2009-01-01

The aberrant expression of the transmembrane protein EpCAM is associated with tumor progression, affecting different cellular processes such as cell–cell adhesion, migration, proliferation, differentiation, signaling, and invasion. However, the in vivo function of EpCAM still remains elusive due to the lack of genetic loss-of-function studies. Here, we describe epcam (tacstd) null mutants in zebrafish. Maternal-zygotic mutants display compromised basal protrusive activity and epithelial morphogenesis in cells of the enveloping layer (EVL) during epiboly. In partial redundancy with E-cadherin (Ecad), EpCAM made by EVL cells is further required for cell–cell adhesion within the EVL and, possibly, for proper attachment of underlying deep cells to the inner surface of the EVL, thereby also affecting deep cell epiboly movements. During later development, EpCAM per se becomes indispensable for epithelial integrity within the periderm of the skin, secondarily leading to disrupted morphology of the underlying basal epidermis and moderate hyper-proliferation of skin cells. On the molecular level, EVL cells of epcam mutant embryos display reduced levels of membranous Ecad, accompanied by an enrichment of tight junction proteins and a basal extension of apical junction complexes (AJCs). Our data suggest that EpCAM acts as a partner of E-cadherin to control adhesiveness and integrity as well as plasticity and morphogenesis within simple epithelia. In addition, EpCAM is required for the interaction of the epithelia with underlying cell layers. PMID:19609345

Cyto-adherence of Mycoplasma mycoides subsp. mycoides to bovine lung epithelial cells.

PubMed

Aye, Racheal; Mwirigi, Martin Kiogora; Frey, Joachim; Pilo, Paola; Jores, Joerg; Naessens, Jan

2015-02-07

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease of cattle, whereas the closely related Mycoplasma mycoides subsp. capri (Mmc) is a goat pathogen. Cyto-adherence is a crucial step in host colonization by mycoplasmas and subsequent pathogenesis. The aim of this study was to investigate the interactions between Mmm and mammalian host cells by establishing a cyto-adherence flow cytometric assay and comparing tissue and species specificity of Mmm and Mmc strains. There were little significant differences in the adherence patterns of eight different Mmm strains to adult bovine lung epithelial cells. However, there was statistically significant variation in binding to different host cells types. Highest binding was observed with lung epithelial cells, intermediate binding with endothelial cells and very low binding with fibroblasts, suggesting the presence of effective adherence of Mmm on cells lining the airways of the lung, which is the target organ for this pathogen, possibly by high expression of a specific receptor. However, binding to bovine fetal lung epithelial cells was comparably low; suggesting that the lack of severe pulmonary disease seen in many infected young calves can be explained by reduced expression of a specific receptor. Mmm bound with high efficiency to adult bovine lung cells and less efficiently to calves or goat lung cells. The data show that cyto-adherence of Mmm is species- and tissue- specific confirming its role in colonization of the target host and subsequent infection and development of CBPP.

Research on Blastocyst Implantation Essential Factors (BIEFs).

PubMed

Yoshinaga, Koji

2010-06-01

Blastocyst implantation is a process of interaction between embryo and the uterus. To understand this process, this review tries to summarize what blastocyst implantation essential factors (BIEFs) play what roles, as well as where in the uterus and at what stage of implantation process. Addition of more new data to this kind of compilation of information will help the development of diagnosis and treatment of infertility caused by implantation failure. The major, important cells of the endometrial cells that interact with invading blastocyst (trophoblast) are luminal epithelial cells, stromal cells (decidual cells) and resident immune cells. BIEFs regulate these cells to successfully maintain pregnancy.

Akap350 Recruits Eb1 to The Spindle Poles, Ensuring Proper Spindle Orientation and Lumen Formation in 3d Epithelial Cell Cultures.

PubMed

Almada, Evangelina; Tonucci, Facundo M; Hidalgo, Florencia; Ferretti, Anabela; Ibarra, Solange; Pariani, Alejandro; Vena, Rodrigo; Favre, Cristián; Girardini, Javier; Kierbel, Arlinet; Larocca, M Cecilia

2017-11-02

The organization of epithelial cells to form hollow organs with a single lumen requires the accurate three-dimensional arrangement of cell divisions. Mitotic spindle orientation is defined by signaling pathways that provide molecular links between specific spots at the cell cortex and astral microtubules, which have not been fully elucidated. AKAP350 is a centrosomal/Golgi scaffold protein, implicated in the regulation of microtubule dynamics. Using 3D epithelial cell cultures, we found that cells with decreased AKAP350 expression (AKAP350KD) formed polarized cysts with abnormal lumen morphology. Analysis of mitotic cells in AKAP350KD cysts indicated defective spindle alignment. We established that AKAP350 interacts with EB1, a microtubule associated protein that regulates spindle orientation, at the spindle poles. Decrease of AKAP350 expression lead to a significant reduction of EB1 levels at spindle poles and astral microtubules. Conversely, overexpression of EB1 rescued the defective spindle orientation induced by deficient AKAP350 expression. The specific delocalization of the AKAP350/EB1complex from the centrosome decreased EB1 levels at astral microtubules and lead to the formation of 3D-organotypic structures which resembled AKAP350KD cysts. We conclude that AKAP350 recruits EB1 to the spindle poles, ensuring EB1 presence at astral microtubules and proper spindle orientation during epithelial morphogenesis.

A role for the pattern recognition receptor Nod2 in promoting recruitment of CD103+ Dendritic Cells to the colon in response to Trichuris muris infection

PubMed Central

Bowcutt, Rowann; Bramhall, Michael; Logunova, Larisa; Wilson, Jim; Booth, Cath; Carding, Simon R.; Grencis, Richard; Cruickshank, Sheena

2014-01-01

The ability of the colon to generate an immune response to pathogens, such as the model pathogen Trichuris muris, is a fundamental and critical defense mechanism. Resistance to T.muris infection is associated with the rapid recruitment of dendritic cells (DCs) to the colonic epithelium via epithelial chemokine production. However, the epithelial-pathogen interactions that drive chemokine production are not known. We addressed the role of the cytosolic pattern recognition receptor Nod2. In response to infection, there was a rapid influx of CD103+CD11c+ DCs into the colonic epithelium in wild type (WT) mice whereas this was absent in Nod2−/− animals. In vitro chemotaxis assays and in vivo experiments using bone marrow chimeras of WT mice reconstituted with Nod2−/− bone marrow and infected with T. muris demonstrated that the migratory function of Nod2−/− DCs was normal. Investigation of colonic epithelial cell (CEC) innate responses revealed a significant reduction in epithelial production of the chemokines CCL2 and CCL5 but not CCL20 by Nod2-deficient CEC. Collectively, these data demonstrate the importance of Nod2 in CEC responses to infection and the requirement for functional Nod2 in initiating host epithelial chemokine mediated responses and subsequent DC recruitment and T cell responses following infection. PMID:24448097

Giardia's Epithelial Cell Interaction In Vitro: Mimicking Asymptomatic Infection?

PubMed Central

Kraft, Martin R.; Klotz, Christian; Bücker, Roland; Schulzke, Jörg-Dieter; Aebischer, Toni

2017-01-01

The protozoan parasite Giardia duodenalis is responsible for more than 280 million cases of gastrointestinal complaints (“giardiasis”) every year, worldwide. Infections are acquired orally, mostly via uptake of cysts in contaminated drinking water. After transformation into the trophozoite stage, parasites start to colonize the duodenum and upper jejunum where they attach to the intestinal epithelium and replicate vegetatively. Outcome of Giardia infections vary between individuals, from self-limiting to chronic, and asymptomatic to severely symptomatic infection, with unspecific gastrointestinal complaints. One proposed mechanism for pathogenesis is the breakdown of intestinal barrier function. This has been studied by analyzing trans-epithelial electric resistances (TEER) or by indicators of epithelial permeability using labeled sugar compounds in in vitro cell culture systems, mouse models or human biopsies and epidemiological studies. Here, we discuss the results obtained mainly with epithelial cell models to highlight contradictory findings. We relate published studies to our own findings that suggest a lack of barrier compromising activities of recent G. duodenalis isolates of assemblage A, B, and E in a Caco-2 model system. We propose that this epithelial cell model be viewed as mimicking asymptomatic infection. This view will likely lead to a more informative use of the model if emphasis is shifted from aiming to identify Giardia virulence factors to defining non-parasite factors that arguably appear to be more decisive for disease. PMID:29018775

Distinct Mutations Led to Inactivation of Type 1 Fimbriae Expression in Shigella spp.

PubMed Central

Bravo, Verónica; Puhar, Andrea; Sansonetti, Philippe; Parsot, Claude; Toro, Cecilia S.

2015-01-01

Shigella spp. are responsible for bacillary dysentery in humans. The acquisition or the modification of the virulence plasmid encoding factors promoting entry of bacteria into and dissemination within epithelial cells was a critical step in the evolution of these bacteria from their Escherichia coli ancestor(s). Incorporation of genomic islands (GI) and gene inactivation also shaped interactions between these pathogens and their human host. Sequence analysis of the GI inserted next to the leuX tRNA gene in S. boydii, S. dysenteriae, S. flexneri, S. sonnei and enteroinvasive E. coli (EIEC) suggests that this region initially carried the fec, yjhATS and fim gene clusters. The fim cluster encoding type I fimbriae is systematically inactivated in both reference strains and clinical isolates and distinct mutations are responsible for this inactivation in at least three phylogenetic groups. To investigate consequences of the presence of fimbriae on the outcome of the interaction of Shigella with host cells, we used a S. flexneri strain harboring a plasmid encoding the E. coli fim operon. Production of fimbriae by this recombinant strain increased the ability of bacteria to adhere to and enter into epithelial cells and had no effect on their ability to disseminate from cell to cell. The observations that production of type I fimbriae increases invasion of epithelial cells and that independent mutations abolish fimbriae production in Shigella suggest that these mutations correspond to pathoadaptive events. PMID:25811616

ICAM-1 Targeting of Doxorubicin-Loaded PLGA Nanoparticles to Lung Epithelial Cells

PubMed Central

Chuda, Chittasupho; Sheng-Xue, Xie; Abdulgader, Baoum; Tatyana, Yakovleva; Teruna, Siahaan J.; Cory, Berkland

2009-01-01

Interaction of leukocyte function associated antigen-1 (LFA-1) on T-lymphoctytes and intercellular adhesion molecule-1 (ICAM-1) on epithelial cells controls leukocyte adhesion, spreading, and extravasation. This process plays an important role in leukocyte recruitment to a specific site of inflammation and has been indentified as a biomarker for certain types of carcinomas. Cyclo-(1,12)-PenITDGEATDSGC (cLABL) has been shown to inhibit LFA-1 and ICAM-1 interaction via binding to ICAM-1. In addition, cLABL has been shown to internalize after binding ICAM-1. The possibility of using cLABL conjugated nanoparticles (cLABL-NP) as a targeted and controlled release drug delivery system has been investigated in this study. The cLABL peptide was conjugated to a modified Pluronic® surfactant on poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles. The cLABL-NP showed more rapid cellular uptake by A549 lung epithelial cells compared to nanoparticles without peptide. The specificity of ICAM-1 mediated internalization was confirmed by blocking the uptake of cLABL-NP to ICAM-1 using free cLABL peptide to block the binding of cLABL-NP to ICAM-1 on the cell surface. Cell studies suggested that cLABL-NPs targeted encapsulated doxorubicin to ICAM-1 expressing cells. Cytotoxicity assay confirmed the activity of the drug incorporated in nanoparticles. Sustained release of doxorubicin afforded by PLGA nanoparticles may enable cLABL-NP as a targeted, controlled release drug delivery system. PMID:19429421

Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

PubMed Central

Klein, Hélène; Garneau, Line; Trinh, Nguyen Thu Ngan; Privé, Anik; Dionne, François; Goupil, Eugénie; Thuringer, Dominique; Parent, Lucie; Brochiero, Emmanuelle; Sauvé, Rémy

2009-01-01

The vectorial transport of ions and water across epithelial cells depends to a large extent on the coordination of the apical and basolateral ion fluxes with energy supply. In this work we provide the first evidence for a regulation by the 5′-AMP-activated protein kinase (AMPK) of the calcium-activated potassium channel KCa3.1 expressed at the basolateral membrane of a large variety of epithelial cells. Inside-out patch-clamp experiments performed on human embryonic kidney (HEK) cells stably transfected with KCa3.1 first revealed a decrease in KCa3.1 activity following the internal addition of AMP at a fixed ATP concentration. This effect was dose dependent with half inhibition at 140 μM AMP in 1 mM ATP. Evidence for an interaction between the COOH-terminal region of KCa3.1 and the γ1-subunit of AMPK was next obtained by two-hybrid screening and pull-down experiments. Our two-hybrid analysis confirmed in addition that the amino acids extending from Asp380 to Ala400 in COOH-terminal were essential for the interaction AMPK-γ1/KCa3.1. Inside-out experiments on cells coexpressing KCa3.1 with the dominant negative AMPK-γ1-R299G mutant showed a reduced sensitivity of KCa3.1 to AMP, arguing for a functional link between KCa3.1 and the γ1-subunit of AMPK. More importantly, coimmunoprecipitation experiments carried out on bronchial epithelial NuLi cells provided direct evidence for the formation of a KCa3.1/AMPK-γ1 complex at endogenous AMPK and KCa3.1 expression levels. Finally, treating NuLi monolayers with the membrane permeant AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) caused a significant decrease of the KCa3.1-mediated short-circuit currents, an effect reversible by coincubation with the AMPK inhibitor Compound C. These observations argue for a regulation of KCa3.1 by AMPK in a functional epithelium through protein/protein interactions involving the γ1-subunit of AMPK. PMID:19052260

Mechanisms and regulation of polymorphonuclear leukocyte and eosinophil adherence to human airway epithelial cells.

PubMed

Jagels, M A; Daffern, P J; Zuraw, B L; Hugli, T E

1999-09-01

Polymorphonuclear leukocytes (PMN) and eosinophils (Eos) are important cellular participants in a variety of acute and chronic inflammatory reactions in the airway. Histologic evidence has implicated direct interactions between these two subsets of leukocytes and airway epithelial cells during inflammation. A comprehensive characterization and comparison of physiologic stimuli and adhesion molecule involvement in granulocyte-epithelial-cell interactions done with nontransformed human airway epithelial cells has not been reported. We therefore examined the regulation and biochemical mechanisms governing granulocyte-epithelial-cell adhesion, using either purified PMN or Eos and primary cultures of human bronchial epithelial cells (HBECs). We investigated the involvement of a number of proinflammatory signals associated with allergic and nonallergic airway inflammation, as well as the contribution of several epithelial and leukocyte adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and members of the beta(1), beta(2), and beta(7) integrin families. ICAM-1 was expressed at low levels on cultured HBECs and was markedly upregulated after stimulation with interferon (IFN)-gamma or, to a lesser extent, with tumor necrosis factor (TNF)-alpha or interleukin (IL)-1. VCAM-1 was not present on resting HBECs, and was not upregulated after stimulation with IFN-gamma, IL-1, IL-4, or TNF-alpha. PMN adhesion to HBECs could be induced either through activation of PMN with IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), or C5a, but not with IL-5 or by preactivation of HBECs with TNF-alpha or IFN-gamma. Blocking antibody studies indicated that PMN-HBEC adherence depended on beta(2) integrins, primarily alpha(M)beta(2) (Mac-1). Adherence of Eos to HBECs could be induced through activation of Eos with IL-5, GM-CSF, or C5a, but not with IL-8 or by prior activation of HBECs with TNF-alpha of IFN-gamma. Maximal adhesion of Eos and PMN required pretreatment of HBECs with either TNF-alpha or IFN-gamma in addition to leukocyte activation. Adherence of Eos to unstimulated HBECs was mediated through both beta(1) and beta(2) integrins, whereas adhesion of Eos to activated HBECs was dominated by beta(2) integrins. Adhesion of both Eos and PMN was inhibited by treatment of HBECs with blocking antibodies to ICAM-1. Differential utilization of beta(1) and beta(2) integrins by Eos, depending on the activation state of the epithelium, is a novel finding and may affect activation and/or recruitment of Eos in airway tissue. Mechanisms of adhesion of HBECs to Eos and PMN, as evidenced by the different responsiveness of the two latter types of cells to IL-8 and IL-5, may account for a prevalence of Eos over PMN in certain airway diseases.

Curcumin targets fibroblast–tumor cell interactions in oral squamous cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dudás, József, E-mail: jozsef.dudas@i-med.ac.at; Fullár, Alexandra, E-mail: fullarsz@gmail.com; 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, 1085 Budapest

Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of OSCC tumor cells. We hypothesized that Curcumin targets this dynamic mutual interaction between CAFs and tumor cells. Normal and 2 μM Curcumin-treated co-culture were performed for 4 days, followed by analysis of tumor cell invasivity, mRNA/protein expression of EMT-markers and mediators, activity measure of matrix metalloproteinase 9 (MMP-9), and western blot analysis of signal transduction in tumor cells and fibroblasts. In Curcumin-treated co-culture, in tumor cells, the levels of nuclear factormore » κB (NFκBα) and early response kinase (ERK)—decreased, in fibroblasts, integrin αv protein synthesis decreased compared to corresponding cells in normal co-culture. The signal modulatory changes induced by Curcumin caused decreased release of EMT-mediators in CAFs and reversal of EMT in tumor cells, which was associated with decreased invasion. These data confirm the palliative potential of Curcumin in clinical application. - Graphical abstract: Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of tumor cells. Curcumin targets this dynamic mutual interaction between CAFs and tumor cells by inhibiting the production of EMT mediators in CAFs and by modification of intracellular signaling in tumor cells. This causes less invasivity and reversal of EMT in tumor cells. Highlights: ► Curcumin targets tumor–fibroblast interaction in head and neck cancer. ► Curcumin suppresses mediators of epithelial–mesenchymal transition. ► Curcumin decreases the invasivity of tumor cells.« less

MUC1 (CD227) interacts with lck tyrosine kinase in Jurkat lymphoma cells and normal T cells.

PubMed

Mukherjee, P; Tinder, T L; Basu, G D; Gendler, S J

2005-01-01

MUC1 (CD227) is a large transmembrane epithelial mucin glycoprotein, which is aberrantly overexpressed in most adenocarcinomas and is a target for immune therapy for epithelial tumors. Recently, MUC1 has been detected in a variety of hematopoietic cell malignancies including T and B cell lymphomas and myelomas; however, its function in these cells is not clearly defined. Using the Jurkat T cell lymphoma cell line and normal human T cells, we demonstrate that MUC1 is not only expressed in these cells but is also phosphorylated upon T cell receptor (TCR) ligation and associates with the Src-related T cell tyrosine kinase, p56lck. Upon TCR-mediated activation of Jurkat cells, MUC1 is found in the low-density membrane fractions, where linker of T cell activation is contained. Abrogation of MUC1 expression in Jurkat cells by MUC1-specific small interfering RNA resulted in defects in TCR-mediated downstream signaling events associated with T cell activation. These include reduction in Ca2+ influx and extracellular signal-regulated kinase 1/2 phosphorylation, leading to a decrease in CD69 expression, proliferation, and interleukin-2 production. These results suggest a regulatory role of MUC1 in modulating proximal signal transduction events through its interaction with proteins of the activation complex.

A Novel Role for Integrin-linked Kinase in Epithelial Sheet Morphogenesis

PubMed Central

Vespa, Alisa; D'Souza, Sudhir J.A.; Dagnino, Lina

2005-01-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca2+ triggers formation of cell–cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca2+ treatment of keratinocytes induces rapid (≤1 h) translocation to the cell membrane of the adherens junction (AJ) proteins E-cadherin and β-catenin. This is followed by slower (>6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell–cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/β-catenin to cell borders, precluding Ca2+-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell–cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell–cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration. PMID:15975904

Maintenance of Taste Organs Is Strictly Dependent on Epithelial Hedgehog/GLI Signaling.

PubMed

Ermilov, Alexandre N; Kumari, Archana; Li, Libo; Joiner, Ariell M; Grachtchouk, Marina A; Allen, Benjamin L; Dlugosz, Andrzej A; Mistretta, Charlotte M

2016-11-01

For homeostasis, lingual taste papilla organs require regulation of epithelial cell survival and renewal, with sustained innervation and stromal interactions. To investigate a role for Hedgehog/GLI signaling in adult taste organs we used a panel of conditional mouse models to manipulate GLI activity within epithelial cells of the fungiform and circumvallate papillae. Hedgehog signaling suppression rapidly led to taste bud loss, papilla disruption, and decreased proliferation in domains of papilla epithelium that contribute to taste cells. Hedgehog responding cells were eliminated from the epithelium but retained in the papilla stromal core. Despite papilla disruption and loss of taste buds that are a major source of Hedgehog ligand, innervation to taste papillae was maintained, and not misdirected, even after prolonged GLI blockade. Further, vimentin-positive fibroblasts remained in the papilla core. However, retained innervation and stromal cells were not sufficient to maintain taste bud cells in the context of compromised epithelial Hedgehog signaling. Importantly taste organ disruption after GLI blockade was reversible in papillae that retained some taste bud cell remnants where reactivation of Hedgehog signaling led to regeneration of papilla epithelium and taste buds. Therefore, taste bud progenitors were either retained during epithelial GLI blockade or readily repopulated during recovery, and were poised to regenerate taste buds once Hedgehog signaling was restored, with innervation and papilla connective tissue elements in place. Our data argue that Hedgehog signaling is essential for adult tongue tissue maintenance and that taste papilla epithelial cells represent the key targets for physiologic Hedgehog-dependent regulation of taste organ homeostasis. Because disruption of GLI transcriptional activity in taste papilla epithelium is sufficient to drive taste organ loss, similar to pharmacologic Hedgehog pathway inhibition, the findings suggest that taste alterations in cancer patients using systemic Hedgehog pathway inhibitors result principally from interruption of signaling activity in taste papillae.

Maintenance of Taste Organs Is Strictly Dependent on Epithelial Hedgehog/GLI Signaling

PubMed Central

Mistretta, Charlotte M.

2016-01-01

For homeostasis, lingual taste papilla organs require regulation of epithelial cell survival and renewal, with sustained innervation and stromal interactions. To investigate a role for Hedgehog/GLI signaling in adult taste organs we used a panel of conditional mouse models to manipulate GLI activity within epithelial cells of the fungiform and circumvallate papillae. Hedgehog signaling suppression rapidly led to taste bud loss, papilla disruption, and decreased proliferation in domains of papilla epithelium that contribute to taste cells. Hedgehog responding cells were eliminated from the epithelium but retained in the papilla stromal core. Despite papilla disruption and loss of taste buds that are a major source of Hedgehog ligand, innervation to taste papillae was maintained, and not misdirected, even after prolonged GLI blockade. Further, vimentin-positive fibroblasts remained in the papilla core. However, retained innervation and stromal cells were not sufficient to maintain taste bud cells in the context of compromised epithelial Hedgehog signaling. Importantly taste organ disruption after GLI blockade was reversible in papillae that retained some taste bud cell remnants where reactivation of Hedgehog signaling led to regeneration of papilla epithelium and taste buds. Therefore, taste bud progenitors were either retained during epithelial GLI blockade or readily repopulated during recovery, and were poised to regenerate taste buds once Hedgehog signaling was restored, with innervation and papilla connective tissue elements in place. Our data argue that Hedgehog signaling is essential for adult tongue tissue maintenance and that taste papilla epithelial cells represent the key targets for physiologic Hedgehog-dependent regulation of taste organ homeostasis. Because disruption of GLI transcriptional activity in taste papilla epithelium is sufficient to drive taste organ loss, similar to pharmacologic Hedgehog pathway inhibition, the findings suggest that taste alterations in cancer patients using systemic Hedgehog pathway inhibitors result principally from interruption of signaling activity in taste papillae. PMID:27893742

Endorsing cellular competitiveness in aberrant epithelium of oral submucous fibrosis progression: neighbourhood analysis of immunohistochemical attributes.

PubMed

Anura, Anji; Kazi, Anees; Pal, Mousumi; Paul, Ranjan Rashmi; Sengupta, Sanghamitra; Chatterjee, Jyotirmoy

2018-04-23

Epithelial abnormality during the transformation of oral submucous fibrosis (OSF) into oral squamous cell carcinoma has been well studied and documented. However, the differential contribution of atrophy and hyperplasia for malignant potentiality of OSF is yet to be resolved. Existing diagnostic conjectures lack precise diagnostic attributes which may be effectively resolved by substantiation of specific molecular pathology signatures. Present study elucidates existence of cellular competitiveness in OSF conditions using computer-assisted neighbourhood analysis in quantitative immunohistochemistry (IHC) framework. The concept of field cancerization was contributory in finding correspondence among neighbouring cells of epithelial layers with reference to differential expression of cardinal cancer-related genes [c-Myc (oncogene), p53 (tumour suppressor), and HIF-1α (hypoxia regulator)] which are known to be important sensors in recognizing cellular competitive interface. Our analyses indicate that different states of OSF condition may be associated with different forms of competitiveness within epithelial neighbouring cells which might be responsible to shape the present and future of the pre-malignant condition. Analytical findings indicated association of atrophic epithelium with stress-driven competitive environment having low c-Myc, high-p53, and stable HIF-1α (the looser cells) which undergo apoptosis. Whereas, the cells with high c-Myc + (winner cells) give rise to hyperplastic epithelium via possible mutation in p53. The epithelial dysplasia plausibly occurs due to clonal expansion of c-Myc and p53 positive supercompetitor cells. Present study proposes quantitative IHC along with neighbourhood analysis which might help us to dig deeper on to the interaction among epithelial cell population to provide a better understanding of field cancerization and malignant transformation of pre-malignancy.

Uropathogenic E. coli Exploit CEA to Promote Colonization of the Urogenital Tract Mucosa

PubMed Central

Muenzner, Petra; Kengmo Tchoupa, Arnaud; Klauser, Benedikt; Brunner, Thomas; Putze, Johannes; Dobrindt, Ulrich; Hauck, Christof R.

2016-01-01

Attachment to the host mucosa is a key step in bacterial pathogenesis. On the apical surface of epithelial cells, members of the human carcinoembryonic antigen (CEA) family are abundant glycoproteins involved in cell-cell adhesion and modulation of cell signaling. Interestingly, several gram-negative bacterial pathogens target these receptors by specialized adhesins. The prototype of a CEACAM-binding pathogen, Neisseria gonorrhoeae, utilizes colony opacity associated (Opa) proteins to engage CEA, as well as the CEA-related cell adhesion molecules CEACAM1 and CEACAM6 on human epithelial cells. By heterologous expression of neisserial Opa proteins in non-pathogenic E. coli we find that the Opa protein-CEA interaction is sufficient to alter gene expression, to increase integrin activity and to promote matrix adhesion of infected cervical carcinoma cells and immortalized vaginal epithelial cells in vitro. These CEA-triggered events translate in suppression of exfoliation and improved colonization of the urogenital tract by Opa protein-expressing E. coli in CEA-transgenic compared to wildtype mice. Interestingly, uropathogenic E. coli expressing an unrelated CEACAM-binding protein of the Afa/Dr adhesin family recapitulate the in vitro and in vivo phenotype. In contrast, an isogenic strain lacking the CEACAM-binding adhesin shows reduced colonization and does not suppress epithelial exfoliation. These results demonstrate that engagement of human CEACAMs by distinct bacterial adhesins is sufficient to blunt exfoliation and to promote host infection. Our findings provide novel insight into mucosal colonization by a common UPEC pathotype and help to explain why human CEACAMs are a preferred epithelial target structure for diverse gram-negative bacteria to establish a foothold on the human mucosa. PMID:27171273

Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties

PubMed Central

Gudjonsson, Thorarinn; Villadsen, René; Nielsen, Helga Lind; Rønnov-Jessen, Lone; Bissell, Mina J.; Petersen, Ole William

2002-01-01

The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC+) and epithelial-specific antigen (ESA+) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC−/ESA+). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC+/ESA+ epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC−/ESA+ epithelial cell line was able to generate itself as well as MUC+/ESA+ epithelial cells and Thy-1+/α-smooth muscle actin+ (ASMA+) myoepithelial cells. The MUC−/ESA+ epithelial cell line further differed from the MUC+/ESA+ epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC+/ESA+ epithelial cell line formed acinus-like spheres. In contrast, the MUC−/ESA+ epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by inoculating the extracellular matrix-embedded cells subcutaneously in nude mice. Thus, MUC−/ESA+ epithelial cells within the luminal epithelial lineage may function as precursor cells of terminal duct lobular units in the human breast. PMID:11914275

Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies.

PubMed

Moura, Marcos de Assis; Amendoeira, Maria Regina Reis; Barbosa, Helene Santos

2009-09-01

The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.

Mesenchymal-epithelial interaction techniques

PubMed Central

Baskin, Lawrence

2016-01-01

This paper reviews the importance of mesenchymal-epithelial interactions in development and gives detailed technical protocols for investigating these interactions. Successful analysis of mesenchymal-epithelial interactions requires knowing the ages in which embryonic, neonatal and adult organs can be separated into mesenchymal and epithelial tissues. Methods for separation of mesenchymal and epithelial and preparation of tissue recombinants are described. PMID:26610327

Factor h and properdin recognize different epitopes on renal tubular epithelial heparan sulfate.

PubMed

Zaferani, Azadeh; Vivès, Romain R; van der Pol, Pieter; Navis, Gerjan J; Daha, Mohamed R; van Kooten, Cees; Lortat-Jacob, Hugues; Seelen, Marc A; van den Born, Jacob

2012-09-07

During proteinuria, renal tubular epithelial cells become exposed to ultrafiltrate-derived serum proteins, including complement factors. Recently, we showed that properdin binds to tubular heparan sulfates (HS). We now document that factor H also binds to tubular HS, although to a different epitope than properdin. Factor H was present on the urinary side of renal tubular cells in proteinuric, but not in normal renal tissues and colocalized with properdin in proteinuric kidneys. Factor H dose-dependently bound to proximal tubular epithelial cells (PTEC) in vitro. Preincubation of factor H with exogenous heparin and pretreatment of PTECs with heparitinase abolished the binding to PTECs. Surface plasmon resonance experiments showed high affinity of factor H for heparin and HS (K(D) values of 32 and 93 nm, respectively). Using a library of HS-like polysaccharides, we showed that chain length and high sulfation density are the most important determinants for glycosaminoglycan-factor H interaction and clearly differ from properdin-heparinoid interaction. Coincubation of properdin and factor H did not hamper HS/heparin binding of one another, indicating recognition of different nonoverlapping epitopes on HS/heparin by factor H and properdin. Finally we showed that certain low anticoagulant heparinoids can inhibit properdin binding to tubular HS, with a minor effect on factor H binding to tubular HS. As a result, these heparinoids can control the alternative complement pathway. In conclusion, factor H and properdin interact with different HS epitopes of PTECs. These interactions can be manipulated with some low anticoagulant heparinoids, which can be important for preventing complement-derived tubular injury in proteinuric renal diseases.

Cold plasma selectivity in the interaction with various types of the cells

NASA Astrophysics Data System (ADS)

Volotskova, Olga; Stepp, Mary Ann; Keidar, Michael

2011-10-01

Present research in the area of cold atmospheric plasma (CAP) demonstrates great potential in various areas including medicine and biology. Depending on their configuration they can be used for wound healing, sterilization, targeted cell/tissue removal, and cancer treatments. Here we explore potential mechanisms by which CAP alters cell migration and influences cell adhesion. The migration studies are focused on the CAP interaction with fibroblasts and corneal epithelial cells. Data show that various types of cells have different thresholds (treatment times) required to achieve maximum inhibition of cell migration which is around ~30-40%. Studies to assess the impact of CAP treatment on the activation state of integrins and focal adhesion size by immunofluorescence showed more active b1 integrin on the cell surface and large focal adhesions after CAP treatment. Based on these data, a thermodynamic model is presented to explain how CAP leads to integrin activation and focal adhesion assembly. Also responses of the various types of the cells to the cold plasma treatment on the example of the epithelial keratinocytes, papilloma and carcinoma cells are studied. Cell cycle, migration and cell vitality analysis were performed. The goal of this study is to understand the mechanism by which the CAP jet alters cell migration, influences adhesion and cell survival.

Pathway-specific effect of caffeine on protection against UV irradiation-induced apoptosis in corneal epithelial cells.

PubMed

Wang, Ling; Lu, Luo

2007-02-01

To define the role of molecular interaction between the UV-induced JNK (c-Jun N-terminal kinase) cascade and corneal epithelial cell apoptosis and protection against apoptosis by caffeine. Rabbit and human corneal epithelial cells were cultured in DMEM/F12 medium containing 10% FBS and 5 microg/mL insulin at 37 degrees C in 5% CO(2). DNA fragmentation and ethidium bromide/acridine orange (EB/AO) nuclear staining were performed to detect cell death. Western blot, immunoprecipitation, and kinase assays were used to measure UV-induced mitogen-activated protein (MAP) kinase activity. UV irradiation-induced apoptosis through apoptosis signal-regulating kinase 1 (ASK1) and MAKK4 (SEK1) upstream from JNK was caffeine sensitive. Caffeine (1,3,7-trimethylxanthine), an agent that is one of the most popular additions to food consumed in the world and a potential enhancer of chemotherapy, effectively protected corneal epithelial cells against apoptosis by its specific effect on the JNK cascade. Theophylline (1,3-dimethylxanthine) exhibited an effect similar to that of caffeine on prevention of UV irradiation-induced apoptosis. However, alterations of either intracellular cAMP or Ca(2+) levels did not alter the effect of caffeine on the JNK signaling pathway. In addition, the blockade of PI3K-like kinases by wortmannin had no impact on the protective effect of caffeine against UV irradiation-induced apoptosis, suggesting that the protective effect of caffeine acts through a specific mechanism involving UV irradiation-induced activation of ASK1 and SEK1. In contrast, caffeine had no effects on melphalan-, hyperosmotic stress-, or IL-1beta-induced activation of the JNK signaling pathway in these cells. UV irradiation stress-induced activation of the ASK1-SEK1-JNK signaling pathway leading to apoptosis is a caffeine-sensitive process, and caffeine, as a multifunctional agent in cells, can specifically interact with the pathway to protect against apoptosis.

Experimental and theoretical investigations concerning a frequency filter behavior of the human retina regarding electric pulse currents. Ph.D. Thesis

NASA Technical Reports Server (NTRS)

Meier-Koll, A.

1979-01-01

Investigation involving patients with injuries in the visual nervous system are discussed. This led to the identification of the epithelial ganglion of the retina as a frequency filter. Threshold curves of the injured visual organs were compared with threshold curves obtained with a control group as a basis for identification. A model which considers the epithelial ganglion as a homogeneous cell layer in which adjacent neurons interact is discussed. It is shown the behavior of the cells against alternating exciting currents can be explained.

Mechanistic insights into the impact of Cold Atmospheric Pressure Plasma on human epithelial cell lines

NASA Astrophysics Data System (ADS)

Dezest, Marlène; Chavatte, Laurent; Bourdens, Marion; Quinton, Damien; Camus, Mylène; Garrigues, Luc; Descargues, Pascal; Arbault, Stéphane; Burlet-Schiltz, Odile; Casteilla, Louis; Clément, Franck; Planat, Valérie; Bulteau, Anne-Laure

2017-01-01

Compelling evidence suggests that Cold Atmospheric Pressure Plasma (CAPP) has potential as a new cancer therapy. However, knowledge about cellular signaling events and toxicity subsequent to plasma treatment is still poorly documented. The aim of this study was to focus on the interaction between 3 different types of plasma (He, He-O2, He-N2) and human epithelial cell lines to gain better insight into plasma-cell interaction. We provide evidence that reactive oxygen and nitrogen species (RONS) are inducing cell death by apoptosis and that the proteasome, a major intracellular proteolytic system which is important for tumor cell growth and survival, is a target of (He or He-N2) CAPP. However, RONS are not the only actors involved in cell death; electric field and charged particles could play a significant role especially for He-O2 CAPP. By differential label-free quantitative proteomic analysis we found that CAPP triggers antioxidant and cellular defense but is also affecting extracellular matrix in keratinocytes. Moreover, we found that malignant cells are more resistant to CAPP treatment than normal cells. Taken together, our findings provide insight into potential mechanisms of CAPP-induced proteasome inactivation and the cellular consequences of these events.

Scribble is required for normal epithelial cell–cell contacts and lumen morphogenesis in the mammalian lung

PubMed Central

Yates, Laura L.; Schnatwinkel, Carsten; Hazelwood, Lee; Chessum, Lauren; Paudyal, Anju; Hilton, Helen; Romero, M. Rosario; Wilde, Jonathan; Bogani, Debora; Sanderson, Jeremy; Formstone, Caroline; Murdoch, Jennifer N.; Niswander, Lee A.; Greenfield, Andy; Dean, Charlotte H.

2013-01-01

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell–cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical–basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, ‘open’ lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in ScribCrc/Crc lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell–cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell–cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion. PMID:23195221

Towards optimization of an organotypic assay system that imitates human hair follicle-like epithelial-mesenchymal interactions.

PubMed

Havlickova, B; Bíró, T; Mescalchin, A; Arenberger, P; Paus, R

2004-10-01

Human hair growth can currently be studied in vitro by the use of organ-cultured scalp hair follicles (HFs). However, simplified organotypic systems are needed for dissecting the underlying epithelial-mesenchymal interactions and as screening tools for candidate hair growth-modulatory agents. To optimize the design and culture conditions of previously published organotypic systems that imitate epithelial-mesenchymal interactions in the human HF as closely as possible. Continuous submerged organotypic 'sandwich' cultures were established. These consist of a pseudodermis (collagen I mixed with and contracted by human interfollicular dermal fibroblasts) on which one of two upper layers is placed: either a mixture of Matrigel basement membrane matrix (BD Biosciences, Bedford, MA, U.S.A.) and follicular dermal papilla fibroblasts (DPC), with outer root sheath keratinocytes (ORSK) layered on the top ('layered' system), or a mixture of Matrigel, DPC and ORSK ('mixed' system). Morphological and functional characteristics of these 'folliculoid sandwiches' were then assessed by routine histology, histomorphometry and immunohistochemistry. In both 'layered' and 'mixed' systems, the ORSK formed spheroid epithelial cell aggregates, which retained their characteristic keratin expression pattern (i.e. cytokeratin 6). In the 'mixed' sandwich model the size of the epithelial cell aggregates was smaller, but the numbers of ORSK were significantly higher than in the 'layered' model at day 14 in the culture. ORSK proliferated better in the 'mixed' than in the 'layered' sandwich system, regardless of the calcium or serum content of the media, whereas apoptosis of ORSK was lowest in the 'mixed' system in serum-free, low calcium medium. The kinetics of proliferation and apoptosis of DPC, which retained their characteristic expression of versican, were similar in both systems. However, proliferation and apoptosis of DPC were higher in the presence of serum and/or under high calcium conditions. Our results underscore the importance of structural design and medium composition for epithelial-mesenchymal interactions as they occur in the human HF. Specifically, we report a new organotypic submerged 'folliculoid sandwich' system with serum-free, low calcium medium and a mixture of interacting human DPC and ORSK, which offers several advantages over previously available assays. This system allows the standardized assessment of the effects of a test agent on the proliferation, apoptosis and key marker expression of human ORSK and DPC under substantially simplified in vitro conditions which approximate the in vivo situation.

A Selected Lactobacillus rhamnosus Strain Promotes EGFR-Independent Akt Activation in an Enterotoxigenic Escherichia coli K88-Infected IPEC-J2 Cell Model.

PubMed

Zhang, Wei; Zhu, Yao-Hong; Yang, Jin-Cai; Yang, Gui-Yan; Zhou, Dong; Wang, Jiu-Feng

2015-01-01

Enterotoxigenic Escherichia coli (ETEC) are important intestinal pathogens that cause diarrhea in humans and animals. Although probiotic bacteria may protect against ETEC-induced enteric infections, the underlying mechanisms are unknown. In this study, porcine intestinal epithelial J2 cells (IPEC-J2) were pre-incubated with and without Lactobacillus rhamnosus ATCC 7469 and then exposed to F4+ ETEC. Increases in TLR4 and NOD2 mRNA expression were observed at 3 h after F4+ ETEC challenge, but these increases were attenuated by L. rhamnosus treatment. Expression of TLR2 and NOD1 mRNA was up-regulated in cells pre-treated with L. rhamnosus. Pre-treatment with L. rhamnosus counteracted F4+ ETEC-induced increases in TNF-α concentration. Increased PGE2. concentrations were observed in cells infected with F4+ ETEC and in cells treated with L. rhamnosus only. A decrease in phosphorylated epidermal growth factor receptor (EGFR) was observed at 3 h after F4+ ETEC challenge in cells treated with L. rhamnosus. Pre-treatment with L. rhamnosus enhanced Akt phosphorylation and increased ZO-1 and occludin protein expression. Our findings suggest that L. rhamnosus protects intestinal epithelial cells from F4+ ETEC-induced damage, partly through the anti-inflammatory response involving synergism between TLR2 and NOD1. In addition, L. rhamnosus promotes EGFR-independent Akt activation, which may activate intestinal epithelial cells in response to bacterial infection, in turn increasing tight junction integrity and thus enhancing the barrier function and restricting pathogen invasion. Pre-incubation with L. rhamnosus was superior to co-incubation in reducing the adhesion of F4+ ETEC to IPEC-J2 cells and subsequently attenuating F4+ ETEC-induced mucin layer destruction and suppressing apoptosis. Our data indicate that a selected L. rhamnosus strain interacts with porcine intestinal epithelial cells to maintain the epithelial barrier and promote intestinal epithelial cell activation in response to bacterial infection, thus protecting cells from the deleterious effects of F4+ ETEC.

A Selected Lactobacillus rhamnosus Strain Promotes EGFR-Independent Akt Activation in an Enterotoxigenic Escherichia coli K88-Infected IPEC-J2 Cell Model

PubMed Central

Yang, Jin-Cai; Yang, Gui-Yan; Zhou, Dong; Wang, Jiu-Feng

2015-01-01

Enterotoxigenic Escherichia coli (ETEC) are important intestinal pathogens that cause diarrhea in humans and animals. Although probiotic bacteria may protect against ETEC-induced enteric infections, the underlying mechanisms are unknown. In this study, porcine intestinal epithelial J2 cells (IPEC-J2) were pre-incubated with and without Lactobacillus rhamnosus ATCC 7469 and then exposed to F4+ ETEC. Increases in TLR4 and NOD2 mRNA expression were observed at 3 h after F4+ ETEC challenge, but these increases were attenuated by L. rhamnosus treatment. Expression of TLR2 and NOD1 mRNA was up-regulated in cells pre-treated with L. rhamnosus. Pre-treatment with L. rhamnosus counteracted F4+ ETEC-induced increases in TNF-α concentration. Increased PGE2. concentrations were observed in cells infected with F4+ ETEC and in cells treated with L. rhamnosus only. A decrease in phosphorylated epidermal growth factor receptor (EGFR) was observed at 3 h after F4+ ETEC challenge in cells treated with L. rhamnosus. Pre-treatment with L. rhamnosus enhanced Akt phosphorylation and increased ZO-1 and occludin protein expression. Our findings suggest that L. rhamnosus protects intestinal epithelial cells from F4+ ETEC-induced damage, partly through the anti-inflammatory response involving synergism between TLR2 and NOD1. In addition, L. rhamnosus promotes EGFR-independent Akt activation, which may activate intestinal epithelial cells in response to bacterial infection, in turn increasing tight junction integrity and thus enhancing the barrier function and restricting pathogen invasion. Pre-incubation with L. rhamnosus was superior to co-incubation in reducing the adhesion of F4+ ETEC to IPEC-J2 cells and subsequently attenuating F4+ ETEC-induced mucin layer destruction and suppressing apoptosis. Our data indicate that a selected L. rhamnosus strain interacts with porcine intestinal epithelial cells to maintain the epithelial barrier and promote intestinal epithelial cell activation in response to bacterial infection, thus protecting cells from the deleterious effects of F4+ ETEC. PMID:25915861

Active tension network model suggests an exotic mechanical state realized in epithelial tissues

NASA Astrophysics Data System (ADS)

Noll, Nicholas; Mani, Madhav; Heemskerk, Idse; Streichan, Sebastian J.; Shraiman, Boris I.

2017-12-01

Mechanical interactions play a crucial role in epithelial morphogenesis, yet understanding the complex mechanisms through which stress and deformation affect cell behaviour remains an open problem. Here we formulate and analyse the active tension network (ATN) model, which assumes that the mechanical balance of cells within a tissue is dominated by cortical tension and introduces tension-dependent active remodelling of the cortex. We find that ATNs exhibit unusual mechanical properties. Specifically, an ATN behaves as a fluid at short times, but at long times supports external tension like a solid. Furthermore, an ATN has an extensively degenerate equilibrium mechanical state associated with a discrete conformal--`isogonal'--deformation of cells. The ATN model predicts a constraint on equilibrium cell geometries, which we demonstrate to approximately hold in certain epithelial tissues. We further show that isogonal modes are observed in the fruit fly embryo, accounting for the striking variability of apical areas of ventral cells and helping understand the early phase of gastrulation. Living matter realizes new and exotic mechanical states, the study of which helps to understand biological phenomena.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes

PubMed Central

Truong-Bolduc, Q. C.; Khan, N. S.

2016-01-01

ABSTRACT We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes. PMID:27956597

Control of epithelial immune-response genes and implications for airway immunity and inflammation.

PubMed

Holtzman, M J; Look, D C; Sampath, D; Castro, M; Koga, T; Walter, M J

1998-01-01

A major goal of our research is to understand how immune cells (especially T cells) infiltrate the pulmonary airway during host defense and inflammatory disease (especially asthma). In that context, we have proposed that epithelial cells lining the airway provide critical biochemical signals for immune-cell influx and activation and that this epithelial-immune cell interaction is a critical feature of airway inflammation and hyperreactivity. In this brief report, we describe our progress in defining a subset of epithelial immune-response genes the expression of which is coordinated for viral defense both directly in response to replicating virus and indirectly under the control of a specific interferon-gamma signal transduction pathway featuring the Stat1 transcription factor as a critical relay signal between cytoplasm and nucleus. Unexpectedly, the same pathway is also activated during asthmatic airway inflammation in a setting where there is no apparent infection and no increase in interferon-gamma levels. The findings provide the first evidence of an overactive Stat1-dependent gene network in asthmatic airways and a novel molecular link between mucosal immunity and inflammation. The findings also offer the possibility that overactivity of Stat1-dependent genes might augment a subsequent T helper cell (Th1)-type response to virus or might combine with a heightened Th2-type response to allergen to account for more severe exacerbations of asthma.

Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion.

PubMed

Kasurinen, Stefanie; Happo, Mikko S; Rönkkö, Teemu J; Orasche, Jürgen; Jokiniemi, Jorma; Kortelainen, Miika; Tissari, Jarkko; Zimmermann, Ralf; Hirvonen, Maija-Riitta; Jalava, Pasi I

2018-01-01

In vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity. We assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups. We detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses. All of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity.

Host-pathogen interactions in bovine mammary epithelial cells and HeLa cells by Staphylococcus aureus isolated from subclinical bovine mastitis.

PubMed

Castilho, Ivana G; Dantas, Stéfani Thais Alves; Langoni, Hélio; Araújo, João P; Fernandes, Ary; Alvarenga, Fernanda C L; Maia, Leandro; Cagnini, Didier Q; Rall, Vera L M

2017-08-01

Staphylococcus aureus is a common pathogen that causes subclinical bovine mastitis due to several virulence factors. In this study, we analyzed S. aureus isolates collected from the milk of cows with subclinical mastitis that had 8 possible combinations of bap, icaA, and icaD genes, to determine their capacity to produce biofilm on biotic (bovine primary mammary epithelial cells and HeLa cells) and abiotic (polystyrene microplates) surfaces, and their ability to adhere to and invade these cells. We also characterized isolates for microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and agr genes, and for their susceptibility to cefquinome sulfate in the presence of biofilm. All isolates adhered to and invaded both cell types, but invasion indexes were higher in bovine primary mammary epithelial cells. Using tryptic soy broth + 1% glucose on abiotic surfaces, 5 out of 8 isolates were biofilm producers, but only the bap + icaA + icaD + isolate was positive in Dulbecco's Modified Eagle's medium. The production of biofilm on biotic surfaces occurred only with this isolate and only on HeLa cells, because the invasion index for bovine primary mammary epithelial cells was too high, making it impossible to use these cells in this assay. Of the 5 biofilm producers in tryptic soy broth + 1% glucose, 4 presented with the bap/fnbA/clfA/clfB/eno/fib/ebpS combination, and all were protected from cefquinome sulfate. We found no predominance of any agr group. The high invasive potential of S. aureus made it impossible to observe biofilm in bovine primary mammary epithelial cells, and we concluded that cells with lower invasion rates, such as HeLa cells, were more appropriate for this assay. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion

PubMed Central

Happo, Mikko S.; Rönkkö, Teemu J.; Orasche, Jürgen; Jokiniemi, Jorma; Kortelainen, Miika; Tissari, Jarkko; Zimmermann, Ralf; Hirvonen, Maija-Riitta; Jalava, Pasi I.

2018-01-01

Background In vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity. Methods We assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups. Results We detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses. Conclusions All of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity. PMID:29466392

Host response to bovine respiratory pathogens.

PubMed

Czuprynski, Charles J

2009-12-01

Bovine respiratory disease (BRD) involves complex interactions amongst viral and bacterial pathogens that can lead to intense pulmonary inflammation (fibrinous pleuropneumonia). Viral infection greatly increases the susceptibility of cattle to secondary infection of the lung with bacterial pathogens like Mannheimia haemolytica and Histophilus somni. The underlying reason for this viral/bacterial synergism, and the manner in which cattle respond to the virulence strategies of the bacterial pathogens, is incompletely understood. Bovine herpesvirus type 1 (BHV-1) infection of bronchial epithelial cells in vitro enhances the binding of M. haemolytica and triggers release of inflammatory mediators that attract and enhance binding of neutrophils. An exotoxin (leukotoxin) released from M. haemolytica further stimulates release of inflammatory mediators and causes leukocyte death. Cattle infected with H. somni frequently display vasculitis. Exposure of bovine endothelial cells to H. somnii or its lipooligosaccharide (LOS) increases endothelium permeability, and makes the surface of the endothelial cells pro-coagulant. These processes are amplified in the presence of platelets. The above findings demonstrate that bovine respiratory pathogens (BHV-1, M. haemolytica and H. somni) interact with leukocytes and other cells (epithelial and endothelial cells) leading to the inflammation that characterizes BRD.

Visualisation and quantification of intracellular interactions of Neisseria meningitidis and human α-actinin by confocal imaging.

PubMed

Murillo, Isabel; Virji, Mumtaz

2010-10-24

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors(1). This process leads to bacterial invasion of endothelial cells(2). More recently, we observed an interaction of Opc with a 100 kDa protein found in whole cell lysates of human cells(3). We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as α-actinin. As no surface expressed α-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and α-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with α-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalra, Rajkumar S., E-mail: renu-wadhwa@aist.go.jp; Wadhwa, Renu, E-mail: renu-wadhwa@aist.go.jp

2015-02-27

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylationmore » target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.« less

Influenza A virus nucleoprotein induces apoptosis in human airway epithelial cells: implications of a novel interaction between nucleoprotein and host protein Clusterin

PubMed Central

Tripathi, S; Batra, J; Cao, W; Sharma, K; Patel, J R; Ranjan, P; Kumar, A; Katz, J M; Cox, N J; Lal, R B; Sambhara, S; Lal, S K

2013-01-01

Apoptosis induction is an antiviral host response, however, influenza A virus (IAV) infection promotes host cell death. The nucleoprotein (NP) of IAV is known to contribute to viral pathogenesis, but its role in virus-induced host cell death was hitherto unknown. We observed that NP contributes to IAV infection induced cell death and heterologous expression of NP alone can induce apoptosis in human airway epithelial cells. The apoptotic effect of IAV NP was significant when compared with other known proapoptotic proteins of IAV. The cell death induced by IAV NP was executed through the intrinsic apoptosis pathway. We screened host cellular factors for those that may be targeted by NP for inducing apoptosis and identified human antiapoptotic protein Clusterin (CLU) as a novel interacting partner. The interaction between IAV NP and CLU was highly conserved and mediated through β-chain of the CLU protein. Also CLU was found to interact specifically with IAV NP and not with any other known apoptosis modulatory protein of IAV. CLU prevents induction of the intrinsic apoptosis pathway by binding to Bax and inhibiting its movement into the mitochondria. We found that the expression of IAV NP reduced the association between CLU and Bax in mammalian cells. Further, we observed that CLU overexpression attenuated NP-induced cell death and had a negative effect on IAV replication. Collectively, these findings indicate a new function for IAV NP in inducing host cell death and suggest a role for the host antiapoptotic protein CLU in this process. PMID:23538443

Breast Cancer Research at NASA

NASA Technical Reports Server (NTRS)

1998-01-01

Epithelial cell monoculture: Long-term growth of human mammary epithelial cells (HMEC) grown in monoculture as 3-dimensional constructions in the presence of attachment beads in the NASA Bioreactor. A: A typical construct about 3.5 mm (less than 1/8th inch) in diameter with slightly dehydrted, crinkled beads contained on the surface as well as within the 3-dimensional structure. B: The center of these constructs is hollow. Crinkling of the beads causes a few to fall out, leaving crater-like impressiions in the construct. The central impression shows a small hole that accesses the hollow center of the construct. C: A closeup view of the cells and the hole the central impression. D: Closer views of cells in the construct showing sell-to-cell interactions. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Robert Richmond, NASA/Marshall Space Flight Center (MSFC).

Microgravity

NASA Image and Video Library

1998-10-10

Epithelial cell monoculture: Long-term growth of human mammary epithelial cells (HMEC) grown in monoculture as 3-dimensional constructions in the presence of attachment beads in the NASA Bioreactor. A: A typical construct about 3.5 mm (less than 1/8th inch) in diameter with slightly dehydrted, crinkled beads contained on the surface as well as within the 3-dimensional structure. B: The center of these constructs is hollow. Crinkling of the beads causes a few to fall out, leaving crater-like impressiions in the construct. The central impression shows a small hole that accesses the hollow center of the construct. C: A closeup view of the cells and the hole the central impression. D: Closer views of cells in the construct showing sell-to-cell interactions. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Robert Richmond, NASA/Marshall Space Flight Center (MSFC).

Bacillus megaterium SF185 induces stress pathways and affects the cell cycle distribution of human intestinal epithelial cells.

PubMed

Di Luccia, B; D'Apuzzo, E; Varriale, F; Baccigalupi, L; Ricca, E; Pollice, A

2016-09-01

The interaction between the enteric microbiota and intestinal cells often involves signal molecules that affect both microbial behaviour and host responses. Examples of such signal molecules are the molecules secreted by bacteria that induce quorum sensing mechanisms in the producing microorganism and signal transduction pathways in the host cells. The pentapeptide competence and sporulation factor (CSF) of Bacillus subtilis is a well characterized quorum sensing factor that controls competence and spore formation in the producing bacterium and induces cytoprotective heat shock proteins in intestinal epithelial cells. We analysed several Bacillus strains isolated from human ileal biopsies of healthy volunteers and observed that some of them were unable to produce CSF but still able to act in a CSF-like fashion on model intestinal epithelial cells. One of those strains belonging to the Bacillus megaterium species secreted at least two factors with effects on intestinal HT29 cells: a peptide smaller than 3 kDa able to induce heat shock protein 27 (hsp27) and p38-MAPK, and a larger molecule able to induce protein kinase B (PKB/Akt) with a pro-proliferative effect.

Solo and keratin filaments regulate epithelial tubule morphology.

PubMed

Nishimura, Ryosuke; Kato, Kagayaki; Fujiwara, Sachiko; Ohashi, Kazumasa; Mizuno, Kensaku

2018-04-28

Epithelial tubules, consisting of the epithelial cell sheet with a central lumen, are the basic structure of many organs. Mechanical forces play an important role in epithelial tubulogenesis; however, little is known about the mechanisms controlling the mechanical forces during epithelial tubule morphogenesis. Solo (also known as ARHGEF40) is a RhoA-targeting guanine-nucleotide exchange factor that is involved in mechanical force-induced RhoA activation and stress fiber formation. Solo binds to keratin-8/keratin-18 (K8/K18) filaments, and this interaction plays a crucial role in mechanotransduction. In this study, we examined the roles of Solo and K8/K18 filaments in epithelial tubulogenesis using MDCK cells cultured in 3D collagen gels. Knockdown of either Solo or K18 resulted in rounder tubules with increased lumen size, indicating that Solo and K8/K18 filaments play critical roles in forming the elongated morphology of epithelial tubules. Moreover, knockdown of Solo or K18 decreased the level of diphosphorylated myosin light chain (a marker of contractile force) at the luminal and outer surfaces of tubules, suggesting that Solo and K8/K18 filaments are involved in the generation of the myosin II-mediated contractile force during epithelial tubule morphogenesis. In addition, K18 filaments were normally oriented along the long axis of the tubule, but knockdown of Solo perturbed their orientation. These results suggest that Solo plays crucial roles in forming the elongated morphology of epithelial tubules and in regulating myosin II activity and K18 filament organization during epithelial tubule formation.

The role of pleiotrophin and β-catenin in fetal lung development

PubMed Central

2010-01-01

Mammalian lung development is a complex biological process, which is temporally and spatially regulated by growth factors, hormones, and extracellular matrix proteins. Abnormal changes of these molecules often lead to impaired lung development, and thus pulmonary diseases. Epithelial-mesenchymal interactions are crucial for fetal lung development. This paper reviews two interconnected pathways, pleiotrophin and Wnt/β-catenin, which are involved in fibroblast and epithelial cell communication during fetal lung development. PMID:20565841

Host-parasite interactions and purifying selection in a microsporidian parasite of honey bees

USDA-ARS?s Scientific Manuscript database

Nosema ceranae is a microsporidian parasite that infects honey bee mid-gut epithelial cells. Infection can impair honey bee cognitive function, shorten lifespan, suppress the innate immune response and inhibit the apoptosis of infected gut cells. However, the virulence factors of this parasite are s...

Alterations of the Bone Marrow Microenvironment Contribute to Prostate Cancer Skeletal Metastasis

DTIC Science & Technology

2012-05-01

potential of tumor cells ( Hiraki et al. 2002, Liao & McCauley 2006). Apoptosis induced by disrupted epithelial cell–matrix interactions was described...enhances survival of chondrocytes under conditions that promote apoptotic cell death. Molecular and Cellular Biology 15 4064–4075. Hiraki A, Ueoka H, Bessho

Interactions between the PDZ domains of Bazooka (Par-3) and phosphatidic acid: in vitro characterization and role in epithelial development.

PubMed

Yu, Cao Guo; Harris, Tony J C

2012-09-01

Bazooka (Par-3) is a conserved polarity regulator that organizes molecular networks in a wide range of cell types. In epithelia, it functions as a plasma membrane landmark to organize the apical domain. Bazooka is a scaffold protein that interacts with proteins through its three PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains and other regions. In addition, Bazooka has been shown to interact with phosphoinositides. Here we show that the Bazooka PDZ domains interact with the negatively charged phospholipid phosphatidic acid immobilized on solid substrates or in liposomes. The interaction requires multiple PDZ domains, and conserved patches of positively charged amino acid residues appear to mediate the interaction. Increasing or decreasing levels of diacylglycerol kinase or phospholipase D-enzymes that produce phosphatidic acid-reveal a role for phosphatidic acid in Bazooka embryonic epithelial activity but not its localization. Mutating residues implicated in phosphatidic acid binding revealed a possible role in Bazooka localization and function. These data implicate a closer connection between Bazooka and membrane lipids than previously recognized. Bazooka polarity landmarks may be conglomerates of proteins and plasma membrane lipids that modify each other's activities for an integrated effect on cell polarity.

Biomechanical regulation of cell orientation and fate

PubMed Central

Lopez, JI; Mouw, JK; Weaver, VM

2009-01-01

Biomechanical regulation of tumor phenotypes have been noted for several decades, yet the function of mechanics in the co-evolution of the tumor epithelium and altered cancer extracellular matrix has not been appreciated until fairly recently. In this review, we examine the dynamic interaction between the developing epithelia and the extracellular matrix, and discuss how similar interactions are exploited by the genetically modified epithelium during tumor progression. We emphasize the process of mechanoreciprocity, which is a phenomenon observed during epithelial transformation, in which tension generated within the extracellular microenvironment induce and cooperate with opposing reactive forces within transformed epithelium to drive tumor progression and metastasis. We highlight the importance of matrix remodeling, and present a new, emerging paradigm that underscores the importance of tissue morphology as a key regulator of epithelial cell invasion and metastasis. PMID:19029939

Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability.

PubMed

Looi, Kevin; Troy, Niamh M; Garratt, Luke W; Iosifidis, Thomas; Bosco, Anthony; Buckley, Alysia G; Ling, Kak-Ming; Martinovich, Kelly M; Kicic-Starcevich, Elizabeth; Shaw, Nicole C; Sutanto, Erika N; Zosky, Graeme R; Rigby, Paul J; Larcombe, Alexander N; Knight, Darryl A; Kicic, Anthony; Stick, Stephen M

2016-10-11

No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function. To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function. Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID 50 ) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT 2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests. HRV-1B infection affected viability that was both time and TCID 50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID 50 , while a significant decrease in all three TJ protein expressions occurred at higher TCID 50 . Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection. HRV-1B infection directly alters human airway epithelial TJ expression leading to increased epithelial permeability potentially via an antiviral response of IL-15.

Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

NASA Technical Reports Server (NTRS)

Goodwin, T. J.; McCarthy, M.; Lin, Y-H

2006-01-01

In vitro three-dimensional (3D) human broncho-epithelial (HBE) tissue-like assemblies (3D HBE TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and parainfluenza virus type 3 (wtPIV3 JS) and the detection of membrane bound glycoproteins over time confirm productive infections with both viruses. Therefore, TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host's immune system.

Mutation in the peb1A Locus of Campylobacter jejuni Reduces Interactions with Epithelial Cells and Intestinal Colonization of Mice

PubMed Central

Pei, Zhiheng; Burucoa, Christophe; Grignon, Bernadette; Baqar, Shahida; Huang, Xiao-Zhe; Kopecko, Dennis J.; Bourgeois, A. L.; Fauchere, Jean-Louis; Blaser, Martin J.

1998-01-01

Campylobacter jejuni is one of the leading causes of bacterial diarrhea throughout the world. We previously found that PEB1 is a homolog of cluster 3 binding proteins of bacterial ABC transporters and that a C. jejuni adhesin, cell-binding factor 1 (CBF1), if not identical to, contains PEB1. A single protein migrating at approximately 27 to 28 kDa was recognized by anti-CBF1 and anti-PEB1. To determine the role that the operon encoding PEB1 plays in C. jejuni adherence, peb1A, the gene encoding PEB1, was disrupted in strain 81-176 by insertion of a kanamycin resistance gene through homologous recombination. Inactivation of this operon completely abolished expression of CBF1, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. In comparison to the wild-type strain, the mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture. Mouse challenge studies showed that the rate and duration of intestinal colonization by the mutant were significantly lower and shorter than with the wild-type strain. In summary, PEB1 is identical to a previously identified cell-binding factor, CBF1, in C. jejuni, and the peb1A locus plays an important role in epithelial cell interactions and in intestinal colonization in a mouse model. PMID:9488379

The role of surface carbohydrates on the interaction of microconidia of Trichophyton mentagrophytes with epithelial cells.

PubMed

Esquenazi, Daniele; de Souza, Wanderley; Alviano, Celuta Sales; Rozental, Sonia

2003-03-20

The presence of carbohydrate-binding adhesins on the microconidia of Trichophyton mentagrophytes surface and their role on cellular interactions were investigated. Flow cytometry showed that this fungus recognizes the sugars mannose and galactose. The binding was inhibited by the addition of methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside, and showed higher fluorescence intensity at 37 degrees C than 28 degrees C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature of the microconidia adhesin. The interaction of the fungus to Chinese hamster ovary epithelial cells and its glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, which express mannose and galactose, respectively, as the terminal carbohydrate on the cell surface. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside to the interaction medium, pretreatment of Lec1 and Lec2 cells with lectins Concanavalina A and Arachis hypogaea and pretreatment with sodium periodate decreased the adhesion and the endocytic index. Examination of thin section by transmission electron microscopy showed that after fungal ingestion by Lec2 cells the fungi are enclosed in a 'loose'-type vacuole while the other cells are found within a 'tight'-type membrane-bound cytoplasmic vacuole. Our results suggest the occurrence of carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. This may have a role in the adhesion process during the infectious process of dermatophytosis.

Adenomatous Polyposis Coli-Mediated Accumulation of Abasic DNA Lesions Lead to Cigarette Smoke Condensate-Induced Neoplastic Transformation of Normal Breast Epithelial Cells1

PubMed Central

Jaiswal, Aruna S; Panda, Harekrushna; Pampo, Christine A; Siemann, Dietmar W; Gairola, C Gary; Hromas, Robert; Narayan, Satya

2013-01-01

Adenomatous polyposis coli (APC) is a multifunctional protein having diverse cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation, and apoptosis. Recently, we found a new role of APC in base excision repair (BER) and showed that it interacts with DNA polymerase β and 5′-flap endonuclease 1 and interferes in BER. Previously, we have also reported that cigarette smoke condensate (CSC) increases expression of APC and enhances the growth of normal human breast epithelial (MCF10A) cells in vitro. In the present study, using APC overexpression and knockdown systems, we have examined the molecular mechanisms by which CSC and its major component, Benzo[α]pyrene, enhances APC-mediated accumulation of abasic DNA lesions, which is cytotoxic and mutagenic in nature, leading to enhanced neoplastic transformation of MCF10A cells in an orthotopic xenograft model. PMID:23555190

PreImplantation Factor in endometriosis: A potential role in inducing immune privilege for ectopic endometrium

PubMed Central

Scarpellini, Fabio; Marconi, Daniela; Rossi, Gabriele; Simmilion, Cedric; Mueller, Michael D.; Barnea, Eytan R.

2017-01-01

Endometriosis is a chronic inflammatory condition characterised by the growth of endometrial epithelial and stromal cells outside the uterine cavity. In addition to Sampson’s theory of retrograde menstruation, endometriosis pathogenesis is facilitated by a privileged inflammatory microenvironment, with T regulatory FoxP3+ expressing T cells (Tregs) being a significant factor. PreImplantation Factor (PIF) is a peptide essential for pregnancy recognition and development. An immune modulatory function of the synthetic PIF analog (sPIF) has been successfully confirmed in multiple animal models. We report that PIF is expressed in the epithelial ectopic cells in close proximity to FoxP3+ stromal cells. We provide evidence that PIF interacts with FoxP3+ cells and modulates cell viability, dependent on cell source and presence of inflammatory mediators. Our finding represent a novel PIF-based mechanism in endometriosis that has potential for novel therapeutics. PMID:28902871

Inflammation and angiogenesis in fibrotic lung disease.

PubMed

Keane, Michael P; Strieter, Robert M; Lynch, Joseph P; Belperio, John A

2006-12-01

The pathogenesis of pulmonary fibrosis is poorly understood. Although inflammation has been presumed to have an important role in the development of fibrosis this has been questioned recently, particularly with regard to idiopathic pulmonary fibrosis (IPF). It is, however, increasingly recognized that the polarization of the inflammatory response toward a type 2 phenotype supports fibroproliferation. Increased attention has been on the role of noninflammatory structural cells such as the fibroblast, myofibroblast, epithelial cell, and endothelial cells. Furthermore, the origin of these cells appears to be multifactorial and includes resident cells, bone marrow-derived cells, and epithelial to mesenchymal transition. Increasing evidence supports the presence of vascular remodeling in fibrotic lung disease, although the precise role in the pathogenesis of fibrosis remains to be determined. Therefore, the pathogenesis of pulmonary fibrosis is complex and involves the interaction of multiple cell types and compartments within the lung.

Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments

PubMed Central

Miller, Daniel P.; Hutcherson, Justin A.; Wang, Yan; Nowakowska, Zuzanna M.; Potempa, Jan; Yoder-Himes, Deborah R.; Scott, David A.; Whiteley, Marvin; Lamont, Richard J.

2017-01-01

Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism. PMID:28900609

Epithelial Control of Gut-Associated Lymphoid Tissue Formation through p38α-Dependent Restraint of NF-κB Signaling.

PubMed

Caballero-Franco, Celia; Guma, Monica; Choo, Min-Kyung; Sano, Yasuyo; Enzler, Thomas; Karin, Michael; Mizoguchi, Atsushi; Park, Jin Mo

2016-03-01

The protein kinase p38α mediates cellular responses to environmental and endogenous cues that direct tissue homeostasis and immune responses. Studies of mice lacking p38α in several different cell types have demonstrated that p38α signaling is essential to maintaining the proliferation-differentiation balance in developing and steady-state tissues. The mechanisms underlying these roles involve cell-autonomous control of signaling and gene expression by p38α. In this study, we show that p38α regulates gut-associated lymphoid tissue (GALT) formation in a noncell-autonomous manner. From an investigation of mice with intestinal epithelial cell-specific deletion of the p38α gene, we find that p38α serves to limit NF-κB signaling and thereby attenuate GALT-promoting chemokine expression in the intestinal epithelium. Loss of this regulation results in GALT hyperplasia and, in some animals, mucosa-associated B cell lymphoma. These anomalies occur independently of luminal microbial stimuli and are most likely driven by direct epithelial-lymphoid interactions. Our study illustrates a novel p38α-dependent mechanism preventing excessive generation of epithelial-derived signals that drive lymphoid tissue overgrowth and malignancy. Copyright © 2016 by The American Association of Immunologists, Inc.

Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells.

PubMed

Lim, HooiCheng; Yu, Chun-Ying; Jou, Tzuu-Shuh

2017-11-01

Establishment of apical-basal polarity, through correct targeting of polarity determinants to distinct domains of the plasma membrane, is a fundamental process for the development of functioning epithelial tubules. Here we report that galectin (Gal)-8 regulates apical-basal polarity of Madin-Darby canine kidney (MDCK) cells via apical targeting of 135-kDa glycoprotein (Gp135). Gal-8 interacts with newly synthesized Gp135 in a glycan-dependent manner. Gal-8 knockdown induces aberrant lumens at the lateral domain and mistargeting of Gp135 to this structure, thus disrupting the kidney epithelial polarity of MDCK cells, which organize lumens at the apical surface. The O -glycosylation deletion mutant of Gp135 phenocopies the effect of Gal-8 knockdown, which suggests that Gal-8 is the decoding machinery for the apical sorting signals of Gp135 residing at its O -glycosylation-rich region. Collectively, our results reveal a new role of Gal-8 in the development of luminal organs by regulating targeting of apical polarity protein Gp135.-Lim, H., Yu, C.-Y., Jou, T.-S. Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells. © FASEB.

Zyxin regulates migration of renal epithelial cells through activation of hepatocyte nuclear factor-1β.

PubMed

Choi, Yun-Hee; McNally, Brian T; Igarashi, Peter

2013-07-01

Hepatocyte nuclear factor-1β (HNF-1β) is an epithelial tissue-specific transcription factor that regulates gene expression in the kidney, liver, pancreas, intestine, and other organs. Mutations of HNF-1β in humans produce renal cysts and congenital kidney anomalies. Here, we identify the LIM-domain protein zyxin as a novel binding partner of HNF-1β in renal epithelial cells. Zyxin shuttles to the nucleus where it colocalizes with HNF-1β. Immunoprecipitation of zyxin in leptomycin B-treated cells results in coprecipitation of HNF-1β. The protein interaction requires the second LIM domain of zyxin and two distinct domains of HNF-1β. Overexpression of zyxin stimulates the transcriptional activity of HNF-1β, whereas small interfering RNA silencing of zyxin inhibits HNF-1β-dependent transcription. Epidermal growth factor (EGF) induces translocation of zyxin into the nucleus and stimulates HNF-1β-dependent promoter activity. The EGF-mediated nuclear translocation of zyxin requires activation of Akt. Expression of dominant-negative mutant HNF-1β, knockdown of zyxin, or inhibition of Akt inhibits EGF-stimulated cell migration. These findings reveal a novel pathway by which extracellular signals are transmitted to the nucleus to regulate the activity of a transcription factor that is essential for renal epithelial differentiation.

Curli Temper Adherence of Escherichia coli O157:H7 to Squamous Epithelial Cells from the Bovine Recto-Anal Junction in a Strain-Dependent Manner

PubMed Central

Carter, Michelle Q.; Sharma, Vijay K.; Stasko, Judith A.; Giron, Jorge A.

2016-01-01

ABSTRACT Our recent studies have shown that intimin and the locus of enterocyte effacement-encoded proteins do not play a role in Escherichia coli O157:H7 (O157) adherence to the bovine recto-anal junction squamous epithelial (RSE) cells. To define factors that play a contributory role, we investigated the role of curli, fimbrial adhesins commonly implicated in adherence to various fomites and plant and human epithelial cells, in O157 adherence to RSE cells. Specifically, we examined (i) wild-type strains of O157; (ii) curli variants of O157 strains; (iii) isogenic curli deletion mutants of O157; and (iv) adherence inhibition of O157 using anti-curlin sera. Results of these experiments conducted under stringent conditions suggest that curli do not solely contribute to O157 adherence to RSE cells and in fact demonstrate a modulating effect on O157 adherence to RSE cells in contrast to HEp-2 cells (human epidermoid carcinoma of the larynx cells with HeLa contamination). The absence of curli and presence of blocking anti-curli antibodies enhanced O157-RSE cell interactions among some strains, thus alluding to a spatial, tempering effect of curli on O157 adherence to RSE cells when present. At the same time, the presence or absence of curli did not alter RSE cell adherence patterns of another O157 strain. These observations are at variance with the reported role of curli in O157 adherence to human cell lines such as HEp-2 and need to be factored in when developing anti-adherence modalities for preharvest control of O157 in cattle. IMPORTANCE This study demonstrated that O157 strains interact with epithelial cells in a host-specific manner. The fimbriae/adhesins that are significant for adherence to human cell lines may not have a role or may have a modulating role in O157 adherence to bovine cells. Targeting such adhesins may not prevent O157 attachment to bovine cells but instead may result in improved adherence. Hence, conducting host-specific evaluations is critical when selecting targets for O157 control strategies. PMID:27742683

Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kikuta, Kazuhiro; Masamune, Atsushi, E-mail: amasamune@med.tohoku.ac.jp; Watanabe, Takashi

2010-12-17

Research highlights: {yields} Recent studies have shown that pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer. {yields} Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and scattered, fibroblast-like appearance. {yields} PSCs decreased the expression of epithelial markers but increased that of mesenchymal markers, along with increased migration. {yields} This study suggests epithelial-mesenchymal transition as a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells. -- Abstract: The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There ismore » accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Because epithelial-mesenchymal transition (EMT) plays a critical role in the progression of pancreatic cancer, we hypothesized that PSCs promote EMT in pancreatic cancer cells. Panc-1 and SUIT-2 pancreatic cancer cells were indirectly co-cultured with human PSCs isolated from patients undergoing operation for pancreatic cancer. The expression of epithelial and mesenchymal markers was examined by real-time PCR and immunofluorescent staining. The migration of pancreatic cancer cells was examined by scratch and two-chamber assays. Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and a scattered, fibroblast-like appearance. The expression of E-cadherin, cytokeratin 19, and membrane-associated {beta}-catenin was decreased, whereas vimentin and Snail (Snai-1) expression was increased more in cancer cells co-cultured with PSCs than in mono-cultured cells. The migration of pancreatic cancer cells was increased by co-culture with PSCs. The PSC-induced decrease of E-cadherin expression was not altered by treatment with anti-TGF-{beta}-neutralizing antibody, excluding a central role of TGF-{beta} in this process. In conclusion, PSCs promoted EMT in pancreatic cancer cells suggesting a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells.« less

Innate cell communication kick-starts pathogen-specific immunity

PubMed Central

Rivera, Amariliz; Siracusa, Mark C.; Yap, George S.; Gause, William C.

2016-01-01

Innate cells are responsible for the rapid recognition of infection and mediate essential mechanisms of pathogen elimination, and also facilitate adaptive immune responses. We review here the numerous intricate interactions among innate cells that initiate protective immunity. The efficient eradication of pathogens depends on the coordinated actions of multiple cells, including innate cells and epithelial cells. Rather than acting as isolated effector cells, innate cells are in constant communication with other responding cells of the immune system, locally and distally. These interactions are critically important for the efficient control of primary infections as well for the development of ‘trained’ innate cells that facilitate the rapid elimination of homologous or heterologous infections. PMID:27002843

Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri.

PubMed

Matsuo, Yosuke; Miyoshi, Yukihiro; Okada, Sanae; Satoh, Eiichi

2012-01-01

A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion.

Transient Overexpression of Gremlin Results in Epithelial Activation and Reversible Fibrosis in Rat Lungs

PubMed Central

Farkas, Laszlo; Farkas, Daniela; Gauldie, Jack; Warburton, David; Shi, Wei; Kolb, Martin

2011-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease of the lung parenchyma, without curative treatment. Gremlin is a bone morphogenic protein (BMP) antagonist, its expression being increased in IPF lungs. It has been implicated in promoting myofibroblast accumulation, likely through inhibited fibroblast apoptosis and epithelial-to-mesenchymal transition. In the current study, we examined the effects of selective adenovirus-mediated overexpression of Gremlin in rat lungs. We show that transient Gremlin overexpression results in activation of alveolar epithelial cells with proliferation and apoptosis, as well as partly reversible lung fibrosis. We found myofibroblasts arranged in fibroblastic foci. Fibroblast proliferation occurred delayed as compared with epithelial changes. Fibrotic pathology significantly declined after Day 14, the reversal being associated with an increase of the epithelium-protective element, fibroblast growth factor (FGF)–10. Our data indicate that Gremlin-mediated BMP inhibition results in activation of epithelial cells and transient fibrosis, but also induction of epithelium-protective FGF10. A Gremlin–BMP–FGF10 loop may explain these results, and demonstrate that the interactions between different factors are quite complex in fibrotic lung disease. Increased Gremlin expression in human IPF tissue may be an expression of continuing epithelial injury, and Gremlin may be part of activated repair mechanisms. PMID:20705941

Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells.

PubMed

Cong, Xin; Zhang, Yan; Li, Jing; Mei, Mei; Ding, Chong; Xiang, Ruo-Lan; Zhang, Li-Wei; Wang, Yun; Wu, Li-Ling; Yu, Guang-Yan

2015-06-15

The epithelial cholinergic system plays an important role in water, ion and solute transport. Previous studies have shown that activation of muscarinic acetylcholine receptors (mAChRs) regulates paracellular transport of epithelial cells; however, the underlying mechanism is still largely unknown. Here, we found that mAChR activation by carbachol and cevimeline reduced the transepithelial electrical resistance (TER) and increased the permeability of paracellular tracers in rat salivary epithelial SMG-C6 cells. Carbachol induced downregulation and redistribution of claudin-4, but not occludin or ZO-1 (also known as TJP1). Small hairpin RNA (shRNA)-mediated claudin-4 knockdown suppressed, whereas claudin-4 overexpression retained, the TER response to carbachol. Mechanistically, the mAChR-modulated claudin-4 properties and paracellular permeability were triggered by claudin-4 phosphorylation through ERK1/2 (also known as MAPK3 and MAPK1, respectively). Mutagenesis assay demonstrated that S195, but not S199, S203 or S207, of claudin-4, was the target for carbachol. Subsequently, the phosphorylated claudin-4 interacted with β-arrestin2 and triggered claudin-4 internalization through the clathrin-dependent pathway. The internalized claudin-4 was further degraded by ubiquitylation. Taken together, these findings suggested that claudin-4 is required for mAChR-modulated paracellular permeability of epithelial cells through an ERK1/2, β-arrestin2, clathrin and ubiquitin-dependent signaling pathway. © 2015. Published by The Company of Biologists Ltd.

Interaction between APC and Fen1 during breast carcinogenesis

PubMed Central

Narayan, Satya; Jaiswal, Aruna S.; Law, Brian K.; Mohammed, Kamal A.; Sharma, Arun K.; Hromas, Robert A.

2016-01-01

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER – adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) – promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents. PMID:27088617

Interaction between APC and Fen1 during breast carcinogenesis.

PubMed

Narayan, Satya; Jaiswal, Aruna S; Law, Brian K; Kamal, Mohammad A; Sharma, Arun K; Hromas, Robert A

2016-05-01

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER - adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) - promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents. Published by Elsevier B.V.

Evaluation and Comparison of the Biopathology of Collagen and Inflammation in the Extracellular Matrix of Oral Epithelial Dysplasias and Inflammatory Fibrous Hyperplasia Using Picrosirius Red Stain and Polarising Microscopy: A Preliminary Study

PubMed Central

Varghese, Soma Susan; Sarojini, Sreenivasan Bargavan; George, Giju Baby; Vinod, Sankar; Mathew, Philips; Babu, Anulekh; Sebastian, Joseph

2015-01-01

Background: The role of tumour inflammation and the dysplastic epithelial-stromal interactions on the nature of collagen fibres in the extracellular matrix of dysplastic epithelium is not fully understood. The present study was aimed to evaluate and compare the inflammation and pathological stromal collagen (loosely packed thin disorganized collagen) present in mild, moderate and severe epithelial dysplasias with that of inflammatory fibrous hyperplasias. The basement membrane intactness of epithelial dysplasias was also evaluated to determine if dysplastic epithelial mesenchymal interaction has any role in the integrity of stromal collagen in epithelial dysplasia. Methods: Oral epithelial dysplasias, inflammatory fibrous hyperplasia and normal oral mucosal samples were used for the study. Packing, thickness and orientation of collagen fibres in mild, moderate and severe grades of oral epithelial dysplasias (n = 24), inflammatory fibrous hyperplasia (n = 8) and normal oral mucosal samples (n = 8) were analysed based on the polarisation of collagen fibres in picrosirius red polarising stain under polarising microscope. Results: All the grades of epithelial dysplasias showed greenish yellow birefringence confirming the presence of loosely arranged pathological collagen in the presence of moderate inflammation. All the cases of inflammatory fibrous hyperplasia showed red polarisation hue and moderate inflammation. A statistically significant difference was found in the packing and orientation of collagen when epithelial dysplasias and inflammatory fibrous hyperplasia were compared (P < 0.01). When the intactness of basement membrane integrity was compared in all the groups of epithelial dysplasia, a statistically significant result was obtained (P < 0.05). Conclusions: Presence of significant amount of loosely packed thin disoriented collagen even in mild epithelial dysplasia suggests that tumourigenic factors are released to connective tissue stroma much earlier than expected. Hence we suggest considering the integrity of extracellular matrix collagen, intactness of basement membrane and inflammation associated with dysplasia along with the anaplasia of epithelial cells in the microscopic assessment of dysplastic epithelium. PMID:26734590

Evaluation and Comparison of the Biopathology of Collagen and Inflammation in the Extracellular Matrix of Oral Epithelial Dysplasias and Inflammatory Fibrous Hyperplasia Using Picrosirius Red Stain and Polarising Microscopy: A Preliminary Study.

PubMed

Varghese, Soma Susan; Sarojini, Sreenivasan Bargavan; George, Giju Baby; Vinod, Sankar; Mathew, Philips; Babu, Anulekh; Sebastian, Joseph

2015-12-01

The role of tumour inflammation and the dysplastic epithelial-stromal interactions on the nature of collagen fibres in the extracellular matrix of dysplastic epithelium is not fully understood. The present study was aimed to evaluate and compare the inflammation and pathological stromal collagen (loosely packed thin disorganized collagen) present in mild, moderate and severe epithelial dysplasias with that of inflammatory fibrous hyperplasias. The basement membrane intactness of epithelial dysplasias was also evaluated to determine if dysplastic epithelial mesenchymal interaction has any role in the integrity of stromal collagen in epithelial dysplasia. Oral epithelial dysplasias, inflammatory fibrous hyperplasia and normal oral mucosal samples were used for the study. Packing, thickness and orientation of collagen fibres in mild, moderate and severe grades of oral epithelial dysplasias (n = 24), inflammatory fibrous hyperplasia (n = 8) and normal oral mucosal samples (n = 8) were analysed based on the polarisation of collagen fibres in picrosirius red polarising stain under polarising microscope. All the grades of epithelial dysplasias showed greenish yellow birefringence confirming the presence of loosely arranged pathological collagen in the presence of moderate inflammation. All the cases of inflammatory fibrous hyperplasia showed red polarisation hue and moderate inflammation. A statistically significant difference was found in the packing and orientation of collagen when epithelial dysplasias and inflammatory fibrous hyperplasia were compared (P < 0.01). When the intactness of basement membrane integrity was compared in all the groups of epithelial dysplasia, a statistically significant result was obtained (P < 0.05). Presence of significant amount of loosely packed thin disoriented collagen even in mild epithelial dysplasia suggests that tumourigenic factors are released to connective tissue stroma much earlier than expected. Hence we suggest considering the integrity of extracellular matrix collagen, intactness of basement membrane and inflammation associated with dysplasia along with the anaplasia of epithelial cells in the microscopic assessment of dysplastic epithelium.

Cellular and molecular mechanisms of tooth root development

PubMed Central

Li, Jingyuan; Parada, Carolina

2017-01-01

ABSTRACT The tooth root is an integral, functionally important part of our dentition. The formation of a functional root depends on epithelial-mesenchymal interactions and integration of the root with the jaw bone, blood supply and nerve innervations. The root development process therefore offers an attractive model for investigating organogenesis. Understanding how roots develop and how they can be bioengineered is also of great interest in the field of regenerative medicine. Here, we discuss recent advances in understanding the cellular and molecular mechanisms underlying tooth root formation. We review the function of cellular structure and components such as Hertwig's epithelial root sheath, cranial neural crest cells and stem cells residing in developing and adult teeth. We also highlight how complex signaling networks together with multiple transcription factors mediate tissue-tissue interactions that guide root development. Finally, we discuss the possible role of stem cells in establishing the crown-to-root transition, and provide an overview of root malformations and diseases in humans. PMID:28143844

Engineering human cell spheroids to model embryonic tissue fusion in vitro

PubMed Central

Wolf, Cynthia J.; Wood, Carmen; Ren, Hongzu; Grindstaff, Rachel; Padgett, William; Swank, Adam; MacMillan, Denise; Fisher, Anna; Winnik, Witold; Abbott, Barbara D.

2017-01-01

Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior in vitro using human cells. We engineered size-controlled human Wharton’s Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications. PMID:28898253

Dlg3 Trafficking and Apical Tight Junction Formation Is Regulated by Nedd4 and Nedd4-2 E3 Ubiquitin Ligases

PubMed Central

Van Campenhout, Claude A.; Eitelhuber, Andrea; Gloeckner, Christian J.; Giallonardo, Patrizia; Gegg, Moritz; Oller, Heide; Grant, Seth G.N.; Krappmann, Daniel; Ueffing, Marius; Lickert, Heiko

2011-01-01

Summary The Drosophila Discs large (Dlg) scaffolding protein acts as a tumor suppressor regulating basolateral epithelial polarity and proliferation. In mammals, four Dlg homologs have been identified; however, their functions in cell polarity remain poorly understood. Here, we demonstrate that the X-linked mental retardation gene product Dlg3 contributes to apical-basal polarity and epithelial junction formation in mouse organizer tissues, as well as to planar cell polarity in the inner ear. We purified complexes associated with Dlg3 in polarized epithelial cells, including proteins regulating directed trafficking and tight junction formation. Remarkably, of the four Dlg family members, Dlg3 exerts a distinct function by recruiting the ubiquitin ligases Nedd4 and Nedd4-2 through its PPxY motifs. We found that these interactions are required for Dlg3 monoubiquitination, apical membrane recruitment, and tight junction consolidation. Our findings reveal an unexpected evolutionary diversification of the vertebrate Dlg family in basolateral epithelium formation. PMID:21920314

Structural centrosome aberrations favor proliferation by abrogating microtubule-dependent tissue integrity of breast epithelial mammospheres

PubMed Central

Schnerch, D; Nigg, E A

2016-01-01

Structural centrosome aberrations are frequently observed in early stage carcinomas, but their role in malignant transformation is poorly understood. Here, we examined the impact of overexpression of Ninein-like protein (Nlp) on the architecture of polarized epithelia in three-dimensional mammospheres. When Nlp was overexpressed to levels resembling those seen in human tumors, it formed striking centrosome-related bodies (CRBs), which sequestered Ninein and affected the kinetics of microtubule (MT) nucleation and release. In turn, the profound reorganization of the MT cytoskeleton resulted in mislocalization of several adhesion and junction proteins as well as the tumor suppressor Scribble, resulting in the disruption of epithelial polarity, cell-cell interactions and mammosphere architecture. Remarkably, cells harboring Nlp-CRBs displayed an enhanced proliferative response to epidermal growth factor. These results demonstrate that structural centrosome aberrations cause not only the disruption of epithelial polarity but also favor overproliferation, two phenotypes typically associated with human carcinomas. PMID:26364601

Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

PubMed

Sun, Zhao; Yu, Wenjie; Sanz Navarro, Maria; Sweat, Mason; Eliason, Steven; Sharp, Thad; Liu, Huan; Seidel, Kerstin; Zhang, Li; Moreno, Myriam; Lynch, Thomas; Holton, Nathan E; Rogers, Laura; Neff, Traci; Goodheart, Michael J; Michon, Frederic; Klein, Ophir D; Chai, Yang; Dupuy, Adam; Engelhardt, John F; Chen, Zhi; Amendt, Brad A

2016-11-15

Sox2 marks dental epithelial stem cells (DESCs) in both mammals and reptiles, and in this article we demonstrate several Sox2 transcriptional mechanisms that regulate dental stem cell fate and incisor growth. Conditional Sox2 deletion in the oral and dental epithelium results in severe craniofacial defects, including impaired dental stem cell proliferation, arrested incisor development and abnormal molar development. The murine incisor develops initially but is absorbed independently of apoptosis owing to a lack of progenitor cell proliferation and differentiation. Tamoxifen-induced inactivation of Sox2 demonstrates the requirement of Sox2 for maintenance of the DESCs in adult mice. Conditional overexpression of Lef-1 in mice increases DESC proliferation and creates a new labial cervical loop stem cell compartment, which produces rapidly growing long tusk-like incisors, and Lef-1 epithelial overexpression partially rescues the tooth arrest in Sox2 conditional knockout mice. Mechanistically, Pitx2 and Sox2 interact physically and regulate Lef-1, Pitx2 and Sox2 expression during development. Thus, we have uncovered a Pitx2-Sox2-Lef-1 transcriptional mechanism that regulates DESC homeostasis and dental development. © 2016. Published by The Company of Biologists Ltd.

Kindlin-2 regulates renal tubular cell plasticity by activation of Ras and its downstream signaling.

PubMed

Wei, Xiaofan; Wang, Xiang; Xia, Yang; Tang, Yan; Li, Feng; Fang, Weigang; Zhang, Hongquan

2014-01-01

Kindlin-2 is an adaptor protein that contributes to renal tubulointerstitial fibrosis (TIF). Epithelial-to-mesenchymal transition (EMT) in tubular epithelial cells was regarded as one of the key events in TIF. To determine whether kindlin-2 is involved in the EMT process, we investigated its regulation of EMT in human kidney tubular epithelial cells (TECs) and explored the underlying mechanism. In this study, we found that overexpression of kindlin-2 suppressed epithelial marker E-cadherin and increased the expression of fibronectin and the myofibroblast marker α-smooth muscle actin (SMA). Kindlin-2 significantly activated ERK1/2 and Akt, and inhibition of ERK1/2 or Akt reversed kindlin-2-induced EMT in human kidney TECs. Mechanistically, kindlin-2 interacted with Ras and son of sevenless (Sos)-1. Furthermore, overexpression of kindlin-2 increased Ras activation through recruiting Sos-1. Treatment with a Ras inhibitor markedly repressed kindlin-2-induced ERK1/2 and Akt activation, leading to restraint of EMT. We further demonstrated that knockdown of kindlin-2 inhibited EGF-induced Ras-Sos-1 interaction, resulting in reduction of Ras activation and suppression of EMT stimulated by EGF. Importantly, we found that depletion of kindlin-2 significantly inhibited activation of ERK1/2 and Akt signaling in mice with unilateral ureteral obstruction. We conclude that kindlin-2, through activating Ras and the downstream ERK1/2 and Akt signaling pathways, plays an important role in regulating renal tubular EMT and could be a potential therapeutic target for the treatment of fibrotic kidney diseases.

RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamaki, Nao; Negishi, Manabu; Katoh, Hironori

2007-08-01

In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as amore » Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85{alpha} and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.« less

MIG-6 negatively regulates STAT3 phosphorylation in uterine epithelial cells

PubMed Central

Yoo, Jung-Yoon; Yang, Woo Sub; Lee, Jae Hee; Kim, Byung Gak; Broaddus, Russell R.; Lim, Jeong M.; Kim, Tae Hoon; Jeong, Jae-Wook

2017-01-01

Endometrial cancer is the most common malignancy of the female genital tract. Progesterone (P4) has been used for several decades in endometrial cancer treatment, especially in women who wish to retain fertility. However, it is unpredictable which patients will respond to P4 treatment and which may have a P4 resistant cancer. Therefore, identifying the mechanism of P4 resistance is essential to improve the therapies for endometrial cancer. Mitogen-inducible gene 6 (Mig-6) is a critical mediator of progesterone receptor (PGR) action in the uterus. In order to study the function of Mig-6 in P4 resistance, we generated a mouse model in which we specifically ablated Mig-6 in uterine epithelial cells using Sprr2f-cre mice (Sprr2fcre+Mig-6f/f). Female mutant mice develop endometrial hyperplasia due to aberrant phosphorylation of STAT3 and proliferation of the endometrial epithelial cells. The results from our immunoprecipitation and cell culture experiments showed that MIG-6 inhibited phosphorylation of STAT3 via protein interactions. Our previous study showed P4 resistance in mice with Mig-6 ablation in Pgr positive cells (Pgrcre/+Mig-6f/f). However, Sprr2fcre+Mig-6f/f mice were P4 responsive. P4 treatment significantly decreased STAT3 phosphorylation and epithelial proliferation in the uterus of mutant mice. We showed that Mig-6 has an important function of tumor suppressor via inhibition of STAT3 phosphorylation in uterine epithelial cells and the anti-tumor effects of P4 are mediated by the endometrial stroma. This data helps to develop a new signaling pathway in the regulation of steroid hormones in the uterus, and to overcome P4 resistance in human reproductive diseases, such as endometrial cancer. PMID:28925396

Bacteria in the vaginal microbiome alter the innate immune response and barrier properties of the human vaginal epithelia in a species-specific manner.

PubMed

Doerflinger, Sylvie Y; Throop, Andrea L; Herbst-Kralovetz, Melissa M

2014-06-15

Bacterial vaginosis increases the susceptibility to sexually transmitted infections and negatively affects women's reproductive health. To investigate host-vaginal microbiota interactions and the impact on immune barrier function, we colonized 3-dimensional (3-D) human vaginal epithelial cells with 2 predominant species of vaginal microbiota (Lactobacillus iners and Lactobacillus crispatus) or 2 prevalent bacteria associated with bacterial vaginosis (Atopobium vaginae and Prevotella bivia). Colonization of 3-D vaginal epithelial cell aggregates with vaginal microbiota was observed with direct attachment to host cell surface with no cytotoxicity. A. vaginae infection yielded increased expression membrane-associated mucins and evoked a robust proinflammatory, immune response in 3-D vaginal epithelial cells (ie, expression of CCL20, hBD-2, interleukin 1β, interleukin 6, interleukin 8, and tumor necrosis factor α) that can negatively affect barrier function. However, P. bivia and L. crispatus did not significantly upregulate pattern-recognition receptor-signaling, mucin expression, antimicrobial peptides/defensins, or proinflammatory cytokines in 3-D vaginal epithelial cell aggregates. Notably, L. iners induced pattern-recognition receptor-signaling activity, but no change was observed in mucin expression or secretion of interleukin 6 and interleukin 8. We identified unique species-specific immune signatures from vaginal epithelial cells elicited by colonization with commensal and bacterial vaginosis-associated bacteria. A. vaginae elicited a signature that is consistent with significant disruption of immune barrier properties, potentially resulting in enhanced susceptibility to sexually transmitted infections during bacterial vaginosis. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cell-matrix adhesion characterization using multiple shear stress zones in single stepwise microchannel

NASA Astrophysics Data System (ADS)

Kim, Min-Ji; Doh, Il; Bae, Gab-Yong; Cha, Hyuk-Jin; Cho, Young-Ho

2014-08-01

This paper presents a cell chip capable to characterize cell-matrix adhesion by monitoring cell detachment rate. The proposed cell chip can supply multiple levels of shear stress in single stepwise microchannel. As epithelial-mesenchymal transition (EMT), one of hallmarks of cancer metastasis is closely associated to the interaction with extracelluar matrix (ECM), we took advantage of two lung cancer cell models with different adhesion properties to ECM depending their epithelial or mesenchymal properties, including the pair of lung cancer cells with (A549sh) or without E-cadherin expression (A549sh-Ecad), which would be optimal model to examine the alteration of adhesion properties after EMT induction. The cell-matrix adhesion resisting to shear stress appeared to be remarkably differed between lung cancer cells. The detachment rate of epithelial-like H358 and mesenchymal-like H460 cells was 53%-80% and 25%-66% in the shear stress range of 34-60 dyn/cm2, respectively. A549sh-Ecad cells exhibits lower detachment rate (5%-9%) compared to A549sh cells (14%-40%). By direct comparison of adhesion between A549sh and A549sh-Ecad, we demonstrated that A549shE-cad to mimic EMT were more favorable to the ECM attachment under the various levels of shear stress. The present method can be applied to quantitative analysis of tumor cell-ECM adhesion.

HTLV-1-infected thymic epithelial cells convey the virus to CD4+ T lymphocytes.

PubMed

Carvalho Barros, Luciana Rodrigues; Linhares-Lacerda, Leandra; Moreira-Ramos, Klaysa; Ribeiro-Alves, Marcelo; Machado Motta, Maria Cristina; Bou-Habib, Dumith Chequer; Savino, Wilson

2017-12-01

The human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD4 + T cells are the main target of HTLV-1, but other cell types are known to be infected, including immature lymphocytes. Developing T cells undergo differentiation in the thymus, through migration and interaction with the thymic microenvironment, in particular with thymic epithelial cells (TEC) the major component of this three dimensional meshwork of non-lymphoid cells. Herein, we show that TEC express the receptors for HTLV-1 and can be infected by this virus through cell-cell contact and by cell-free virus suspensions. The expression of anti-apoptosis, chemokine and adhesion molecules genes are altered in HTLV-1-infected TEC, although gene expression of antigen presentation molecules remained unchanged. Furthermore, HTLV-1-infected TEC transmitted the virus to a CD4 + T cell line and to CD4 + T cells from healthy donors, during in vitro cellular co-cultures. Altogether, our data point to the possibility that the human thymic epithelial cells play a role in the establishment and progression of HTLV-1 infection, functioning as a reservoir and transmitting the virus to maturing CD4 + T lymphocytes, which in turn will cause disease in the periphery. Copyright © 2017. Published by Elsevier GmbH.

Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells

PubMed Central

Ashili, Shashanka P.; Houkal, Jeff; Smith, Dean; Mohammadreza, Aida; Lee, Kristen B.; Forrester, Jessica; Kumar, Ashok; Anis, Yasser H.; Paulson, Thomas G.; Youngbull, Cody A.; Tian, Yanqing; Holl, Mark R.; Johnson, Roger H.; Meldrum, Deirdre R.

2012-01-01

Abstract. Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system’s capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates. PMID:22502580

Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells.

PubMed

Kelbauskas, Laimonas; Ashili, Shashanka P; Houkal, Jeff; Smith, Dean; Mohammadreza, Aida; Lee, Kristen B; Forrester, Jessica; Kumar, Ashok; Anis, Yasser H; Paulson, Thomas G; Youngbull, Cody A; Tian, Yanqing; Holl, Mark R; Johnson, Roger H; Meldrum, Deirdre R

2012-03-01

Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system's capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates. © 2012 Society of Photo-Optical Instrumentation Engineers (SPIE).

Capsule expression in Streptococcus mitis modulates interaction with oral keratinocytes and alters susceptibility to human antimicrobial peptides.

PubMed

Rukke, H V; Engen, S A; Schenck, K; Petersen, F C

2016-08-01

Streptococcus mitis is a colonizer of the oral cavity and the nasopharynx, and is closely related to Streptococcus pneumoniae. Both species occur in encapsulated and unencapsulated forms, but in S. mitis the role of the capsule in host interactions is mostly unknown. Therefore, the aim of this study was to examine how capsule expression in S. mitis can modulate interactions with the host with relevance for colonization. The S. mitis type strain, as well as two mutants of the type strain, an isogenic capsule deletion mutant, and a capsule switch mutant expressing the serotype 4 capsule of S. pneumoniae TIGR4, were used. Wild-type and capsule deletion strains of S. pneumoniae TIGR4 were included for comparison. We found that capsule production in S. mitis reduced adhesion to oral and lung epithelial cells. Further, exposure of oral epithelial cells to encapsulated S. mitis resulted in higher interleukin-6 and CXCL-8 transcription levels relative to the unencapsulated mutant. Capsule expression in S. mitis increased the sensitivity to human neutrophil peptide 1-3 but reduced the sensitivity to human β-defensin-3 and cathelicidin. This was in contrast with S. pneumoniae in which capsule expression has been generally associated with increased sensitivity to human antimicrobial peptides (AMPs). Collectively, these findings indicate that capsule expression in S. mitis is important in modulating interactions with epithelial cells, and is associated with increased or reduced susceptibility to AMPs depending on the nature of the AMP. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Akt recruits Dab2 to albumin endocytosis in the proximal tubule.

PubMed

Koral, Kelly; Li, Hui; Ganesh, Nandita; Birnbaum, Morris J; Hallows, Kenneth R; Erkan, Elif

2014-12-15

Proximal tubule epithelial cells have a highly sophisticated endocytic machinery to retrieve the albumin in the glomerular filtrate. The megalin-cubilin complex and the endocytic adaptor disabled-2 (Dab2) play a pivotal role in albumin endocytosis. We previously demonstrated that protein kinase B (Akt) regulates albumin endocytosis in the proximal tubule through an interaction with Dab2. Here, we examined the nature of Akt-Dab2 interaction. The pleckstrin homology (PH) and catalytic domains (CD) of Akt interacted with the proline-rich domain (PRD) of Dab2 based on yeast-two hybrid (Y2H) experiments. Pull-down experiments utilizing the truncated constructs of Dab2 demonstrated that the initial 11 amino acids of Dab2-PRD were sufficient to mediate the interaction between Akt and Dab2. Endocytosis experiments utilizing Akt1- and Akt2-silencing RNA revealed that both Akt1 and Akt2 mediate albumin endocytosis in proximal tubule epithelial cells; therefore, Akt1 and Akt2 may play a compensatory role in albumin endocytosis. Furthermore, both Akt isoforms phosphorylated Dab2 at Ser residues 448 and 449. Ser-to-Ala mutations of these Dab2 residues inhibited albumin endocytosis and resulted in a shift in location of Dab2 from the peripheral to the perinuclear area, suggesting the physiological relevance of these phosphorylation sites in albumin endocytosis. We conclude that both Akt1 and Akt2 are involved in albumin endocytosis, and phosphorylation of Dab2 by Akt induces albumin endocytosis in proximal tubule epithelial cells. Further delineation of how Akt affects expression/phosphorylation of endocytic adaptors and receptors will enhance our understanding of the molecular network triggered by albumin overload in the proximal tubule. Copyright © 2014 the American Physiological Society.

The influence of the microenvironment on the malignant phenotype

NASA Technical Reports Server (NTRS)

Park, C. C.; Bissell, M. J.; Barcellos-Hoff, M. H.

2000-01-01

Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. As tissue becomes cancerous, there are reciprocal interactions between neoplastic cells, adjacent normal cells such as stroma and endothelium, and their microenvironments. The current dominant paradigm wherein multiple genetic lesions provide both the impetus for, and the Achilles heel of, cancer might be inadequate to understand cancer as a disease process. In the following brief review, we will use selected examples to illustrate the influence of the microenvironment in the evolution of the malignant phenotype. We will also discuss recent studies that suggest novel therapeutic interventions might be derived from focusing on microenvironment and tumor cells interactions.

The Pathogenesis of Human Cervical Epithelium Cells Induced by Interacting with Trichomonas vaginalis

PubMed Central

Lin, Wei-Chen; Chang, Wei-Ting; Chang, Tsuey-Yu; Shin, Jyh-Wei

2015-01-01

Background Trichomonas vaginalis is a protozoan parasite that occurs in the urogenital-vaginal tract and is the primary causative agent of trichomoniasis, a common sexually transmitted disease in humans. The aggregation of this protozoan tends to destroy epithelial cells and induce pathogenesis. Principal Findings This study cultured T. vaginalis and human cervical epithelial cells (Z172) under the same conditions in the experiments. Following co-culturing for ten hours, the protozoans became attached to Z172, such that the cells presented a round shape and underwent shrinkage. Time-lapse recording and flow cytometry on interacted Z172 revealed that 70% had been disrupted, 18% presented a necrosis-like morphology and 8% showed signs of apoptosis. Gene expression profiling revealed in the seven inflammatory Z172 genes as well as in T. vaginalis genes that code for adhesion proteins 65 and 65-1. Significance These results suggest that cytopathogenic effects progress while Z172 is in contact with T. vaginalis, and the resulting morphological changes can be categorized as disruption. PMID:25901354

Infection of Polarized Cultures of Human Intestinal Epithelial Cells with Hepatitis A Virus: Vectorial Release of Progeny Virions through Apical Cellular Membranes

PubMed Central

Blank, Christian A.; Anderson, David A.; Beard, Michael; Lemon, Stanley M.

2000-01-01

Although hepatitis A virus (HAV) is typically transmitted by the fecal-oral route, little is known of its interactions with cells of the gastrointestinal tract. We studied the replication of HAV in polarized cultures of Caco-2 cells, a human cell line which retains many differentiated functions of small intestinal epithelial cells. Virus uptake was 30- to 40-fold more efficient when the inoculum was placed on the apical rather than the basolateral surface of these cells, suggesting a greater abundance of the cellular receptor for HAV on the apical surface. Infection proceeded without cytopathic effect and did not influence transepithelial resistance or the diffusion of inulin across cell monolayers. Nonetheless, there was extensive release of progeny virus, which occurred almost exclusively into apical supernatant fluids (36.4% ± 12.5% of the total virus yield compared with 0.23% ± 0.13% release into basolateral fluids). Brefeldin A caused a profound inhibition of HAV replication, but also selectively reduced apical release of virus. These results indicate that polarized human epithelial cell cultures undergo vectorial infection with HAV and that virus release is largely restricted to the apical membrane. Virus release occurs in the absence of cytopathic effect and may involve cellular vesicular transport mechanisms. PMID:10864660

Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

PubMed

Kameishi, Sumako; Umemoto, Terumasa; Matsuzaki, Yu; Fujita, Masako; Okano, Teruo; Kato, Takashi; Yamato, Masayuki

2016-05-06

Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laiko, Marina; Murtazina, Rakhilya; Malyukova, Irina

Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cellsmore » in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.« less

Resolvin E1-induced intestinal alkaline phosphatase promotes resolution of inflammation through LPS detoxification

PubMed Central

Campbell, Eric L.; MacManus, Christopher F.; Kominsky, Douglas J.; Keely, Simon; Glover, Louise E.; Bowers, Brittelle E.; Scully, Melanie; Bruyninckx, Walter J.; Colgan, Sean P.

2010-01-01

Resolvin-E1 (RvE1) has been demonstrated to promote inflammatory resolution in numerous disease models. Given the importance of epithelial cells to coordination of mucosal inflammation, we hypothesized that RvE1 elicits an epithelial resolution signature. Initial studies revealed that the RvE1-receptor (ChemR23) is expressed on intestinal epithelial cells (IECs) and that microarray profiling of cells exposed to RvE1 revealed regulation of inflammatory response gene expression. Notably, RvE1 induced intestinal alkaline phosphatase (ALPI) expression and significantly enhanced epithelial ALPI enzyme activity. One role recently attributed to ALPI is the detoxification of bacterial LPS. In our studies, RvE1-exposed epithelia detoxified LPS (assessed by attenuation of NF-κB signaling). Furthermore, in epithelial-bacterial interaction assays, we determined that ALPI retarded the growth of Escherichia coli. To define these features in vivo, we used a murine dextran sulfate sodium (DSS) model of colitis. Compared with vehicle controls, administration of RvE1 resulted in significant improvement of disease activity indices (e.g., body weight, colon length) concomitant with increased ALPI expression in the intestinal epithelium. Moreover, inhibition of ALPI activity resulted in increased severity of colitis in DSS-treated animals and partially abrogated the protective influence of RvE1. Together, these data implicate a previously unappreciated role for ALPI in RvE1-mediated inflammatory resolution. PMID:20660763

Resolvin E1-induced intestinal alkaline phosphatase promotes resolution of inflammation through LPS detoxification.

PubMed

Campbell, Eric L; MacManus, Christopher F; Kominsky, Douglas J; Keely, Simon; Glover, Louise E; Bowers, Brittelle E; Scully, Melanie; Bruyninckx, Walter J; Colgan, Sean P

2010-08-10

Resolvin-E1 (RvE1) has been demonstrated to promote inflammatory resolution in numerous disease models. Given the importance of epithelial cells to coordination of mucosal inflammation, we hypothesized that RvE1 elicits an epithelial resolution signature. Initial studies revealed that the RvE1-receptor (ChemR23) is expressed on intestinal epithelial cells (IECs) and that microarray profiling of cells exposed to RvE1 revealed regulation of inflammatory response gene expression. Notably, RvE1 induced intestinal alkaline phosphatase (ALPI) expression and significantly enhanced epithelial ALPI enzyme activity. One role recently attributed to ALPI is the detoxification of bacterial LPS. In our studies, RvE1-exposed epithelia detoxified LPS (assessed by attenuation of NF-kappaB signaling). Furthermore, in epithelial-bacterial interaction assays, we determined that ALPI retarded the growth of Escherichia coli. To define these features in vivo, we used a murine dextran sulfate sodium (DSS) model of colitis. Compared with vehicle controls, administration of RvE1 resulted in significant improvement of disease activity indices (e.g., body weight, colon length) concomitant with increased ALPI expression in the intestinal epithelium. Moreover, inhibition of ALPI activity resulted in increased severity of colitis in DSS-treated animals and partially abrogated the protective influence of RvE1. Together, these data implicate a previously unappreciated role for ALPI in RvE1-mediated inflammatory resolution.

Parallels between Global Transcriptional Programs of Polarizing Caco-2 Intestinal Epithelial Cells In Vitro and Gene Expression Programs in Normal Colon and Colon Cancer

PubMed Central

Sääf, Annika M.; Halbleib, Jennifer M.; Chen, Xin; Yuen, Siu Tsan; Leung, Suet Yi

2007-01-01

Posttranslational mechanisms are implicated in the development of epithelial cell polarity, but little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized temporal patterns of gene expression during cell–cell adhesion-initiated polarization of cultured human Caco-2 cells, which develop structural and functional polarity resembling enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell–cell contacts. Comparison to gene expression patterns in normal human colon and colon tumors revealed that the pattern in proliferating, nonpolarized Caco-2 cells paralleled patterns seen in human colon cancer in vivo, including expression of genes involved in cell proliferation. The pattern switched in polarized Caco-2 cells to one more closely resembling that in normal colon tissue, indicating that regulation of transcription underlying Caco-2 cell polarization is similar to that during enterocyte differentiation in vivo. Surprisingly, the temporal program of gene expression in polarizing Caco-2 cells involved changes in signaling pathways (e.g., Wnt, Hh, BMP, FGF) in patterns similar to those during migration and differentiation of intestinal epithelial cells in vivo, despite the absence of morphogen gradients and interactions with stromal cells characteristic of enterocyte differentiation in situ. The full data set is available at http://microarray-pubs.stanford.edu/CACO2. PMID:17699589

The role of genomic islands in Escherichia coli K1 interactions with intestinal and kidney epithelial cells.

PubMed

Yousuf, Farzana Abubakar; Rafiq, Sahar; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

2016-04-01

The completion of Escherichia coli K1 genome has identified several genomic islands that are present in meningitis-causing E. coli RS218 but absent in the non-pathogenic E. coli MG1655. In this study, the role of various genomic islands in E. coli K1 interactions with intestinal epithelial cells (Caco-2) and kidney epithelial cells (MA104) was determined. Using association assays, invasion assays, and intracellular survival assays, the findings revealed that the genomic island deletion mutants of RS218 related to P fimbriae, S fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, protein secretion system (T1SS for hemolysin; T2SS; T5SS for antigen 43), Iro system and hmu system), invasins (CNF1, IbeA), toxins (α-hemolysin), K1 capsule biosynthesis, metabolism (d-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism), prophage genes, showed reduced interactions with both cell types. Next, we determined the role of various genomic islands in E. coli K1 resistance to serum. When exposed to the normal human serum, the viability of the genomic island deletion mutants related to adhesins such as S fimbriae, P fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, antigen 43 and T5SS for antigen 43, T2SS, and T1SS for hemolysin, Iro system and hmu system, prophage genes, metabolism (sugar metabolism and d-serine catabolism), K1 capsule biosynthesis, and invasins such as CNF1 was affected, suggesting their role in bacteremia. The characterization of these genomic islands should reveal mechanisms of E. coli K1 pathogenicity that could be of value as therapeutic targets. Copyright © 2016 Elsevier Ltd. All rights reserved.

Three-Dimensional, Transgenic Cell Models to Quantify Space Genotoxic Effects

NASA Technical Reports Server (NTRS)

Gonda, S. R.; Sognier, M. A.; Wu, H.; Pingerelli, P. L.; Glickman, B. W.; Dawson, David L. (Technical Monitor)

1999-01-01

The space environment contains radiation and chemical agents known to be mutagenic and carcinogenic to humans. Additionally, microgravity is a complicating factor that may modify or synergize induced genotoxic effects. Most in vitro models fail to use human cells (making risk extrapolation to humans more difficult), overlook the dynamic effect of tissue intercellular interactions on genotoxic damage, and lack the sensitivity required to measure low-dose effects. Currently a need exists for a model test system that simulates cellular interactions present in tissue, and can be used to quantify genotoxic damage induced by low levels of radiation and chemicals, and extrapolate assessed risk to humans. A state-of-the-art, three-dimensional, multicellular tissue equivalent cell culture model will be presented. It consists of mammalian cells genetically engineered to contain multiple copies of defined target genes for genotoxic assessment,. NASA-designed bioreactors were used to coculture mammalian cells into spheroids, The cells used were human mammary epithelial cells (H184135) and Stratagene's (Austin, Texas) Big Blue(TM) Rat 2 lambda fibroblasts. The fibroblasts were genetically engineered to contain -a high-density target gene for mutagenesis (60 copies of lacl/LacZ per cell). Tissue equivalent spheroids were routinely produced by inoculation of 2 to 7 X 10(exp 5) fibroblasts with Cytodex 3 beads (150 micrometers in diameter). at a 20:1 cell:bead ratio, into 50-ml HARV bioreactors (Synthecon, Inc.). Fibroblasts were cultured for 5 days, an equivalent number of epithelial cells added, and the fibroblast/epithelial cell coculture continued for 21 days. Three-dimensional spheroids with diameters ranging from 400 to 600 micrometers were obtained. Histological and immunohistochemical Characterization revealed i) both cell types present in the spheroids, with fibroblasts located primarily in the center, surrounded by epithelial cells; ii) synthesis of extracellular matrix; and iii,, mitotic cells located throughout the spheroids. Spheroidal integrity and cell viability were retained for the 30-day test period after removal of spheroids from the bioreactor. Potential utility of this three-dimensional, transgenic model for genotoxicity was initially assessed by exposure of spheroids to 0-2 Gy neon at dose rates of 0.3 to 1.5 Gy/min (National Institute of Radiological Sciences, Chiba, Japan). Quantification of mutation at the lacl gene revealed a linear dose response for mutation induction. Limited sequencing analysis of mutant clones revealed higher frequencies of deletions and multiple base sequence changes with increasing dose. These results suggest that our three-dimensional, transgenic model is applicable to a wide variety of studies involving the quantification, identification, and characterization of genotoxicity incurred in space and on Earth. This model uniquely allows investigation of the interaction of relevant factors, namely cell-to-cell interactions and the mechanistic interaction of microgravity with radiation insults and DNA repair. Using this three-dimensional model will allow us to obtain dual genotoxic information (i.e., mutation rate plus chromosome aberration data) from the same system so that one endpoint can be used to reference the other, thereby increasing the fidelity of the data set. Moreover, the tissue-equivalent nature of the three-dimensional model provides high confidence for relevance of risk assessment, i.e., the establishment of quality factors directly applicable to the microgravity environment.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes.

PubMed

Truong-Bolduc, Q C; Khan, N S; Vyas, J M; Hooper, D C

2017-02-01

We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes. Copyright © 2017 American Society for Microbiology.

Listeria monocytogenes InlP interacts with afadin and facilitates basement membrane crossing.

PubMed

Faralla, Cristina; Bastounis, Effie E; Ortega, Fabian E; Light, Samuel H; Rizzuto, Gabrielle; Nocadello, Salvatorre; Anderson, Wayne F; Robbins, Jennifer R; Theriot, Julie A; Bakardjiev, Anna I

2018-05-30

During pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such as Listeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the L. monocytogenes protein Internalin P (InlP) as a secreted virulence factor critical for placental infection. Here, we show that InlP, but not the highly similar L. monocytogenes internalin Lmo2027, binds to human afadin (encoded by AF-6), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca2+-binding site. Despite afadin's involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited by AF-6 knock-out MDCK cells. L. monocytogenes ΔinlP mutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion in inlP (inlP ΔLRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promote L. monocytogenes transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.

Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits adhesion of human endometriotic epithelial and stromal cells through suppression of integrin-mediated mechanisms.

PubMed

Lee, JeHoon; Banu, Sakhila K; Burghardt, Robert C; Starzinski-Powitz, Anna; Arosh, Joe A

2013-03-01

Endometriosis is a chronic gynecological disease of reproductive age women characterized by the presence of functional endometrial tissues outside the uterine cavity. Interactions between the endometriotic cells and the peritoneal extracellular matrix proteins (ECM) are crucial mechanisms that allow adhesion of the endometriotic cells into peritoneal mesothelia. Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. In previous studies, we have reported that selective inhibition of PGE2 receptors PTGER2 and PTGER4 decreases survival and invasion of human endometriotic epithelial and stromal cells through multiple mechanisms. Results of the present study indicates that selective inhibition of PTGER2- and PTGER4-mediated PGE2 signaling 1) decreases the expression and/or activity of specific integrin receptor subunits Itgb1 (beta1) and Itgb3 (beta3) but not Itgb5 (beta5), Itga1 (alpha1), Itga2 (alpha2), Itga5 (alpha5), and Itgav (alphav); 2) decreases integrin-signaling components focal adhesion kinase or protein kinase 2 (PTK2) and talin proteins; 3) inhibits interactions between Itgb1/Itgb3 subunits, PTK2, and talin and PTGER2/PTGER4 proteins through beta-arrestin-1 and Src kinase protein complex in human endometriotic epithelial cells 12Z and stromal cells 22B; and 4) decreases adhesion of 12Z and 22B cells to ECM collagen I, collagen IV, fibronectin, and vitronectin in a substrate-specific manner. These novel findings provide an important molecular framework for further evaluation of selective inhibition of PTGER2 and PTGER4 as potential nonsteroidal therapy to expand the spectrum of currently available treatment options for endometriosis in child-bearing age women.

Nitric oxide and superoxide mediate diesel particle effects in cytokine-treated mice and murine lung epithelial cells — implications for susceptibility to traffic-related air pollution

PubMed Central

2012-01-01

Background Epidemiologic studies associate childhood exposure to traffic-related air pollution with increased respiratory infections and asthmatic and allergic symptoms. The strongest associations between traffic exposure and negative health impacts are observed in individuals with respiratory inflammation. We hypothesized that interactions between nitric oxide (NO), increased during lung inflammatory responses, and reactive oxygen species (ROS), increased as a consequence of traffic exposure ─ played a key role in the increased susceptibility of these at-risk populations to traffic emissions. Methods Diesel exhaust particles (DEP) were used as surrogates for traffic particles. Murine lung epithelial (LA-4) cells and BALB/c mice were treated with a cytokine mixture (cytomix: TNFα, IL-1β, and IFNγ) to induce a generic inflammatory state. Cells were exposed to saline or DEP (25 μg/cm2) and examined for differential effects on redox balance and cytotoxicity. Likewise, mice undergoing nose-only inhalation exposure to air or DEP (2 mg/m3 × 4 h/d × 2 d) were assessed for differential effects on lung inflammation, injury, antioxidant levels, and phagocyte ROS production. Results Cytomix treatment significantly increased LA-4 cell NO production though iNOS activation. Cytomix +  DEP-exposed cells incurred the greatest intracellular ROS production, with commensurate cytotoxicity, as these cells were unable to maintain redox balance. By contrast, saline + DEP-exposed cells were able to mount effective antioxidant responses. DEP effects were mediated by: (1) increased ROS including superoxide anion (O2˙-), related to increased xanthine dehydrogenase expression and reduced cytosolic superoxide dismutase activity; and (2) increased peroxynitrite generation related to interaction of O2˙- with cytokine-induced NO. Effects were partially reduced by superoxide dismutase (SOD) supplementation or by blocking iNOS induction. In mice, cytomix +  DEP-exposure resulted in greater ROS production in lung phagocytes. Phagocyte and epithelial effects were, by and large, prevented by treatment with FeTMPyP, which accelerates peroxynitrite catalysis. Conclusions During inflammation, due to interactions of NO and O2˙-, DEP-exposure was associated with nitrosative stress in surface epithelial cells and resident lung phagocytes. As these cell types work in concert to provide protection against inhaled pathogens and allergens, dysfunction would predispose to development of respiratory infection and allergy. Results provide a mechanism by which individuals with pre-existing respiratory inflammation are at increased risk for exposure to traffic-dominated urban air pollution. PMID:23151036

Protective effect of NSA on intestinal epithelial cells in a necroptosis model

PubMed Central

Dong, Wei; Zhang, Min; Zhu, Yaxi; Chen, Yuanhan; Zhao, Xingchen; Li, Ruizhao; Zhang, Li; Ye, Zhiming; Liang, Xingling

2017-01-01

Objective This study aimed to investigate the protective effect of the necroptosis inhibitor necrosulfonamide (NSA) on intestinal epithelial cells using a novel in vitro necroptosis model that mimics inflammatory bowel disease (IBD). Methods 2,4,6-trinitrobenzenesulfonic acid (TNBS) was perfused into the rectum of BALB/c mice to established a colitis model. Pathologic injury and cell death were evaluated. A novel in vitro model of necroptosis was established in Caco-2 cells using TNF-α and Z-VAD-fmk, and the cells were treated with or without NSA. Morphologic changes, manner of cell death and the levels of phosphorylation of receptor-interacting protein kinase 3 (p-RIPK3) and mixed-lineage kinase domain-like (p-MLKL) were detected. Results In the TNBS-induced colitis in mice, TUNEL-positive and caspase-3-negative cells were observed in the intestinal mucosa, and p-RIPK3 was found to be elevated. Under the stimulation of TNF-α and Z-VAD-fmk, the morphologic damage in the Caco-2 cells was aggravated, the proportion of necrosis was increased, and the level of p-RIPK3 and p-MLKL were increased, confirming that the regulated cell death was necroptosis. NSA reversed the morphological abnormalities and reduced necrotic cell death induced by TNF-α and Z-VAD-fmk. Conclusion NSA can inhibit necroptosis in intestinal epithelial cells in vitro and might confer a potential protective effect against IBD. PMID:29156831

Protective effect of NSA on intestinal epithelial cells in a necroptosis model.

PubMed

Dong, Wei; Zhang, Min; Zhu, Yaxi; Chen, Yuanhan; Zhao, Xingchen; Li, Ruizhao; Zhang, Li; Ye, Zhiming; Liang, Xingling

2017-10-17

This study aimed to investigate the protective effect of the necroptosis inhibitor necrosulfonamide (NSA) on intestinal epithelial cells using a novel in vitro necroptosis model that mimics inflammatory bowel disease (IBD). 2,4,6-trinitrobenzenesulfonic acid (TNBS) was perfused into the rectum of BALB/c mice to established a colitis model. Pathologic injury and cell death were evaluated. A novel in vitro model of necroptosis was established in Caco-2 cells using TNF- α and Z-VAD-fmk, and the cells were treated with or without NSA. Morphologic changes, manner of cell death and the levels of phosphorylation of receptor-interacting protein kinase 3 (p-RIPK3) and mixed-lineage kinase domain-like (p-MLKL) were detected. In the TNBS-induced colitis in mice, TUNEL-positive and caspase-3-negative cells were observed in the intestinal mucosa, and p-RIPK3 was found to be elevated. Under the stimulation of TNF- α and Z-VAD-fmk, the morphologic damage in the Caco-2 cells was aggravated, the proportion of necrosis was increased, and the level of p-RIPK3 and p-MLKL were increased, confirming that the regulated cell death was necroptosis. NSA reversed the morphological abnormalities and reduced necrotic cell death induced by TNF- α and Z-VAD-fmk. NSA can inhibit necroptosis in intestinal epithelial cells in vitro and might confer a potential protective effect against IBD.

Rifaximin decreases virulence of Crohn's disease-associated Escherichia coli and epithelial inflammatory responses.

PubMed

Dogan, Belgin; Fu, Jing; Zhang, Shiying; Scherl, Ellen J; Simpson, Kenneth W

2018-05-01

Escherichia coli with an adherent and invasive pathotype (AIEC) is implicated in the pathogenesis of Crohn's disease (CD). Rifaximin improves symptoms in mild-to-moderate CD. It is unclear if this outcome is due to its effects on bacteria or intestinal epithelial inflammatory responses. We examined the effects of rifaximin on the growth and virulence of CD-associated E. coli and intestinal epithelial inflammatory responses. Seven well-characterized CD-associated E. coli strains (six AIEC, one non-AIEC; four rifaximin-resistant, three sensitive) were evaluated. We assessed the effects of rifaximin on CD-associated E. coli growth, adhesion to, and invasion of epithelial cells, virulence gene expression, motility, and survival in macrophages. Additionally, we determined the effects of rifaximin on intestinal epithelial inflammatory responses. In vitro rifaximin exerted a dose-dependent effect on the growth of sensitive strains but did not affect the growth of resistant strains. Rifaximin reduced adhesion, invasion, virulence gene expression and motility of CD-associated E. coli in a manner that was independent of its antimicrobial effect. Furthermore, rifaximin reduced IL-8 secretion from pregnane X receptor-expressing T84 colonic epithelial cells. The effect of rifaximin on adhesion was largely attributable to its action on bacteria, whereas decreases in invasion and cytokine secretion were due to its effect on the epithelium. In conclusion, our results show that rifaximin interferes with multiple steps implicated in host-AIEC interactions related to CD, including adhesion to, and invasion of epithelial cells, virulence gene expression, motility, and pro-inflammatory cytokine secretion. Further study is required to determine the relationship of these effects to clinical responses in CD patients.

Interactions between bacteria and the gut mucosa: Do enteric neurotransmitters acting on the mucosal epithelium influence intestinal colonization or infection?

USDA-ARS?s Scientific Manuscript database

The intestinal epithelium is a critical barrier between the internal and external milieux of the mammalian host. Epithelial interactions between these two host environments have been shown to be modulated by several different, cross-communicating cell types residing in the gut mucosa. These includ...

Interactions between bacteria and the gut mucosa: Do enteric neurotransmitters acting on the mucosal epithelium influence intestinal colonization or infection?

USDA-ARS?s Scientific Manuscript database

The intestinal epithelium is a critical barrier between the internal and external milieux of the mammalian host. Epithelial interactions between these two host environments have been shown to be modulated by several different, cross-communicating cell types residing in the gut mucosa. These include ...

The carboxyl terminus of the alpha-subunit of the amiloride-sensitive epithelial sodium channel binds to F-actin.

PubMed

Mazzochi, Christopher; Bubien, James K; Smith, Peter R; Benos, Dale J

2006-03-10

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.

The PDZ Ligand Domain of the Human Papillomavirus Type 16 E6 Protein Is Required for E6's Induction of Epithelial Hyperplasia In Vivo

PubMed Central

Nguyen, Marie L.; Nguyen, Minh M.; Lee, Denis; Griep, Anne E.; Lambert, Paul F.

2003-01-01

Human papillomaviruses (HPVs) are the causative agent of warts. Infections with high-risk HPVs are associated with anogenital and head and neck cancers. One of the viral genes responsible for HPV's oncogenic activity is E6. Mice expressing the HPV-16 E6 protein in their epidermis (K14E6WT) develop epithelial hyperplasia and squamous carcinomas. Numerous cellular proteins interact with E6, some of which can be grouped based on common amino acid motifs in their E6-binding domains. One such group, the PDZ partners, including hDLG, hSCRIBBLE, MUPP1, and MAGI, bind to the carboxy-terminal four amino acids of E6 through their PDZ domains. E6's interaction with the PDZ partners leads to their degradation. Additionally, E6's binding to PDZ proteins has been correlated with its ability to transform baby rat kidney cells in tissue culture and to confer tumorigenicity onto cells in xenograft experiments. To address whether the ability of E6 to bind PDZ domain partners is necessary for E6 to confer epithelial hyperproliferation in vivo, we generated transgenic mice that express in stratified squamous epithelia a mutant of E6 lacking the last six amino acids at its carboxyl terminus, E6Δ146-151, from the human keratin 14 (K14) promoter. The K14E6Δ146-151 mice exhibit a radiation response similar to that of the K14E6WT mice, demonstrating that this protein, as predicted, retains an ability to inactivate p53. However, the K14E6Δ146-151 mice fail to display epithelial hyperplasia. These results indicate that an interaction of E6 with PDZ partners is necessary for its induction of epithelial hyperplasia. PMID:12768014

Fabrication of a multi-layer three-dimensional scaffold with controlled porous micro-architecture for application in small intestine tissue engineering.

PubMed

Knight, Toyin; Basu, Joydeep; Rivera, Elias A; Spencer, Thomas; Jain, Deepak; Payne, Richard

2013-01-01

Various methods can be employed to fabricate scaffolds with characteristics that promote cell-to-material interaction. This report examines the use of a novel technique combining compression molding with particulate leaching to create a unique multi-layered scaffold with differential porosities and pore sizes that provides a high level of control to influence cell behavior. These cell behavioral responses were primarily characterized by bridging and penetration of two cell types (epithelial and smooth muscle cells) on the scaffold in vitro. Larger pore sizes corresponded to an increase in pore penetration, and a decrease in pore bridging. In addition, smaller cells (epithelial) penetrated further into the scaffold than larger cells (smooth muscle cells). In vivo evaluation of a multi-layered scaffold was well tolerated for 75 d in a rodent model. This data shows the ability of the components of multi-layered scaffolds to influence cell behavior, and demonstrates the potential for these scaffolds to promote desired tissue outcomes in vivo.

Chemo-elastic modeling of invasive carcinoma development accompanied by oncogenic epithelial-mesenchymal transition

NASA Astrophysics Data System (ADS)

Bratsun, D. A.; Krasnyakov, I. V.; Pismen, L.

2017-09-01

We present a further development of a multiscale chemo-mechanical model of carcinoma growth in the epithelium tissue proposed earlier. The epithelium is represented by an elastic 2D array of polygonal cells, each with its own gene regulation dynamics. The model allows the simulation of evolution of multiple cells interacting via the chemical signaling or mechanically induced strain. The algorithm takes into account the division and intercalation of cells. The latter is most important since, first of all, carcinoma cells lose cell-cell adhesion and polarity via the oncogenic variant of the epithelial-mesenchymal transition (EMT) at which cells gain migratory and invasive properties. This process is mediated by E-cadherin repression and requires the differentiation of tumor cells with respect to the edge of the tumor that means that front cells should be most mobile. Taking into account this suggestion, we present the results of simulations demonstrating different patterns of carcinoma invasion. The comparison of our results with recent experimental observations is given and discussed.

Herpes Simplex Virus Dances with Amyloid Precursor Protein while Exiting the Cell

PubMed Central

Cheng, Shi-Bin; Ferland, Paulette; Webster, Paul; Bearer, Elaine L.

2011-01-01

Herpes simplex type 1 (HSV1) replicates in epithelial cells and secondarily enters local sensory neuronal processes, traveling retrograde to the neuronal nucleus to enter latency. Upon reawakening newly synthesized viral particles travel anterograde back to the epithelial cells of the lip, causing the recurrent cold sore. HSV1 co-purifies with amyloid precursor protein (APP), a cellular transmembrane glycoprotein and receptor for anterograde transport machinery that when proteolyzed produces A-beta, the major component of senile plaques. Here we focus on transport inside epithelial cells of newly synthesized virus during its transit to the cell surface. We hypothesize that HSV1 recruits cellular APP during transport. We explore this with quantitative immuno-fluorescence, immuno-gold electron-microscopy and live cell confocal imaging. After synchronous infection most nascent VP26-GFP-labeled viral particles in the cytoplasm co-localize with APP (72.8+/−6.7%) and travel together with APP inside living cells (81.1+/−28.9%). This interaction has functional consequences: HSV1 infection decreases the average velocity of APP particles (from 1.1+/−0.2 to 0.3+/−0.1 µm/s) and results in APP mal-distribution in infected cells, while interplay with APP-particles increases the frequency (from 10% to 81% motile) and velocity (from 0.3+/−0.1 to 0.4+/−0.1 µm/s) of VP26-GFP transport. In cells infected with HSV1 lacking the viral Fc receptor, gE, an envelope glycoprotein also involved in viral axonal transport, APP-capsid interactions are preserved while the distribution and dynamics of dual-label particles differ from wild-type by both immuno-fluorescence and live imaging. Knock-down of APP with siRNA eliminates APP staining, confirming specificity. Our results indicate that most intracellular HSV1 particles undergo frequent dynamic interplay with APP in a manner that facilitates viral transport and interferes with normal APP transport and distribution. Such dynamic interactions between APP and HSV1 suggest a mechanistic basis for the observed clinical relationship between HSV1 seropositivity and risk of Alzheimer's disease. PMID:21483850

Transcriptome analysis of Cronobacter sakazakii ATCC BAA-894 after interaction with human intestinal epithelial cell line HCT-8.

PubMed

Jing, Chun-e; Du, Xin-jun; Li, Ping; Wang, Shuo

2016-01-01

Cronobacter spp. are opportunistic pathogens that are responsible for infections including severe meningitis, septicemia, and necrotizing enterocolitis in neonates and infants. To date, questions still remain regarding the mechanisms of pathogenicity and virulence determinants for each bacterial strain. In this study, we established an in vitro model for Cronobacter sakazakii ATCC BAA-894 infection of HCT-8 human colorectal epithelial cells. The transcriptome profile of C. sakazakii ATCC BAA-894 after interaction with HCT-8 cells was determined using high-throughput whole-transcriptome sequencing (RNA sequencing (RNA-seq)). Gene expression profiles indicated that 139 genes were upregulated and 72 genes were downregulated in the adherent C. sakazakii ATCC BAA-894 strain on HCT-8 cells compared to the cultured bacteria in the cell-free medium. Expressions of some flagella genes and virulence factors involved in adherence were upregulated. High osmolarity and osmotic stress-associated genes were highly upregulated, as well as genes responsible for the synthesis of lipopolysaccharides and outer membrane proteins, iron acquisition systems, and glycerol and glycerophospholipid metabolism. In sum, our study provides further insight into the mechanisms underlying C. sakazakii pathogenesis in the human gastrointestinal tract.

Herpes simplex virus type 2 glycoprotein H interacts with integrin αvβ3 to facilitate viral entry and calcium signaling in human genital tract epithelial cells.

PubMed

Cheshenko, Natalia; Trepanier, Janie B; González, Pablo A; Eugenin, Eliseo A; Jacobs, William R; Herold, Betsy C

2014-09-01

Herpes simplex virus (HSV) entry requires multiple interactions at the cell surface and activation of a complex calcium signaling cascade. Previous studies demonstrated that integrins participate in this process, but their precise role has not been determined. These studies were designed to test the hypothesis that integrin αvβ3 signaling promotes the release of intracellular calcium (Ca2+) stores and contributes to viral entry and cell-to-cell spread. Transfection of cells with small interfering RNA (siRNA) targeting integrin αvβ3, but not other integrin subunits, or treatment with cilengitide, an Arg-Gly-Asp (RGD) mimetic, impaired HSV-induced Ca2+ release, viral entry, plaque formation, and cell-to-cell spread of HSV-1 and HSV-2 in human cervical and primary genital tract epithelial cells. Coimmunoprecipitation studies and proximity ligation assays indicated that integrin αvβ3 interacts with glycoprotein H (gH). An HSV-2 gH-null virus was engineered to further assess the role of gH in the virus-induced signaling cascade. The gH-2-null virus bound to cells and activated Akt to induce a small Ca2+ response at the plasma membrane, but it failed to trigger the release of cytoplasmic Ca2+ stores and was impaired for entry and cell-to-cell spread. Silencing of integrin αvβ3 and deletion of gH prevented phosphorylation of focal adhesion kinase (FAK) and the transport of viral capsids to the nuclear pore. Together, these findings demonstrate that integrin signaling is activated downstream of virus-induced Akt signaling and facilitates viral entry through interactions with gH by activating the release of intracellular Ca2+ and FAK phosphorylation. These findings suggest a new target for HSV treatment and suppression. Herpes simplex viruses are the leading cause of genital disease worldwide, the most common infection associated with neonatal encephalitis, and a major cofactor for HIV acquisition and transmission. There is no effective vaccine. These epidemiological findings underscore the urgency to develop novel HSV treatment or prevention strategies. This study addresses this gap by further defining the signaling pathways the virus usurps to enter human genital tract epithelial cells. Specifically, the study defines the role played by integrins and by the viral envelope glycoprotein H in entry and cell-to-cell spread. This knowledge will facilitate the identification of new targets for the development of treatment and prevention. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Epithelial-mesenchymal transition and tumor suppression are controlled by a reciprocal feedback loop between ZEB1 and Grainyhead-like-2

PubMed Central

Cieply, Benjamin; Farris, Joshua; Denvir, James; Ford, Heide; Frisch, Steven M.

2013-01-01

Epithelial-mesenchymal transition (EMT) in carcinoma cells enhances malignant progression by promoting invasion and survival. EMT is induced by microenvironmental factors including TGF-β and Wnt agonists, and by the E-box-binding transcription factors Twist, Snail and ZEB. Grainyhead-like-2 (GRHL2), a member of the mammalian Grainyhead family of wound healing regulatory transcription factors, suppresses EMT and restores sensitivity to anoikis by repressing ZEB1 expression and inhibiting TGF-β signaling. In this study, we elucidate the functional relationship between GRHL2 and ZEB1 in EMT/MET and tumor biology. At least three homeodomain proteins, Six1, LBX1, and HoxA5, transactivated the ZEB1 promoter, in the case of Six1, through direct protein-promoter interaction. GRHL2 altered the Six1-DNA complex, inhibiting this transactivation. Correspondingly, GRHL2 expression prevented tumor initiation in xenograft assays, sensitized breast cancer cells to paclitaxel and suppressed the emergence of CD44highCD24low cells (defining the cancer stem cell phenotype in the cell type studied). GRHL2 was down-regulated in recurrent mouse tumors that had evolved to an oncogene-independent, EMT-like state, supporting a role for GRHL2 down-regulation in this phenotypic transition, modeling disease recurrence. The combination of TGF-β and Wnt activation repressed GRHL2 expression by direct interaction of ZEB1 with the GRHL2 promoter, inducing EMT. Together, our observations indicate that a reciprocal feedback loop between GRHL2 and ZEB1 controls epithelial vs. mesenchymal phenotypes and EMT-driven tumor progression. PMID:23943797

Epithelial-mesenchymal transition abolishes the susceptibility of polarized epithelial cell lines to measles virus.

PubMed

Shirogane, Yuta; Takeda, Makoto; Tahara, Maino; Ikegame, Satoshi; Nakamura, Takanori; Yanagi, Yusuke

2010-07-02

Measles virus (MV), an enveloped negative-strand RNA virus, remains a major cause of morbidity and mortality in developing countries. MV predominantly infects immune cells by using signaling lymphocyte activation molecule (SLAM; also called CD150) as a receptor, but it also infects polarized epithelial cells, forming tight junctions in a SLAM-independent manner. Although the ability of MV to infect polarized epithelial cells is thought to be important for its transmission, the epithelial cell receptor for MV has not been identified. A transcriptional repressor, Snail, induces epithelial-mesenchymal transition (EMT), in which epithelial cells lose epithelial cell phenotypes, such as adherens and tight junctions. In this study, EMT was induced by expressing Snail in a lung adenocarcinoma cell line, II-18, which is highly susceptible to wild-type MV. Snail-expressing II-18 cells lost adherens and tight junctions. Microarray analysis confirmed the induction of EMT in II-18 cells and suggested a novel function of Snail in protein degradation and distribution. Importantly, wild-type MV no longer entered EMT-induced II-18 cells, suggesting that the epithelial cell receptor is down-regulated by the induction of EMT. Other polarized cell lines, NCI-H358 and HT-29, also lost susceptibility to wild-type MV when EMT was induced. However, the complete formation of tight junctions rather reduced MV entry into HT-29 cells. Taken together, these data suggest that the unidentified epithelial cell receptor for MV is involved in the formation of epithelial intercellular junctions.

Single-cell heterogeneity in ductal carcinoma in situ of breast.

PubMed

Gerdes, Michael J; Gökmen-Polar, Yesim; Sui, Yunxia; Pang, Alberto Santamaria; LaPlante, Nicole; Harris, Adrian L; Tan, Puay-Hoon; Ginty, Fiona; Badve, Sunil S

2018-03-01

Heterogeneous patterns of mutations and RNA expression have been well documented in invasive cancers. However, technological challenges have limited the ability to study heterogeneity of protein expression. This is particularly true for pre-invasive lesions such as ductal carcinoma in situ of the breast. Cell-level heterogeneity in ductal carcinoma in situ was analyzed in a single 5 μm tissue section using a multiplexed immunofluorescence analysis of 11 disease-related markers (EGFR, HER2, HER4, S6, pmTOR, CD44v6, SLC7A5 and CD10, CD4, CD8 and CD20, plus pan-cytokeratin, pan-cadherin, DAPI, and Na+K+ATPase for cell segmentation). Expression was quantified at cell level using a single-cell segmentation algorithm. K-means clustering was used to determine co-expression patterns of epithelial cell markers and immune markers. We document for the first time the presence of epithelial cell heterogeneity within ducts, between ducts and between patients with ductal carcinoma in situ. There was moderate heterogeneity in a distribution of eight clusters within each duct (average Shannon index 0.76; range 0-1.61). Furthermore, within each patient, the average Shannon index across all ducts ranged from 0.33 to 1.02 (s.d. 0.09-0.38). As the distribution of clusters within ducts was uneven, the analysis of eight ducts might be sufficient to represent all the clusters ie within- and between-duct heterogeneity. The pattern of epithelial cell clustering was associated with the presence and type of immune infiltrates, indicating a complex interaction between the epithelial tumor and immune system for each patient. This analysis also provides the first evidence that simultaneous analysis of both the epithelial and immune/stromal components might be necessary to understand the complex milieu in ductal carcinoma in situ lesions.

The spectraplakin Short stop is an essential microtubule regulator involved in epithelial closure in Drosophila

PubMed Central

Takács, Zsanett; Vilmos, Péter; Lénárt, Péter; Röper, Katja; Erdélyi, Miklós

2017-01-01

ABSTRACT Dorsal closure of the Drosophila embryonic epithelium provides an excellent model system for the in vivo analysis of molecular mechanisms regulating cytoskeletal rearrangements. In this study, we investigated the function of the Drosophila spectraplakin Short stop (Shot), a conserved cytoskeletal structural protein, during closure of the dorsal embryonic epithelium. We show that Shot is essential for the efficient final zippering of the opposing epithelial margins. By using isoform-specific mutant alleles and genetic rescue experiments with truncated Shot variants, we demonstrate that Shot functions as an actin–microtubule cross-linker in mediating zippering. At the leading edge of epithelial cells, Shot regulates protrusion dynamics by promoting filopodia formation. Fluorescence recovery after photobleaching (FRAP) analysis and in vivo imaging of microtubule growth revealed that Shot stabilizes dynamic microtubules. The actin- and microtubule-binding activities of Shot are simultaneously required in the same molecule, indicating that Shot is engaged as a physical crosslinker in this process. We propose that Shot-mediated interactions between microtubules and actin filaments facilitate filopodia formation, which promotes zippering by initiating contact between opposing epithelial cells. PMID:28062848

Investigating the Link between Molecular Subtypes of Glioblastoma, Epithelial-Mesenchymal Transition, and CD133 Cell Surface Protein

PubMed Central

Zarkoob, Hadi; Taube, Joseph H.; Singh, Sheila K.; Mani, Sendurai A.; Kohandel, Mohammad

2013-01-01

In this manuscript, we use genetic data to provide a three-faceted analysis on the links between molecular subclasses of glioblastoma, epithelial-to-mesenchymal transition (EMT) and CD133 cell surface protein. The contribution of this paper is three-fold: First, we use a newly identified signature for epithelial-to-mesenchymal transition in human mammary epithelial cells, and demonstrate that genes in this signature have significant overlap with genes differentially expressed in all known GBM subtypes. However, the overlap between genes up regulated in the mesenchymal subtype of GBM and in the EMT signature was more significant than other GBM subtypes. Second, we provide evidence that there is a negative correlation between the genetic signature of EMT and that of CD133 cell surface protein, a putative marker for neural stem cells. Third, we study the correlation between GBM molecular subtypes and the genetic signature of CD133 cell surface protein. We demonstrate that the mesenchymal and neural subtypes of GBM have the strongest correlations with the CD133 genetic signature. While the mesenchymal subtype of GBM displays similarity with the signatures of both EMT and CD133, it also exhibits some differences with each of these signatures that are partly due to the fact that the signatures of EMT and CD133 are inversely related to each other. Taken together these data shed light on the role of the mesenchymal transition and neural stem cells, and their mutual interaction, in molecular subtypes of glioblastoma multiforme. PMID:23734191

Effects of high glucose on the production of heparan sulfate proteoglycan by mesangial and epithelial cells.

PubMed

van Det, N F; van den Born, J; Tamsma, J T; Verhagen, N A; Berden, J H; Bruijn, J A; Daha, M R; van der Woude, F J

1996-04-01

Changes in heparan sulfate metabolism may be important in the pathogenesis of diabetic nephropathy. Recent studies performed on renal biopsies from patients with diabetic nephropathy revealed a decrease in heparan sulfate glycosaminoglycan staining in the glomerular basement membrane without changes in staining for heparan sulfate proteoglycan-core protein. To understand this phenomenon at the cellular level, we investigated the effect of high glucose conditions on the synthesis of heparan sulfate proteoglycan by glomerular cells in vitro. Human adult mesangial and glomerular visceral epithelial cells were cultured under normal (5 mM) and high glucose (25 mM) conditions. Immunofluorescence performed on cells cultured in 25 mM glucose confirmed and extended the in vivo histological observations. Using metabolic labeling we observed an altered proteoglycan production under high glucose conditions, with predominantly a decrease in heparan sulfate compared to dermatan sulfate or chondroitin sulfate proteoglycan. N-sulfation analysis of heparan sulfate proteoglycan produced under high glucose conditions revealed less di- and tetrasaccharides compared to larger oligosaccharides, indicating an altered sulfation pattern. Furthermore, with quantification of glomerular basement membrane heparan sulfate by ELISA, a significant decrease was observed when mesangial and visceral epithelial cells were cultured in high glucose conditions. We conclude that high glucose concentration induces a significant alteration of heparan sulfate production by mesangial cells and visceral epithelial cells. Changes in sulfation and changes in absolute quantities are both observed and may explain the earlier in vivo observations. These changes may be of importance for the altered integrity of the glomerular charge-dependent filtration barrier and growth-factor matrix interactions in diabetic nephropathy.

Quantification of regenerative potential in primary human mammary epithelial cells

PubMed Central

Linnemann, Jelena R.; Miura, Haruko; Meixner, Lisa K.; Irmler, Martin; Kloos, Uwe J.; Hirschi, Benjamin; Bartsch, Harald S.; Sass, Steffen; Beckers, Johannes; Theis, Fabian J.; Gabka, Christian; Sotlar, Karl; Scheel, Christina H.

2015-01-01

We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. PMID:26071498

Interactions between the PDZ domains of Bazooka (Par-3) and phosphatidic acid: in vitro characterization and role in epithelial development

PubMed Central

Yu, Cao Guo; Harris, Tony J. C.

2012-01-01

Bazooka (Par-3) is a conserved polarity regulator that organizes molecular networks in a wide range of cell types. In epithelia, it functions as a plasma membrane landmark to organize the apical domain. Bazooka is a scaffold protein that interacts with proteins through its three PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains and other regions. In addition, Bazooka has been shown to interact with phosphoinositides. Here we show that the Bazooka PDZ domains interact with the negatively charged phospholipid phosphatidic acid immobilized on solid substrates or in liposomes. The interaction requires multiple PDZ domains, and conserved patches of positively charged amino acid residues appear to mediate the interaction. Increasing or decreasing levels of diacylglycerol kinase or phospholipase D—enzymes that produce phosphatidic acid—reveal a role for phosphatidic acid in Bazooka embryonic epithelial activity but not its localization. Mutating residues implicated in phosphatidic acid binding revealed a possible role in Bazooka localization and function. These data implicate a closer connection between Bazooka and membrane lipids than previously recognized. Bazooka polarity landmarks may be conglomerates of proteins and plasma membrane lipids that modify each other's activities for an integrated effect on cell polarity. PMID:22833561

Collective and single cell behavior in epithelial contact inhibition.

PubMed

Puliafito, Alberto; Hufnagel, Lars; Neveu, Pierre; Streichan, Sebastian; Sigal, Alex; Fygenson, D Kuchnir; Shraiman, Boris I

2012-01-17

Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing, and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as "contact inhibition." The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured Madin-Darby canine kidney cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size, and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition.

Re-establishment of gap junctional intercellular communication (GJIC) between human endometrial carcinomas by prostaglandin E(2).

PubMed

Schlemmer, Scott R; Kaufman, David G

2012-12-01

Reduced intercellular communication via gap junctions is correlated with carcinogenesis. Gap junctional intercellular communication (GJIC), between normal human endometrial epithelial cells is enhanced when endometrial stromal cells were present in culture. This enhancement of GJIC between normal epithelial cells also occurs when they are cultured in medium conditioned by stromal cells. This observation indicated that a soluble compound (or compounds) produced and secreted by stromal cells mediates GJIC in epithelial cells. Previous studies have shown that endometrial stromal cells release prostaglandin E(2) (PGE(2)) and prostaglandin F(2α) (PGF(2α)) under physiological conditions. When we evaluated the response of normal endometrial epithelial cells to various concentrations of PGE(2,) we found enhanced GJIC with 1nM PGE(2). This is a smaller increase in GJIC than that induced by medium conditioned by stromal cells. When the extracellular concentration of PGE(2) was measured after incubation with stromal cells, it was found to be similar to the concentrations showing maximal GJIC between the normal epithelial cells. When indomethacin was used to inhibit prostaglandin synthesis by stromal cells, GJIC was reduced but not eliminated between normal endometrial epithelial cells. These observations suggest that although PGE(2) secreted by stromal cells is an important mediator of GJIC between the epithelial cells, it is not the sole mediator. Transformed endometrial epithelial cells did not demonstrate GJIC even in the presence of stromal cells. However, we were able to re-establish GJIC in transformed epithelial cells when we added PGE(2) to the cells. Our findings show that PGE(2) may serve as an intercellular mediator between stromal and epithelial cells that regulates GJIC in normal and malignant epithelial cells. This suggests that maintenance of GJIC by preserving or replacing PGE(2) secretion by endometrial stromal cells may have the potential to suppress carcinogenesis in endometrial epithelial cells. Copyright © 2012 Elsevier Inc. All rights reserved.

The role of the intestinal microvasculature in inflammatory bowel disease: studies with a modified Caco-2 model including endothelial cells resembling the intestinal barrier in vitro.

PubMed

Kasper, Jennifer Y; Hermanns, Maria Iris; Cavelius, Christian; Kraegeloh, Annette; Jung, Thomas; Danzebrink, Rolf; Unger, Ronald E; Kirkpatrick, Charles James

The microvascular endothelium of the gut barrier plays a crucial role during inflammation in inflammatory bowel disease. We have modified a commonly used intestinal cell model based on the Caco-2 cells by adding microvascular endothelial cells (ISO-HAS-1). Transwell filters were used with intestinal barrier-forming Caco-2 cells on top and the ISO-HAS-1 on the bottom of the filter. The goal was to determine whether this coculture mimics the in vivo situation more closely, and whether the model is suitable to evaluate interactions of, for example, prospective nanosized drug vehicles or contrast agents with this coculture in a physiological and inflamed state as it would occur in inflammatory bowel disease. We monitored the inflammatory responsiveness of the cells (release of IL-8, soluble intercellular adhesion molecule 1, and soluble E-selectin) after exposure to inflammatory stimuli (lipopolysaccharide, TNF-α, INF-γ, IL1-β) and a nanoparticle (Ba/Gd: coprecipitated BaSO 4 and Gd(OH) 3 ), generally used as contrast agents. The barrier integrity of the coculture was evaluated via the determination of transepithelial electrical resistance and the apparent permeability coefficient (P app ) of NaFITC. The behavior of the coculture Caco-1/ISO-HAS-1 was compared to the respective monocultures Caco-2 and ISO-HAS-1. Based on transepithelial electrical resistance, the epithelial barrier integrity of the coculture remained stable during incubation with all stimuli, whereas the P app decreased after exposure to the cytokine mixture (TNF-α, INF-γ, IL1-β, and Ba/Gd). Both the endothelial and epithelial monocultures showed a high inflammatory response in both the upper and lower transwell-compartments. However, in the coculture, inflammatory mediators were only detected on the epithelial side and not on the endothelial side. Thus in the coculture, based on the P app , the epithelial barrier appears to prevent a potential inflammatory overreaction in the underlying endothelial cells. In summary, this coculture model exhibits in vivo-like features, which cannot be observed in conventional monocultures, making the former more suitable to study interactions with external stimuli.

Autoinducer-2 Production in Campylobacter jejuni Contributes to Chicken Colonization ▿

PubMed Central

Quiñones, Beatriz; Miller, William G.; Bates, Anna H.; Mandrell, Robert E.

2009-01-01

Inactivation of luxS, encoding an AI-2 biosynthesis enzyme, in Campylobacter jejuni strain 81-176 significantly reduced colonization of the chick lower gastrointestinal tract, chemotaxis toward organic acids, and in vitro adherence to LMH chicken hepatoma cells. Thus, AI-2 production in C. jejuni contributes to host colonization and interactions with epithelial cells. PMID:19011073

Pathogenesis of human diffusely adhering Escherichia coli expressing Afa/Dr adhesins (Afa/Dr DAEC): current insights and future challenges.

PubMed

Servin, Alain L

2014-10-01

The pathogenicity and clinical pertinence of diffusely adhering Escherichia coli expressing the Afa/Dr adhesins (Afa/Dr DAEC) in urinary tract infections (UTIs) and pregnancy complications are well established. In contrast, the implication of intestinal Afa/Dr DAEC in diarrhea is still under debate. These strains are age dependently involved in diarrhea in children, are apparently not involved in diarrhea in adults, and can also be asymptomatic intestinal microbiota strains in children and adult. This comprehensive review analyzes the epidemiology and diagnosis and highlights recent progress which has improved the understanding of Afa/Dr DAEC pathogenesis. Here, I summarize the roles of Afa/Dr DAEC virulence factors, including Afa/Dr adhesins, flagella, Sat toxin, and pks island products, in the development of specific mechanisms of pathogenicity. In intestinal epithelial polarized cells, the Afa/Dr adhesins trigger cell membrane receptor clustering and activation of the linked cell signaling pathways, promote structural and functional cell lesions and injuries in intestinal barrier, induce proinflammatory responses, create angiogenesis, instigate epithelial-mesenchymal transition-like events, and lead to pks-dependent DNA damage. UTI-associated Afa/Dr DAEC strains, following adhesin-membrane receptor cell interactions and activation of associated lipid raft-dependent cell signaling pathways, internalize in a microtubule-dependent manner within urinary tract epithelial cells, develop a particular intracellular lifestyle, and trigger a toxin-dependent cell detachment. In response to Afa/Dr DAEC infection, the host epithelial cells generate antibacterial defense responses. Finally, I discuss a hypothetical role of intestinal Afa/Dr DAEC strains that can act as "silent pathogens" with the capacity to emerge as "pathobionts" for the development of inflammatory bowel disease and intestinal carcinogenesis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Pathogenesis of Human Diffusely Adhering Escherichia coli Expressing Afa/Dr Adhesins (Afa/Dr DAEC): Current Insights and Future Challenges

PubMed Central

2014-01-01

SUMMARY The pathogenicity and clinical pertinence of diffusely adhering Escherichia coli expressing the Afa/Dr adhesins (Afa/Dr DAEC) in urinary tract infections (UTIs) and pregnancy complications are well established. In contrast, the implication of intestinal Afa/Dr DAEC in diarrhea is still under debate. These strains are age dependently involved in diarrhea in children, are apparently not involved in diarrhea in adults, and can also be asymptomatic intestinal microbiota strains in children and adult. This comprehensive review analyzes the epidemiology and diagnosis and highlights recent progress which has improved the understanding of Afa/Dr DAEC pathogenesis. Here, I summarize the roles of Afa/Dr DAEC virulence factors, including Afa/Dr adhesins, flagella, Sat toxin, and pks island products, in the development of specific mechanisms of pathogenicity. In intestinal epithelial polarized cells, the Afa/Dr adhesins trigger cell membrane receptor clustering and activation of the linked cell signaling pathways, promote structural and functional cell lesions and injuries in intestinal barrier, induce proinflammatory responses, create angiogenesis, instigate epithelial-mesenchymal transition-like events, and lead to pks-dependent DNA damage. UTI-associated Afa/Dr DAEC strains, following adhesin-membrane receptor cell interactions and activation of associated lipid raft-dependent cell signaling pathways, internalize in a microtubule-dependent manner within urinary tract epithelial cells, develop a particular intracellular lifestyle, and trigger a toxin-dependent cell detachment. In response to Afa/Dr DAEC infection, the host epithelial cells generate antibacterial defense responses. Finally, I discuss a hypothetical role of intestinal Afa/Dr DAEC strains that can act as “silent pathogens” with the capacity to emerge as “pathobionts” for the development of inflammatory bowel disease and intestinal carcinogenesis. PMID:25278576

Ultraviolet B Radiation Stimulates the Interaction between Nuclear Factor of Activated T Cells 5 (NFAT5) and Nuclear Factor-Kappa B (NF-κB) in Human Lens Epithelial Cells.

PubMed

Chung, Inyoung; Hah, Young-Sool; Ju, SunMi; Kim, Ji-Hye; Yoo, Woong-Sun; Cho, Hee-Young; Yoo, Ji-Myong; Seo, Seong-Wook; Choi, Wan-Sung; Kim, Seong-Jae

2017-07-01

Nuclear factor-kappa B (NF-κB) has been proposed as a therapeutic target for the treatment of cataracts. The authors investigated the relationship between nuclear factor of activated T cells 5 (NFAT5) and NF-κB in ultraviolet B (UVB)-irradiated human lens epithelial (HLE) cells. Human lens epithelial B-3 (HLE-B3) cells were exposed to UVB light at a dose of 10 mJ/cm 2 and then incubated for 24 h. Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) assay. Gene expression level of NFAT5 was determined using real-time quantitative polymerase chain reaction (qPCR). Protein expression levels of NFAT5, NF-κB p65, and α-smooth muscle actin (α-SMA) and the association of NFAT5 with the NF-κB p65 subunit were measured by Western blot analysis and a co-immunoprecipitation assay, respectively. The cellular distribution of NFAT5 and NF-κB p65 was examined by triple immunofluorescence staining. At 24 h after UVB exposure, cell viability significantly decreased in a dose-dependent manner, and UVB light (15 and 20 mJ/cm 2 ) significantly increased the ROS generation. UVB irradiation increased NFAT5 mRNA and protein levels and increased phosphorylation of NF-κB in HLE-B3 cells. α-SMA protein levels were increased in the irradiated cells. In addition, NFAT5 and NF-κB translocated from the cytoplasm to the nucleus, and binding between the p65 subunit and NFAT5 was increased. Exposure to UVB radiation induces nuclear translocation and stimulates binding between NFAT5 and NF-κB proteins in HLE-B3 cells. These interactions may form part of the biochemical mechanism of cataractogenesis in UVB-irradiated HLECs.

Nonwoven-Based Gelatin/Polycaprolactone Membrane Proves Suitability in a Preclinical Assessment for Treatment of Soft Tissue Defects

PubMed Central

Schulz, Simon; Angarano, Marco; Fabritius, Martin; Mülhaupt, Rolf; Dard, Michel; Obrecht, Marcel; Tomakidi, Pascal

2014-01-01

Standard preclinical assessments in vitro often have limitations regarding their transferability to human beings, mainly evoked by their nonhuman and tissue-different/nontissue-specific source. Here, we aimed at employing tissue-authentic simple and complex interactive fibroblast-epithelial cell systems and their in vivo-relevant biomarkers for preclinical in vitro assessment of nonwoven-based gelatin/polycaprolactone membranes (NBMs) for treatment of soft tissue defects. NBMs were composed of electrospun gelatin and polycaprolactone nanofiber nonwovens. Scanning electron microscopy in conjunction with actin/focal contact integrin fluorescence revealed successful adhesion and proper morphogenesis of keratinocytes and fibroblasts, along with cells' derived extracellular matrix deposits. The “feel-good factor” of cells under study on the NBM was substantiated by forming a confluent connective tissue entity, which was concomitant with a stratified epithelial equivalent. Immunohistochemistry proved tissue authenticity over time by abundance of the biomarker vimentin in the connective tissue entity, and chronological increase of keratins KRT1/10 and involucrin expression in epithelial equivalents. Suitability of the novel NBM as wound dressing was evidenced by an almost completion of epithelial wound closure in a pilot mini-pig study, after a surgical intervention-caused gingival dehiscence. In summary, preclinical assessment by tissue-authentic cell systems and the animal pilot study revealed the NBM as an encouraging therapeutic medical device for prospective clinical applications. PMID:24494668

An epithelial armamentarium to sense the microbiota.

PubMed

Prescott, David; Lee, Jooeun; Philpott, Dana J

2013-11-30

Intestinal epithelial cells were once thought to be inert, non-responsive cells that simply acted as a physical barrier that prevents the contents of the intestinal lumen from accessing the underlying tissue. However, it is now clear that these cells express a full repertoire of Toll- and Nod-like receptors, and that their activation by components of the microbiota is vital for the development of a functional epithelium, maintenance of barrier integrity, and defense against pathogenic organisms. Additionally, mounting evidence suggests that epithelial sensing of bacteria plays a significant role in the management of the numbers and types of microbes present in the gut microbiota via the production of antimicrobial peptides and other microbe-modulatory products. This is a critical process, as it is now becoming apparent that alterations in the composition of the microbiota can predispose an individual to a wide variety of chronic diseases. In this review, we will discuss the bacterial pattern recognition receptors that are known to be expressed by the intestinal epithelium, and how each of them individually contributes to these vital protective functions. Moreover, we will review what is known about the communication between epithelial cells and various classes of underlying leukocytes, and discuss how they interact with the microbiota to form a three-part relationship that maintains homeostasis in the gut. Copyright © 2013 Elsevier Ltd. All rights reserved.

Capsular Polysaccharide is a Main Component of Mycoplasma ovipneumoniae in the Pathogen-Induced Toll-Like Receptor-Mediated Inflammatory Responses in Sheep Airway Epithelial Cells

PubMed Central

Jiang, Zhongjia; Song, Fuyang; Li, Yanan; Xue, Di; Deng, Guangcun; Li, Min

2017-01-01

Mycoplasma ovipneumoniae (M. ovipneumoniae) is characterized as an etiological agent of primary atypical pneumonia that specifically infects sheep and goat. In an attempt to better understand the pathogen-host interaction between the invading M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory responses against capsular polysaccharide (designated as CPS) of M. ovipneumoniae using sheep bronchial epithelial cells cultured in an air-liquid interface (ALI) model. Results showed that CPS derived from M. ovipneumoniae could activate toll-like receptor- (TLR-) mediated inflammatory responses, along with an elevated expression of nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor 3 (IRF3) as well as various inflammatory-associated mediators, representatively including proinflammatory cytokines, such as IL1β, TNFα, and IL8, and anti-inflammatory cytokines such as IL10 and TGFβ of TLR signaling cascade. Mechanistically, the CPS-induced inflammation was TLR initiated and was mediated by activations of both MyD88-dependent and MyD88-independent signaling pathways. Of importance, a blockage of CPS with specific antibody led a significant reduction of M. ovipneumoniae-induced inflammatory responses in sheep bronchial epithelial cells. These results suggested that CPS is a key virulent component of M. ovipneumoniae, which may play a crucial role in the inflammatory response induced by M. ovipneumoniae infections. PMID:28553017

Altered prostate epithelial development and IGF-1 signal in mice lacking the androgen receptor in stromal smooth muscle cells.

PubMed

Yu, Shengqiang; Zhang, Caixia; Lin, Chiu-Chun; Niu, Yuanjie; Lai, Kuo-Pao; Chang, Hong-chiang; Yeh, Shauh-Der; Chang, Chawnshang; Yeh, Shuyuan

2011-04-01

Androgens and the androgen receptor (AR) play critical roles in the prostate development via mesenchymal-epithelial interactions. Smooth muscle cells (SMC), differentiated from mesenchyme, are one of the basic components of the prostate stroma. However, the roles of smooth muscle AR in prostate development are still obscure. We established the smooth muscle selective AR knockout (SM-ARKO) mouse model using the Cre-loxP system, and confirmed the ARKO efficiency at RNA, DNA and protein levels. Then, we observed the prostate morphology changes, and determined the epithelial proliferation, apoptosis, and differentiation. We also knocked down the AR in a prostate smooth muscle cell line (PS-1) to confirm the in vivo findings and to probe the mechanism. The AR was selectively and efficiently knocked out in the anterior prostates of SM-ARKO mouse. The SM-ARKO prostates have defects with loss of infolding structures, and decrease of epithelial proliferation, but with little change of apoptosis and differentiation. The mechanism studies showed that IGF-1 expression level decreased in the SM-ARKO prostates and AR-knockdown PS-1 cells. The decreased IGF-1 expression might contribute to the defective development of SM-ARKO prostates. The AR in SMCs plays important roles in the prostate development via the regulation of IGF-1 signal. Copyright © 2010 Wiley-Liss, Inc.

Epithelial Cell–Derived Secreted and Transmembrane 1a Signals to Activated Neutrophils during Pneumococcal Pneumonia

PubMed Central

Kamata, Hirofumi; Yamamoto, Kazuko; Wasserman, Gregory A.; Zabinski, Mary C.; Yuen, Constance K.; Lung, Wing Yi; Gower, Adam C.; Belkina, Anna C.; Ramirez, Maria I.; Deng, Jane C.; Quinton, Lee J.; Jones, Matthew R.

2016-01-01

Airway epithelial cell responses are critical to the outcome of lung infection. In this study, we aimed to identify unique contributions of epithelial cells during lung infection. To differentiate genes induced selectively in epithelial cells during pneumonia, we compared genome-wide expression profiles from three sorted cell populations: epithelial cells from uninfected mouse lungs, epithelial cells from mouse lungs with pneumococcal pneumonia, and nonepithelial cells from those same infected lungs. Of 1,166 transcripts that were more abundant in epithelial cells from infected lungs compared with nonepithelial cells from the same lungs or from epithelial cells of uninfected lungs, 32 genes were identified as highly expressed secreted products. Especially strong signals included two related secreted and transmembrane (Sectm) 1 genes, Sectm1a and Sectm1b. Refinement of sorting strategies suggested that both Sectm1 products were induced predominantly in conducting airway epithelial cells. Sectm1 was induced during the early stages of pneumococcal pneumonia, and mutation of NF-κB RelA in epithelial cells did not diminish its expression. Instead, type I IFN signaling was necessary and sufficient for Sectm1 induction in lung epithelial cells, mediated by signal transducer and activator of transcription 1. For target cells, Sectm1a bound to myeloid cells preferentially, in particular Ly6GbrightCD11bbright neutrophils in the infected lung. In contrast, Sectm1a did not bind to neutrophils from uninfected lungs. Sectm1a increased expression of the neutrophil-attracting chemokine CXCL2 by neutrophils from the infected lung. We propose that Sectm1a is an epithelial product that sustains a positive feedback loop amplifying neutrophilic inflammation during pneumococcal pneumonia. PMID:27064756

The influence of surface carbohydrates during in vitro infection of mammalian cells by the dermatophyte Trichophyton rubrum.

PubMed

Esquenazi, Daniele; Alviano, Celuta S; de Souza, Wanderley; Rozental, Sonia

2004-04-01

In order to better understand the role played by surface glycoconjugates during host cell adhesion and endocytosis of Trichophyton rubrum, we looked for the presence of carbohydrate-binding adhesins on the microconidia surface and their role on cellular interaction with epithelial and macrophages cells. The interaction of T. rubrum with chinese hamster ovary epithelial cells and their glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, that express mannose and galactose, respectively. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates to the interaction medium, pretreatment with lectins and with sodium periodate decreased the adhesion and endocytic index for all mutants. The ability of the fungus to penetrate into mammalian cells was confirmed in experiments using macrophages treated with cytochalasin D. Flow cytometric analysis showed that this fungus recognizes mannose and galactose. The binding was inhibited by the addition of methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside, and showed higher fluorescence intensity at 37 than at 28 degrees C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature for the microconidia adhesins. The presence of lectin-like molecules in fungus cell could be observed by scanning electron microscopy of the fungus incubated with colloidal-gold labeled neoglycoproteins. Our results suggest that T. rubrum has the ability to invade mammalian cells and expresses carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. These adhesins may play an important role on the adhesion and invasion of the fungus during the infectious process of dermatophytosis.

Cross talk between primary human renal tubular cells and endothelial cells in cocultures.

PubMed

Tasnim, Farah; Zink, Daniele

2012-04-15

Interactions between renal tubular epithelial cells and adjacent endothelial cells are essential for normal renal functions but also play important roles in renal disease and repair. Here, we investigated cocultures of human primary renal proximal tubular cells (HPTC) and human primary endothelial cells to address the cross talk between these cell types. HPTC showed improved proliferation, marker gene expression, and enzyme activity in cocultures. Also, the long-term maintenance of epithelia formed by HPTC was improved, which was due to the secretion of transforming growth factor-β1 and its antagonist α2-macroglobulin. HPTC induced endothelial cells to secrete increased amounts of these factors, which balanced each other functionally and only displayed in combination the observed positive effects. In addition, in the presence of HPTC endothelial cells expressed increased amounts of hepatocyte growth factor and vascular endothelial growth factor, which have well-characterized effects on renal tubular epithelial cells as well as on endothelial cells. Together, the results showed that HPTC stimulated endothelial cells to express a functionally balanced combination of various factors, which in turn improved the performance of HPTC. The results give new insights into the cross talk between renal epithelial and endothelial cells and suggest that cocultures could be also useful models for the analysis of cellular communication in renal disease and repair. Furthermore, the characterization of defined microenvironments, which positively affect HPTC, will be helpful for improving the performance of this cell type in in vitro applications including in vitro toxicology and kidney tissue engineering.

Kinetics of antibody-induced modulation of respiratory syncytial virus antigens in a human epithelial cell line

PubMed Central

Sarmiento, Rosa E; Tirado, Rocio G; Valverde, Laura E; Gómez-Garcia, Beatriz

2007-01-01

Background The binding of viral-specific antibodies to cell-surface antigens usually results in down modulation of the antigen through redistribution of antigens into patches that subsequently may be internalized by endocytosis or may form caps that can be expelled to the extracellular space. Here, by use of confocal-laser-scanning microscopy we investigated the kinetics of the modulation of respiratory syncytial virus (RSV) antigen by RSV-specific IgG. RSV-infected human epithelial cells (HEp-2) were incubated with anti-RSV polyclonal IgG and, at various incubation times, the RSV-cell-surface-antigen-antibody complexes (RSV Ag-Abs) and intracellular viral proteins were detected by indirect immunoflourescence. Results Interaction of anti-RSV polyclonal IgG with RSV HEp-2 infected cells induced relocalization and aggregation of viral glycoproteins in the plasma membrane formed patches that subsequently produced caps or were internalized through clathrin-mediated endocytosis participation. Moreover, the concentration of cell surface RSV Ag-Abs and intracellular viral proteins showed a time dependent cyclic variation and that anti-RSV IgG protected HEp-2 cells from viral-induced death. Conclusion The results from this study indicate that interaction between RSV cell surface proteins and specific viral antibodies alter the expression of viral antigens expressed on the cells surface and intracellular viral proteins; furthermore, interfere with viral induced destruction of the cell. PMID:17608950

Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

PubMed

Radtke, Andrea L; Herbst-Kralovetz, Melissa M

2012-04-03

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties. The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type(1, 7-13). Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri

PubMed Central

MATSUO, Yosuke; MIYOSHI, Yukihiro; OKADA, Sanae; SATOH, Eiichi

2012-01-01

A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion. PMID:24936355

Evaluation of a novel label-free photonic-crystal biosensor imaging system for the detection of prostate cancer cells

NASA Astrophysics Data System (ADS)

DeLuna, Frank; Ding, XiaoFie; Sun, Lu-Zhe; Ye, Jing Yong

2017-02-01

Biomarker screening for prostate-specific antigen (PSA) is the current clinical standard for detection of prostate cancer. However this method has shown many limitations, mainly in its specificity, which can lead to a high false positive rate. Thus, there is a growing need in developing a more specific detection system for prostate cancer. Using a Photonic- Crystal-based biosensor in a Total-Internal-Reflection (PC-TIR) configuration, we demonstrate the use of refractive index (RI) to accomplish label-free detection of prostate cancer cells against non-cancerous prostate epithelial cells. The PC-TIR biosensor possesses an open microcavity, which in contrast to traditional closed microcavities, allows for easier access of analyte molecules or cells to interact with its sensing surface. In this study, an imaging system was designed using the PC-TIR biosensor to quantify cell RI as the contrast parameter for prostate cancer detection. Non-cancerous BPH-1 prostate epithelial cells and prostate cancer PC-3 cells were placed on a single biosensor and measured concurrently. Recorded image data was then analyzed through a home-built MatLab program. Results demonstrate that RI is a suitable variable for differentiation between prostate cancer cells and non-cancerous prostate epithelial cells. Our study shows clinical potential in utilizing RI test for the detection of prostate cancer.

Hertwig's Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars.

PubMed

Yamamoto, Tsuneyuki; Yamada, Tamaki; Yamamoto, Tomomaya; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

2015-06-29

To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis.

Hertwig’s Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars

PubMed Central

Yamamoto, Tsuneyuki; Yamada, Tamaki; Yamamoto, Tomomaya; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

2015-01-01

To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis. PMID:26160988

Multiple Cellular Responses to Serotonin Contribute to Epithelial Homeostasis

PubMed Central

Pai, Vaibhav P.; Horseman, Nelson D.

2011-01-01

Epithelial homeostasis incorporates the paradoxical concept of internal change (epithelial turnover) enabling the maintenance of anatomical status quo. Epithelial cell differentiation and cell loss (cell shedding and apoptosis) form important components of epithelial turnover. Although the mechanisms of cell loss are being uncovered the crucial triggers that modulate epithelial turnover through regulation of cell loss remain undetermined. Serotonin is emerging as a common autocrine-paracine regulator in epithelia of multiple organs, including the breast. Here we address whether serotonin affects epithelial turnover. Specifically, serotonin's roles in regulating cell shedding, apoptosis and barrier function of the epithelium. Using in vivo studies in mouse and a robust model of differentiated human mammary duct epithelium (MCF10A), we show that serotonin induces mammary epithelial cell shedding and disrupts tight junctions in a reversible manner. However, upon sustained exposure, serotonin induces apoptosis in the replenishing cell population, causing irreversible changes to the epithelial membrane. The staggered nature of these events induced by serotonin slowly shifts the balance in the epithelium from reversible to irreversible. These finding have very important implications towards our ability to control epithelial regeneration and thus address pathologies of aberrant epithelial turnover, which range from degenerative disorders (e.g.; pancreatitis and thyrioditis) to proliferative disorders (e.g.; mastitis, ductal ectasia, cholangiopathies and epithelial cancers). PMID:21390323

Epithelial Cells in Urine: MedlinePlus Lab Test Information

MedlinePlus

... page: https://medlineplus.gov/labtests/epithelialcellsinurine.html Epithelial Cells in Urine To use the sharing features on ... page, please enable JavaScript. What is an Epithelial Cells in Urine Test? Epithelial cells are a type ...

Production of the Escherichia coli Common Pilus by Uropathogenic E. coli Is Associated with Adherence to HeLa and HTB-4 Cells and Invasion of Mouse Bladder Urothelium

PubMed Central

Carrillo-Casas, Erika Margarita; Durán, Laura; Zhang, Yushan; Hernández-Castro, Rigoberto; Puente, José L.; Daaka, Yehia; Girón, Jorge A.

2014-01-01

Uropathogenic Escherichia coli (UPEC) strains cause urinary tract infections and employ type 1 and P pili in colonization of the bladder and kidney, respectively. Most intestinal and extra-intestinal E. coli strains produce a pilus called E. coli common pilus (ECP) involved in cell adherence and biofilm formation. However, the contribution of ECP to the interaction of UPEC with uroepithelial cells remains to be elucidated. Here, we report that prototypic UPEC strains CFT073 and F11 mutated in the major pilin structural gene ecpA are significantly deficient in adherence to cultured HeLa (cervix) and HTB-4 (bladder) epithelial cells in vitro as compared to their parental strains. Complementation of the ecpA mutant restored adherence to wild-type levels. UPEC strains produce ECP upon growth in Luria-Bertani broth or DMEM tissue culture medium preferentially at 26°C, during incubation with cultured epithelial cells in vitro at 37°C, and upon colonization of mouse bladder urothelium ex vivo. ECP was demonstrated on and inside exfoliated bladder epithelial cells present in the urine of urinary tract infection patients. The ability of the CFT073 ecpA mutant to invade the mouse tissue was significantly reduced. The presence of ECP correlated with the architecture of the biofilms produced by UPEC strains on inert surfaces. These data suggest that ECP can potentially be produced in the bladder environment and contribute to the adhesive and invasive capabilities of UPEC during its interaction with the host bladder. We propose that along with other known adhesins, ECP plays a synergistic role in the multi-step infection of the urinary tract. PMID:25036370

Production of the Escherichia coli common pilus by uropathogenic E. coli is associated with adherence to HeLa and HTB-4 cells and invasion of mouse bladder urothelium.

PubMed

Saldaña, Zeus; De la Cruz, Miguel A; Carrillo-Casas, Erika Margarita; Durán, Laura; Zhang, Yushan; Hernández-Castro, Rigoberto; Puente, José L; Daaka, Yehia; Girón, Jorge A

2014-01-01

Uropathogenic Escherichia coli (UPEC) strains cause urinary tract infections and employ type 1 and P pili in colonization of the bladder and kidney, respectively. Most intestinal and extra-intestinal E. coli strains produce a pilus called E. coli common pilus (ECP) involved in cell adherence and biofilm formation. However, the contribution of ECP to the interaction of UPEC with uroepithelial cells remains to be elucidated. Here, we report that prototypic UPEC strains CFT073 and F11 mutated in the major pilin structural gene ecpA are significantly deficient in adherence to cultured HeLa (cervix) and HTB-4 (bladder) epithelial cells in vitro as compared to their parental strains. Complementation of the ecpA mutant restored adherence to wild-type levels. UPEC strains produce ECP upon growth in Luria-Bertani broth or DMEM tissue culture medium preferentially at 26°C, during incubation with cultured epithelial cells in vitro at 37°C, and upon colonization of mouse bladder urothelium ex vivo. ECP was demonstrated on and inside exfoliated bladder epithelial cells present in the urine of urinary tract infection patients. The ability of the CFT073 ecpA mutant to invade the mouse tissue was significantly reduced. The presence of ECP correlated with the architecture of the biofilms produced by UPEC strains on inert surfaces. These data suggest that ECP can potentially be produced in the bladder environment and contribute to the adhesive and invasive capabilities of UPEC during its interaction with the host bladder. We propose that along with other known adhesins, ECP plays a synergistic role in the multi-step infection of the urinary tract.

Gene Expression Profiles of Human Dendritic Cells Interacting with Aspergillus fumigatus in a Bilayer Model of the Alveolar Epithelium/Endothelium Interface

PubMed Central

Morton, Charles Oliver; Fliesser, Mirjam; Dittrich, Marcus; Mueller, Tobias; Bauer, Ruth; Kneitz, Susanne; Hope, William; Rogers, Thomas Richard; Einsele, Hermann; Loeffler, Juergen

2014-01-01

The initial stages of the interaction between the host and Aspergillus fumigatus at the alveolar surface of the human lung are critical in the establishment of aspergillosis. Using an in vitro bilayer model of the alveolus, including both the epithelium (human lung adenocarcinoma epithelial cell line, A549) and endothelium (human pulmonary artery epithelial cells, HPAEC) on transwell membranes, it was possible to closely replicate the in vivo conditions. Two distinct sub-groups of dendritic cells (DC), monocyte-derived DC (moDC) and myeloid DC (mDC), were included in the model to examine immune responses to fungal infection at the alveolar surface. RNA in high quantity and quality was extracted from the cell layers on the transwell membrane to allow gene expression analysis using tailored custom-made microarrays, containing probes for 117 immune-relevant genes. This microarray data indicated minimal induction of immune gene expression in A549 alveolar epithelial cells in response to germ tubes of A. fumigatus. In contrast, the addition of DC to the system greatly increased the number of differentially expressed immune genes. moDC exhibited increased expression of genes including CLEC7A, CD209 and CCL18 in the absence of A. fumigatus compared to mDC. In the presence of A. fumigatus, both DC subgroups exhibited up-regulation of genes identified in previous studies as being associated with the exposure of DC to A. fumigatus and exhibiting chemotactic properties for neutrophils, including CXCL2, CXCL5, CCL20, and IL1B. This model closely approximated the human alveolus allowing for an analysis of the host pathogen interface that complements existing animal models of IA. PMID:24870357

Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

PubMed

Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

2016-08-01

The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland. © 2016 International Federation for Cell Biology.

Canine corneal epithelial cells possess a sustained proliferative capacity and generate a spontaneously derived cell line.

PubMed

Morita, Maresuke; Fujita, Naoki; Abe, Momoko; Hayashimoto, Koji; Nakagawa, Takayuki; Nishimura, Ryohei; Tsuzuki, Keiko

2018-06-01

We have previously reported characteristics of canine corneal epithelial cells in vitro and found that canine corneal epithelial cells could maintain their proliferative capacity even after continuous culture without the use of feeder cells and growth promoting additives. The objective of this study was to elucidate proliferative characteristics of canine corneal epithelial cells independent of feeder cells and growth promoting additives, with the aim of developing a spontaneously derived corneal epithelial cell line. Canine and rabbit corneal epithelial cells were harvested from the limbus and cultured with, or without, feeder cells and growth promoting additives, and both were passaged continuously until growth arrest. Canine corneal epithelial cells could proliferate independently, and could be passaged more times than rabbit cells. A canine corneal epithelial cell line, cCEpi, which could be passaged more than 100 times without using feeder cells and growth promoting additives, was established. cCEpi cells maintained a cell morphology close to the primary culture and expressed p63, cytokeratin 15 (K15), and K3. Although changes in colony morphology, shortening of the population doubling time and a heteroploid karyotype were observed, cCEpi was not tumorigenic. Stratified cell sheets cultured from cCEpi were morphologically and immunohistologically similar to sheets cultivated from early passage cells. In conclusion, canine corneal epithelial cells can proliferate independent of feeder cells and growth promoting additives. cCEpi maintains properties similar to normal corneal epithelial cells and could be a useful source for studies in cellular biology and for developing novel therapies. Copyright © 2018 Elsevier Ltd. All rights reserved.

MUC1, the renaissance molecule.

PubMed

Gendler, S J

2001-07-01

MUC1 is a large, heavily glycosylated mucin expressed on the apical surfaces of most simple, secretory epithelia including the mammary gland, gastrointestinal, respiratory, urinary and reproductive tracts. Although MUC1 was thought to be an epithelial-specific protein, it is now known to be expressed on a variety of hematopoietic cells as well. Mucins function in protection and lubrication of epithelial surfaces. Transmembrane mucins, which contain cytoplasmic tail domains, appear to have additional functions through their abilities to interact with many proteins involved in signal transduction and cell adhesion. The goal of this review is to highlight recent discoveries that suggest that MUC1 may be a multifunctional protein, located on the surfaces of cells as a sensor of the environment, poised to signal to the interior when things go awry.

Percutaneous connectors

NASA Technical Reports Server (NTRS)

Picha, G. J.; Taylor, S. R.

1981-01-01

A surface possessing a regular array of micro-pillars was evaluated with regard to its ability to control epithelial downgrowth at the percutaneous interface. A range of pillar sizes were applied to the vertical segment of T shaped Biomer (R) implants. These percutaneous tabs were implanted into the dorsum of cats for a period of 6 weeks using a standardized surgical technique. Comments were made post-operatively and at the time of retrieval. A quantitative scoring system was applied to these observations as well as histological results. As observed, the pillar morphology used displayed the ability to control epithelial downgrowth. Collagen ingrowth into the interpillar spaces and possibly direct interactions of the epithelial cells with the morphology may account for the inhibition. The reproducibility of epithelial inhibition is, however, limited by other factors which are currently not well understood. These factors and potential methods of assessment are discussed.

Galectin-3 modulates the polarized surface delivery of β1-integrin in epithelial cells.

PubMed

Hönig, Ellena; Ringer, Karina; Dewes, Jenny; von Mach, Tobias; Kamm, Natalia; Kreitzer, Geri; Jacob, Ralf

2018-05-10

Epithelial cells require a precise intracellular transport and sorting machinery in order to establish and maintain their polarized architecture. This machinery includes beta-galactoside binding galectins for glycoprotein targeting to the apical membrane. Galectin-3 sorts cargo destined for the apical plasma membrane into vesicular carriers. After delivery of cargo to the apical milieu, galectin-3 recycles back into sorting organelles. We analyzed the role of galectin-3 in the polarized distribution of β1-integrin in MDCK cells. Integrins are located primarily at the basolateral domain of epithelial cells. We demonstrate that a minor pool of β1-integrin interacts with galectin-3 at the apical plasma membrane. Knockdown of galectin-3 decreases apical delivery of β1-integrin. This loss is restored by supplementation with recombinant galectin-3 and galectin-3 overexpression. Our data suggest that galectin-3 targets newly synthesized β1-integrin to the apical membrane and promotes apical delivery of β1-integrin internalized from the basolateral membrane. In parallel, galectin-3 knockout results in a reduction in cell proliferation and an impairment in proper cyst development. Our results suggest that galectin-3 modulates the surface distribution of β1-integrin and affects the morphogenesis of polarized cells. © 2018. Published by The Company of Biologists Ltd.

Transformation of Epithelial Ovarian Cancer Stemlike Cells into Mesenchymal Lineage via EMT Results in Cellular Heterogeneity and Supports Tumor Engraftment

PubMed Central

Jiang, Hua; Lin, Xiaolong; Liu, Yingtao; Gong, Wenjia; Ma, Xiaoling; Yu, Yinhua; Xie, Yi; Sun, Xiaoxi; Feng, Youji; Janzen, Viktor; Chen, Tong

2012-01-01

Ovarian cancers are heterogeneous and contain stemlike cells that are able to self-renew and are responsible for sustained tumor growth. Metastasis in the peritoneal cavity occurs more frequently in ovarian cancer than in other malignancies, but the underlying mechanism remains largely unknown. We have identified that ovarian cancer stemlike cells (CSCs), which were defined as side population (SP) cells, were present in patients’ ascitic fluid and mesenchymally transformed cell lines, ES-2 and HO-8910PM. SP cells, which were sorted from both cell lines and implanted into immunocompromised mice, were localized to the xenografted tumor boundary. In addition, SP cells exhibited an epithelial phenotype and showed a distinct gene expression profile with reduced expression of cell adhesion molecules (CAMs), indicating that SP cells exert an important role in ovarian cancer progression on the basis of their delicate interaction with the surrounding microenvironment and anatomical localization in tumors. In contrast, non-SP cells exhibited a more mesenchymal phenotype and showed more increased invasive potential than SP cells. This heterogeneity was observed as an endogenous transformation via the epithelial–mesenchymal transition (EMT) process. Inhibition of the EMT process by Snail1 silencing reduced the SP cell frequency, and affected their invasive capacity and engraftment. These findings illustrate the interplay between epithelial ovarian CSCs and the EMT, and exert a link to explain tumor heterogeneity and its necessity for ovarian cancer maintenance, metastasis and progression. PMID:22801793

Heparanase Localization during Palatogenesis in Mice

PubMed Central

Hirata, Azumi; Katayama, Kentaro; Tsuji, Takehito; Natsume, Nagato; Sugahara, Toshio; Koga, Yuichi; Otsuki, Yoshinori; Nakamura, Hiroaki

2013-01-01

Palatogenesis is directed by epithelial-mesenchymal interactions and results partly from remodeling of the extracellular matrix (ECM) of the palatal shelves. Here, we assessed heparanase distribution in developing mouse palates. No heparanase was observed in the vertically oriented palatal shelves in early stages of palate formation. As palate formation progressed, the palatal shelves were reorganized and arranged horizontally above the tongue, and heparanase localized to the epithelial cells of these shelves. When the palatal bilateral shelves first made contact, the heparanase localized to epithelial cells at the tips of shelves. Later in fusing palatal shelves, the cells of the medial epithelial seam (MES) were labeled with intense heparanase signal. In contrast, the basement membrane heparan sulfate (HS) was scarcely observed in the palatal shelves in contact. Moreover, perlecan labeling was sparse in the basement membrane of the MES, on which laminin and type IV collagen were observed. Moreover, we assessed the distribution of matrix metalloproteinase- (MMP-) 9, MMP-2, and MMP-3 in developing mouse palates and these MMPs were observed in the MES. Our findings indicated that heparanase was important for palate formation because it mediated degradation of the ECM of palatal shelves. Heparanase may, in concert with other proteases, participate in the regression of the MES. PMID:23509775

Rooster feathering, androgenic alopecia, and hormone dependent tumor growth: What is in common?

PubMed Central

Mayer, Julie Ann; Chuong, Cheng-Ming; Widelitz, Randall

2015-01-01

Different epithelial organs form as a result of epithelial - mesenchymal interactions and share a common theme modulated by variations (Chuong edit. In Molecular Basis of Epithelial Appendage Morphogenesis, 1998). One of the major modulators is the sex hormone pathway that acts on the prototype signaling pathway to alter organ phenotypes. Here we focus on how the sex hormone pathway interfaces with epithelia morphogenesis related signaling pathways. We first survey these sex hormone regulated morphogenetic processes in various epithelial organs. Sexual dimorphism of hairs and feathers has implications in sexual selection. Diseases of these pathways result in androgenic alopecia, hirsutism, henny feathering, etc. The growth and development of mammary glands, prostate glands and external genitalia essential for reproductive function are also dependent on sex hormones. Diseases affecting these organs include congenital anomalies and hormone dependent type of breast and prostate cancers. To study the role of sex hormones in new growth in the context of system biology / pathology, an in vivo model in which organ formation starts from stem cells is essential. With recent developments (Yu et al., The morphogenesis of feathers. Nature 420:308–312, 2002), the growth of tail feathers in roosters and hens has become a testable model in which experimental manipulations are possible. We show exemplary data of differences in their growth rate, proliferative cell population and signaling molecule expression. Working hypotheses are proposed on how the sex hormone pathways may interact with growth pathways. It is now possible to test these hypotheses using the chicken model to learn fundamental mechanisms on how sex hormones affect organogenesis, epithelial organ cycling, and growth related tumorigenesis. PMID:15617560

Helicobacter pylori: bacterial factors and interaction with the epithelial cells.

PubMed Central

Mégraud, F.

1996-01-01

Helicobacter pylori has been recognized as a major cause of most of the diseases of the stomach. These diseases are preceded by lesions of gastritis induced by H. pylori. This long-standing infection gives us a very good model of interaction between a bacterium and its host. We will review the direct and indirect effects of H. pylori. PMID:9041687

The interactions of single-wall carbon nanohorns with polar epithelium.

PubMed

Shi, Yujie; Shi, Zujin; Li, Suxin; Zhang, Yuan; He, Bing; Peng, Dong; Tian, Jie; Zhao, Ming; Wang, Xueqing; Zhang, Qiang

2017-01-01

Single-wall carbon nanohorns (SWCNHs), which have multitudes of horn interstices, an extensive surface area, and a spherical aggregate structure, offer many advantages over other carbon nanomaterials being used as a drug nanovector. The previous studies on the interaction between SWCNHs and cells have mostly emphasized on cellular uptake and intracellular trafficking, but seldom on epithelial cells. Polar epithelium as a typical biological barrier constitutes the prime obstacle for the transport of therapeutic agents to target site. This work tried to explore the permeability of SWCNHs through polar epithelium and their abilities to modulate transcellular transport, and evaluate the potential of SWCNHs in drug delivery. Madin-Darby canine kidney (MDCK) cell monolayer was used as a polar epithelial cell model, and as-grown SWCNHs, together with oxidized and fluorescein isothiocyanate-conjugated bovine serum albumin-labeled forms, were constructed and comprehensively investigated in vitro and in vivo. Various methods such as transmission electron microscopy and confocal imaging were used to visualize their intracellular uptake and localization, as well as to investigate the potential transcytotic process. The related mechanism was explored by specific inhibitors. Additionally, fast multispectral optoacoustic tomography imaging was used for monitoring the distribution and transport process of SWCNHs in vivo after oral administration in nude mice, as an evidence for their interaction with the intestinal epithelium. The results showed that SWCNHs had a strong bioadhesion property, and parts of them could be uptaken and transcytosed across the MDCK monolayer. Multiple mechanisms were involved in the uptake and transcytosis of SWCNHs with varying degrees. After oral administration, oxidized SWCNHs were distributed in the gastrointestinal tract and retained in the intestine for up to 36 h probably due to their surface adhesion and endocytosis into the intestinal epithelium. Overall, this comprehensive investigation demonstrated that SWCNHs can serve as a promising nanovector that can cross the barrier of polar epithelial cells and deliver drugs effectively.

Immune regulation by CD40-CD40-l interactions - 2; Y2K update.

PubMed

van Kooten, C

2000-11-01

CD40 is a cell surface receptor, which belongs to the TNF-R family, and which was first identified and functionally characterized on B lymphocytes. However, in recent years it has become clear that CD40 is expressed much broader, including expression on monocytes, dendritic cells, endothelial cells and epithelial cells. Therefore it is now thought that CD40 plays a more general role in immune regulation. The present paper reviews recent developments in this field of research, with main emphasis on CD40 signal transduction and on in vivo functions of CD40/CD40-L interactions.