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Sample records for acinar epithelial cells

  1. Metabotropic glutamate receptor 1 disrupts mammary acinar architecture and initiates malignant transformation of mammary epithelial cells

    PubMed Central

    Teh, Jessica L. F.; Shah, Raj; La Cava, Stephanie; Dolfi, Sonia C.; Mehta, Madhura S.; Kongara, Sameera; Price, Sandy; Ganesan, Shridar; Reuhl, Kenneth R.; Hirshfield, Kim M.

    2016-01-01

    Metabotropic glutamate receptor 1 (mGluR1/Grm1) is a member of the G-protein-coupled receptor superfamily, which was once thought to only participate in synaptic transmission and neuronal excitability, but has more recently been implicated in non-neuronal tissue functions. We previously described the oncogenic properties of Grm1 in cultured melanocytes in vitro and in spontaneous melanoma development with 100 % penetrance in vivo. Aberrant mGluR1 expression was detected in 60–80 % of human melanoma cell lines and biopsy samples. As most human cancers are of epithelial origin, we utilized immortalized mouse mammary epithelial cells (iMMECs) as a model system to study the transformative properties of Grm1. We introduced Grm1 into iMMECs and isolated several stable mGluR1-expressing clones. Phenotypic alterations in mammary acinar architecture were assessed using three-dimensional morphogenesis assays. We found that mGluR1-expressing iMMECs exhibited delayed lumen formation in association with decreased central acinar cell death, disrupted cell polarity, and a dramatic increase in the activation of the mitogen-activated protein kinase pathway. Orthotopic implantation of mGluR1-expressing iMMEC clones into mammary fat pads of immunodeficient nude mice resulted in mammary tumor formation in vivo. Persistent mGluR1 expression was required for the maintenance of the tumorigenic phenotypes in vitro and in vivo, as demonstrated by an inducible Grm1-silencing RNA system. Furthermore, mGluR1 was found be expressed in human breast cancer cell lines and breast tumor biopsies. Elevated levels of extracellular glutamate were observed in mGluR1-expressing breast cancer cell lines and concurrent treatment of MCF7 xenografts with glutamate release inhibitor, riluzole, and an AKT inhibitor led to suppression of tumor progression. Our results are likely relevant to human breast cancer, highlighting a putative role of mGluR1 in the pathophysiology of breast cancer and the potential

  2. The role of alpha 6 beta 1 integrin and EGF in normal and malignant acinar morphogenesis of human prostatic epithelial cells.

    PubMed

    Bello-DeOcampo, D; Kleinman, H K; Webber, M M

    2001-09-01

    Complex multiple interactions between cells and extracellular matrix occur during acinar morphogenesis involving integrin receptors and growth factors. Changes in these interactions occur during carcinogenesis as cells progress from a normal to a malignant, invasive phenotype. We have developed human prostatic epithelial cell lines of the same lineage, which represent multiple steps in carcinogenesis, similar to prostatic intraepithelial neoplasia and subsequent tumor progression. The non-tumorigenic, RWPE-1 and the tumorigenic WPE1-NB27 and WPE1-NB26 cell lines were used to examine their ability to undergo acinar morphogenesis in a 3-D cell culture model and its relationship to invasion, integrin expression and EGF presence. An inverse relationship between the degree of acinar formation and invasive ability was observed. The non-tumorigenic, non-invasive RWPE-1 and the low tumorigenic, low invasive, WPE1-NB27 cells show high and decreased acinar forming ability, respectively, while the more invasive WPE1-NB26 cells show a loss of acinar formation. While RWPE-1 acini show basal expression of alpha 6 beta 1 integrin, which correlates with their ability to polarize and form acini, WPE1-NB27 cells lack alpha 6 but show basal, but weaker expression of beta 1 integrin. WPE1-NB26 cells show loss alpha 6 and abnormal, diffused beta 1 integrin expression. A dose-dependent decrease in acinar formation was observed in RWPE-1 cells when cell proliferation was induced by EGF. Anti-functional antibody to EGF caused an increase in acinar formation in RWPE-1 cells. These results suggest that malignant cells lose the ability to undergo acinar morphogenesis and that the degree of this loss appears to be related to invasive ability, EGF levels and alterations in laminin-specific integrin expression. This model system mimics different steps in prostate carcinogenesis and has applications in the secondary and tertiary prevention of prostate cancer.

  3. Primary retroperitoneal acinar cell cystadenoma.

    PubMed

    Pesci, Anna; Castelli, Paola; Facci, Enrico; Romano, Luigi; Zamboni, Giuseppe

    2012-03-01

    In this report, we describe a case of hitherto unreported primary retroperitoneal acinar cell cystadenoma that morphologically and immunophenotypically resembled pancreatic acinar cell cystadenoma. Pancreatic acinar cell cystadenoma is a very uncommon benign lesion characterized by acinar cell differentiation, the evidence of pancreatic exocrine enzyme production, and the absence of cellular atypia. Our case occurred in a 55-year-old woman presenting a 10-cm multilocular cystic lesion in the retroperitoneum thought to be a mucinous cystic neoplasm. At laparotomy, the cystic mass, which showed no connection with any organ, was completely resected with a clinical diagnosis of cystic lymphangioma. The diagnosis of retroperitoneal acinar cell cystadenoma was based on the recognition of morphological acinar differentiation, the immunohistochemical demonstration of the acinar marker trypsin, and the absence of cellular atypia. These peculiar features can be used in the differential diagnosis with all the other cystic lesions of the retroperitoneum.

  4. Comparison of several radiation effects in human MCF10A mammary epithelial cells cultured as 2D monolayers or 3D acinar stuctures in matrigel.

    PubMed

    Lin, Yu-Fen; Nagasawa, Hatsumi; Peng, Yuanlin; Chuang, Eric Y; Bedford, Joel S

    2009-06-01

    It has been argued that the cell-cell and cell-matrix interaction networks in normal tissues are disrupted by radiation and that this largely controls many of the most important cellular radiation responses. This has led to the broader assertion that individual cells in normal tissue or a 3D normal-tissue-like culture will respond to radiation very differently than the same cells in a 2D monolayer culture. While many studies have shown that, in some cases, cell-cell contact in spheroids of transformed or tumor cell lines can alter radiation responses relative to those for the same cells in monolayer cultures, a question remains regarding the possible effect of the above-mentioned disruption of signaling networks that operate more specifically for cells in normal tissues or in a 3D tissue-like context. To test the generality of this notion, we used human MCF-10A cells, an immortalized mammary epithelial cell line that produces acinar structures in culture with many properties of human mammary ducts. We compared the dose responses for these cells in the 2D monolayer and in 3D ductal or acinar structures. The responses examined were reproductive cell death, induction of chromosomal aberrations, and the levels of gamma-H2AX foci in cells after single acute gamma-ray doses and immediately after 20 h of irradiation at a dose rate of 0.0017 Gy/min. We found no significant differences in the dose responses of these cells in 2D or 3D growth conditions. While this does not mean that such differences cannot occur in other situations, it does mean that they do not generally or necessarily occur.

  5. Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion

    PubMed Central

    Vanni, Cristina; Ognibene, Marzia; Finetti, Federica; Mancini, Patrizia; Cabodi, Sara; Segalerba, Daniela; Torrisi, Maria Rosaria; Donnini, Sandra; Bosco, Maria Carla; Varesio, Luigi; Eva, Alessandra

    2015-01-01

    The proteins of the Dbl family are guanine nucleotide exchange factors (GEFs) of Rho GTPases and are known to be involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders, neoplastic transformation, and tumor metastasis. We have previously demonstrated that expression of Dbl oncogene in lens epithelial cells modulates genes encoding proteins involved in epithelial-mesenchymal-transition (EMT) and induces angiogenesis in the lens. Our present study was undertaken to investigate the role of Dbl oncogene in epithelial cells transformation, providing new insights into carcinoma progression.To assess how Dbl oncogene can modulate EMT, cell migration, morphogenesis, and expression of pro-apoptotic and angiogenic factors we utilized bi- and 3-dimensional cultures of MCF-10 A cells. We show that upon Dbl expression MCF-10 A cells undergo EMT. In addition, we found that Dbl overexpression sustains Cdc42 and Rac activation inducing morphological alterations, characterized by the presence of lamellipodia and conferring a high migratory capacity to the cells. Moreover, Dbl expressing MCF-10 A cells form altered 3D structures and can induce angiogenesis by producing proangiogenic factors such as CCL2. These results support a role for Dbl oncogene in epithelial cell differentiation and transformation and suggest the relevance of GEF deregulation in tumor onset and progression. PMID:25723869

  6. Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion.

    PubMed

    Vanni, Cristina; Ognibene, Marzia; Finetti, Federica; Mancini, Patrizia; Cabodi, Sara; Segalerba, Daniela; Torrisi, Maria Rosaria; Donnini, Sandra; Bosco, Maria Carla; Varesio, Luigi; Eva, Alessandra

    2015-01-01

    The proteins of the Dbl family are guanine nucleotide exchange factors (GEFs) of Rho GTPases and are known to be involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders, neoplastic transformation, and tumor metastasis. We have previously demonstrated that expression of Dbl oncogene in lens epithelial cells modulates genes encoding proteins involved in epithelial-mesenchymal-transition (EMT) and induces angiogenesis in the lens. Our present study was undertaken to investigate the role of Dbl oncogene in epithelial cells transformation, providing new insights into carcinoma progression.To assess how Dbl oncogene can modulate EMT, cell migration, morphogenesis, and expression of pro-apoptotic and angiogenic factors we utilized bi- and 3-dimensional cultures of MCF-10 A cells. We show that upon Dbl expression MCF-10 A cells undergo EMT. In addition, we found that Dbl overexpression sustains Cdc42 and Rac activation inducing morphological alterations, characterized by the presence of lamellipodia and conferring a high migratory capacity to the cells. Moreover, Dbl expressing MCF-10 A cells form altered 3D structures and can induce angiogenesis by producing proangiogenic factors such as CCL2. These results support a role for Dbl oncogene in epithelial cell differentiation and transformation and suggest the relevance of GEF deregulation in tumor onset and progression.

  7. The ryanodine receptor is expressed in human pancreatic acinar cells and contributes to acinar cell injury.

    PubMed

    Lewarchik, Christopher M; Orabi, Abrahim I; Jin, Shunqian; Wang, Dong; Muili, Kamaldeen A; Shah, Ahsan U; Eisses, John F; Malik, Adeel; Bottino, Rita; Jayaraman, Thottala; Husain, Sohail Z

    2014-09-01

    Physiological calcium (Ca(2+)) signals within the pancreatic acinar cell regulate enzyme secretion, whereas aberrant Ca(2+) signals are associated with acinar cell injury. We have previously identified the ryanodine receptor (RyR), a Ca(2+) release channel on the endoplasmic reticulum, as a modulator of these pathological signals. In the present study, we establish that the RyR is expressed in human acinar cells and mediates acinar cell injury. We obtained pancreatic tissue from cadaveric donors and identified isoforms of RyR1 and RyR2 by qPCR. Immunofluorescence staining of the pancreas showed that the RyR is localized to the basal region of the acinar cell. Furthermore, the presence of RyR was confirmed from isolated human acinar cells by tritiated ryanodine binding. To determine whether the RyR is functionally active, mouse or human acinar cells were loaded with the high-affinity Ca(2+) dye (Fluo-4 AM) and stimulated with taurolithocholic acid 3-sulfate (TLCS) (500 μM) or carbachol (1 mM). Ryanodine (100 μM) pretreatment reduced the magnitude of the Ca(2+) signal and the area under the curve. To determine the effect of RyR blockade on injury, human acinar cells were stimulated with pathological stimuli, the bile acid TLCS (500 μM) or the muscarinic agonist carbachol (1 mM) in the presence or absence of the RyR inhibitor ryanodine. Ryanodine (100 μM) caused an 81% and 47% reduction in acinar cell injury, respectively, as measured by lactate dehydrogenase leakage (P < 0.05). Taken together, these data establish that the RyR is expressed in human acinar cells and that it modulates acinar Ca(2+) signals and cell injury.

  8. Duct Cells Contribute to Regeneration of Endocrine and Acinar Cells Following Pancreatic Damage in Adult Mice

    PubMed Central

    CRISCIMANNA, ANGELA; SPEICHER, JULIE A.; HOUSHMAND, GOLBAHAR; SHIOTA, CHIYO; PRASADAN, KRISHNA; Ji, BAOAN; LOGSDON, CRAIG D.; GITTES, GEORGE K.; ESNI, FARZAD

    2015-01-01

    BACKGROUND & AIMS There have been conflicting results on a cell of origin in pancreatic regeneration. These discrepancies predominantly stem from lack of specific markers for the pancreatic precursors/stem cells, as well as differences in the targeted cells and severity of tissue injury in the experimental models so far proposed. We attempted to create a model that used diphtheria toxin receptor (DTR) to ablate specific cell populations, control the extent of injury, and avoid induction of the inflammatory response. METHODS To target specific types of pancreatic cells, we crossed R26DTR or R26dtR/lacZ mice with transgenic mice that express the Cre recombinase in the pancreas, under control of the Pdx1 (global pancreatic) or elastase (acinar-specific) promoters. RESULTS Exposure of PdxCre;R26DTR mice to diphtheria toxin resulted in extensive ablation of acinar and endocrine tissues but not ductal cells. Surviving cells within the ductal compartment contributed to regeneration of endocrine and acinar cells via recapitulation of the embryonic pancreatic developmental program. However, following selective ablation of acinar tissue in ElaCre-ERT2;R26DTR mice, regeneration likely occurred by reprogramming of ductal cells to acinar lineage. CONCLUSIONS In the pancreas of adult mice, epithelial cells within the ductal compartment contribute to regeneration of endocrine and acinar cells. The severity of injury determines the regenerative mechanisms and cell types that contribute to this process. PMID:21763240

  9. Basal autophagy maintains pancreatic acinar cell homeostasis and protein synthesis and prevents ER stress

    PubMed Central

    Antonucci, Laura; Fagman, Johan B.; Kim, Ju Youn; Todoric, Jelena; Gukovsky, Ilya; Mackey, Mason; Ellisman, Mark H.; Karin, Michael

    2015-01-01

    Pancreatic acinar cells possess very high protein synthetic rates as they need to produce and secrete large amounts of digestive enzymes. Acinar cell damage and dysfunction cause malnutrition and pancreatitis, and inflammation of the exocrine pancreas that promotes development of pancreatic ductal adenocarcinoma (PDAC), a deadly pancreatic neoplasm. The cellular and molecular mechanisms that maintain acinar cell function and whose dysregulation can lead to tissue damage and chronic pancreatitis are poorly understood. It was suggested that autophagy, the principal cellular degradative pathway, is impaired in pancreatitis, but it is unknown whether impaired autophagy is a cause or a consequence of pancreatitis. To address this question, we generated Atg7Δpan mice that lack the essential autophagy-related protein 7 (ATG7) in pancreatic epithelial cells. Atg7Δpan mice exhibit severe acinar cell degeneration, leading to pancreatic inflammation and extensive fibrosis. Whereas ATG7 loss leads to the expected decrease in autophagic flux, it also results in endoplasmic reticulum (ER) stress, accumulation of dysfunctional mitochondria, oxidative stress, activation of AMPK, and a marked decrease in protein synthetic capacity that is accompanied by loss of rough ER. Atg7Δpan mice also exhibit spontaneous activation of regenerative mechanisms that initiate acinar-to-ductal metaplasia (ADM), a process that replaces damaged acinar cells with duct-like structures. PMID:26512112

  10. Expression of claudin-5 in canine pancreatic acinar cell carcinoma - An immunohistochemical study.

    PubMed

    Jakab, Csaba; Rusvai, Miklós; Gálfi, Péter; Halász, Judit; Kulka, Janina

    2011-03-01

    Claudin-5 is an endothelium-specific tight junction protein. The aim of the present study was to detect the expression pattern of this molecule in intact pancreatic tissues and in well-differentiated and poorly differentiated pancreatic acinar cell carcinomas from dogs by the use of cross-reactive humanised anticlaudin-5 antibody. The necropsy samples taken from dogs included 10 nonneoplastic pancreatic tissues, 10 well-differentiated pancreatic acinar cell carcinomas, 10 poorly differentiated pancreatic acinar cell carcinomas, 5 intrahepatic metastases of well-differentiated and 5 intrahepatic metastases of poorly differentiated acinar cell carcinomas. A strong lateral membrane claudin-5 positivity was detected in exocrine cells in all intact pancreas samples. The endocrine cells of the islets of Langerhans and the epithelial cells of the ducts were negative for claudin-5. The endothelial cells of vessels and lymphatic channels in the stroma of the intact pancreas showed strong membrane positivity for this claudin. All well-differentiated exocrine pancreas carcinomas and all poorly-differentiated pancreatic acinar cell carcinoma samples showed a diffuse loss of claudin-5 expression. The claudin-5-positive peritumoural vessels and lymphatic channels facilitated the detection of vascular invasion of the claudin-5-negative cancer cells. In liver metastasis samples, the pancreatic carcinomas were negative for claudin-5. It seems that the loss of expression of claudin-5 may lead to carcinogenesis in canine exocrine pancreatic cells.

  11. Case report. Acinar cell carcinoma with fatty change arising from the pancreas.

    PubMed

    Chung, W-S; Park, M-S; Kim, D W; Kim, K W

    2011-12-01

    Acinar cell carcinoma of the pancreas is a rare malignant tumour developing from acinar cells, accounting for approximately 1% of pancreatic exocrine tumours. We experienced a case of an acinar cell carcinoma with fatty change. To the best of our knowledge, this is the first case report of an acinar cell carcinoma with fatty change in the clinical literature.

  12. CFTR-Mediated Cl− Transport in the Acinar and Duct Cells of Rabbit Lacrimal Gland

    PubMed Central

    Lu, Michael; Ding, Chuanqing

    2013-01-01

    Purpose We investigated the role that the cystic fibrosis transmembrane conductance regulator (CFTR) may play in Cl− transport in the acinar and ductal epithelial cells of rabbit lacrimal gland (LG). Methods Primary cultured LG acinar cells were processed for whole-cell patch-clamp electrophysiological recording of Cl− currents by using perfusion media with high and low [Cl−], 10 μM forskolin and 100 μM 3-isobutyl-1-methylxanthine (IBMX), the non-specific Cl− channel blocker 4,4′-disothiocyanostilbene-2, 2′ sulphonic acid (DIDS; 100 μM) and CFTRinh-172 (10 μM), a specific blocker for CFTR. Ex vivo live cell imaging of [Cl−] changes in duct cells was performed on freshly dissected LG duct with a multiphoton confocal laser scanning microscope using a Cl− sensitive fluorescence dye, N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide. Results Whole-cell patch-clamp studies demonstrated the presence of Cl− current in isolated acinar cells and revealed that this Cl− current was mediated by CFTR channel. Live cell imaging also showed the presence of CFTR-mediated Cl− transport across the plasma membrane of duct cells. Conclusions Our previous data showed the presence of CFTR in all acinar and duct cells within the rabbit LG, with expression most prominent in the apical membranes of duct cells. The present study demonstrates that CFTR is actively involved in Cl− transport in both acinar cells and epithelial cells from duct segments, suggesting that CFTR may play a significant role in LG secretion. PMID:22578307

  13. TGF-β1 promotes acinar to ductal metaplasia of human pancreatic acinar cells

    PubMed Central

    Liu, Jun; Akanuma, Naoki; Liu, Chengyang; Naji, Ali; Halff, Glenn A.; Washburn, William K.; Sun, Luzhe; Wang, Pei

    2016-01-01

    Animal studies suggest that pancreatitis-induced acinar-to-ductal metaplasia (ADM) is a key event for pancreatic ductal adenocarcinoma (PDAC) initiation. However, there has not been an adequate system to explore the mechanisms of human ADM induction. We have developed a flow cytometry-based, high resolution lineage tracing method and 3D culture system to analyse ADM in human cells. In this system, well-known mouse ADM inducers did not promote ADM in human cells. In contrast, TGF-β1 efficiently converted human acinar cells to duct-like cells (AD) in a SMAD-dependent manner, highlighting fundamental differences between the species. Functionally, AD cells gained transient proliferative capacity. Furthermore, oncogenic KRAS did not induce acinar cell proliferation, but did sustain the proliferation of AD cells, suggesting that oncogenic KRAS requires ADM-associated-changes to promote PDAC initiation. This ADM model provides a novel platform to explore the mechanisms involved in the development of human pancreatic diseases. PMID:27485764

  14. Role and regulation of autophagy in the development of acinar structures formed by bovine BME-UV1 mammary epithelial cells.

    PubMed

    Sobolewska, Agnieszka; Motyl, Tomasz; Gajewska, Malgorzata

    2011-10-01

    Autophagy is a catabolic process providing an alternative energy source for cells under stressful conditions such as starvation, growth factor deprivation or hypoxia. During involution of the bovine mammary gland autophagy is induced in mammary epithelial cells (MECs) as a survival mechanism, and is tightly regulated by hormones and growth factors necessary for gland development. In the present study we adapted the three-dimensional culture model to investigate the role of autophagy during formation of alveoli-like structures by bovine BME-UV1 MECs grown on extracellular matrix (ECM) components. Using confocal microscopy and Western-blot analyses of autophagic and apoptotic markers: LC3, and cleaved caspase-3, we showed that autophagy was induced in centrally localized cells within the developing acini. These cells lacked a direct contact with ECM, and formed a distinct population from the outer layer of cells. Induction of autophagy preceded apoptosis, but did not inhibit the formation of a hollow lumen. In the presence of steroid hormones: 17β-estradiol and progesterone, although autophagy was augmented, acini formation proceeded normally. In contrast, the major lactogenic hormone: prolactin, which supports functional differentiation of alveoli, did not alter induction of autophagy within the spheroids. BME-UV1 cells cultured on Matrigel in the presence of growth factors IGF-I and EGF formed larger, underdeveloped acini without lumens due to caspase-3 inhibition, and sustained autophagy in the centre of the spheroids, while TGF-β1 accelerated apoptosis, and increased autophagy significantly. Our observations suggest that sex steroids 17β-estradiol and progesterone, as well as growth factor TGF-β1 may regulate the development of the bovine mammary gland by inducing autophagy in addition to regulating proliferation and apoptosis of MECs. These data indicate that autophagy may play an important role during alveolargenesis.

  15. Therapeutic potential of targeting acinar cell reprogramming in pancreatic cancer.

    PubMed

    Wong, Chi-Hin; Li, You-Jia; Chen, Yang-Chao

    2016-08-21

    Pancreatic ductal adenocarcinoma (PDAC) is a common pancreatic cancer and the fourth leading cause of cancer death in the United States. Treating this life-threatening disease remains challenging due to the lack of effective prognosis, diagnosis and therapy. Apart from pancreatic duct cells, acinar cells may also be the origin of PDAC. During pancreatitis or combined with activating KRas(G12D) mutation, acinar cells lose their cellular identity and undergo a transdifferentiation process called acinar-to-ductal-metaplasia (ADM), forming duct cells which may then transform into pancreatic intraepithelial neoplasia (PanIN) and eventually PDAC. During ADM, the activation of mitogen-activated protein kinases, Wnt, Notch and phosphatidylinositide 3-kinases/Akt signaling inhibits the transcription of acinar-specific genes, including Mist and amylase, but promotes the expression of ductal genes, such as cytokeratin-19. Inhibition of this transdifferentiation process hinders the development of PanIN and PDAC. In addition, the transdifferentiated cells regain acinar identity, indicating ADM may be a reversible process. This provides a new therapeutic direction in treating PDAC through cancer reprogramming. Many studies have already demonstrated the success of switching PanIN/PDAC back to normal cells through the use of PD325901, the expression of E47, and the knockdown of Dickkopf-3. In this review, we discuss the signaling pathways involved in ADM and the therapeutic potential of targeting reprogramming in order to treat PDAC.

  16. Effects of Benzodiazepines on Acinar and Myoepithelial Cells

    PubMed Central

    Mattioli, Tatiana M. F.; Alanis, Luciana R. A.; Sapelli, Silvana da Silva; de Lima, Antonio A. S.; de Noronha, Lucia; Rosa, Edvaldo A. R.; Althobaiti, Yusuf S.; Almalki, Atiah H.; Sari, Youssef; Ignacio, Sergio A.; Johann, Aline C. B. R.; Gregio, Ana M. T.

    2016-01-01

    Background: Benzodiazepines (BZDs), the most commonly prescribed psychotropic drugs with anxiolytic action, may cause hyposalivation. It has been previously shown that BZDs can cause hypertrophy and decrease the acini cell number. In this study, we investigated the effects of BZDs and pilocarpine on rat parotid glands, specifically on acinar, ductal, and myoepithelial cells. Methods: Ninety male Wistar rats were divided into nine groups. Control groups received a saline solution for 30 days (C30) and 60 days (C60), and pilocarpine (PILO) for 60 days. Experimental groups received lorazepam (L30) and midazolam (M30) for 30 days. Another group (LS60 or MS60) received lorazepam or midazolam for 30 days, respectively, and saline for additional 30 days. Finally, other groups (LP60 or MP60) received either lorazepam or midazolam for 30 days, respectively, and pilocarpine for additional 30 days. The expression of calponin in myoepithelial cells and the proliferating cell nuclear antigen (PCNA) in acinar and ductal cells were evaluated. Results: Animals treated with lorazepam showed an increase in the number of positive staining cells for calponin as compared to control animals (p < 0.05). Midazolam administered with pilocarpine (MP60) induced an increase in the proliferation of acinar and ductal cells and a decrease in the positive staining cells for calponin as compared to midazolam administered with saline (MS60). Conclusion: We found that myoepithelial cells might be more sensitive to the effects of BZD than acinar and ductal cells in rat parotid glands. PMID:27445812

  17. Loss of acinar cell IKKα triggers spontaneous pancreatitis in mice

    PubMed Central

    Li, Ning; Wu, Xuefeng; Holzer, Ryan G.; Lee, Jun-Hee; Todoric, Jelena; Park, Eek-Joong; Ogata, Hisanobu; Gukovskaya, Anna S.; Gukovsky, Ilya; Pizzo, Donald P.; VandenBerg, Scott; Tarin, David; Atay, Çiǧdem; Arkan, Melek C.; Deerinck, Thomas J.; Moscat, Jorge; Diaz-Meco, Maria; Dawson, David; Erkan, Mert; Kleeff, Jörg; Karin, Michael

    2013-01-01

    Chronic pancreatitis is an inflammatory disease that causes progressive destruction of pancreatic acinar cells and, ultimately, loss of pancreatic function. We investigated the role of IκB kinase α (IKKα) in pancreatic homeostasis. Pancreas-specific ablation of IKKα (IkkαΔpan) caused spontaneous and progressive acinar cell vacuolization and death, interstitial fibrosis, inflammation, and circulatory release of pancreatic enzymes, clinical signs resembling those of human chronic pancreatitis. Loss of pancreatic IKKα causes defective autophagic protein degradation, leading to accumulation of p62-mediated protein aggregates and enhanced oxidative and ER stress in acinar cells, but none of these effects is related to NF-κB. Pancreas-specific p62 ablation prevented ER and oxidative stresses and attenuated pancreatitis in IkkαΔpan mice, suggesting that cellular stress induced by p62 aggregates promotes development of pancreatitis. Importantly, downregulation of IKKα and accumulation of p62 aggregates were also observed in chronic human pancreatitis. Our studies demonstrate that IKKα, which may control autophagic protein degradation through its interaction with ATG16L2, plays a critical role in maintaining pancreatic acinar cell homeostasis, whose dysregulation promotes pancreatitis through p62 aggregate accumulation. PMID:23563314

  18. Encapsulation of primary salivary gland cells in enzymatically degradable poly(ethylene glycol) hydrogels promotes acinar cell characteristics.

    PubMed

    Shubin, Andrew D; Felong, Timothy J; Schutrum, Brittany E; Joe, Debria S L; Ovitt, Catherine E; Benoit, Danielle S W

    2017-03-01

    Radiation therapy for head and neck cancers leads to permanent xerostomia due to the loss of secretory acinar cells in the salivary glands. Regenerative treatments utilizing primary submandibular gland (SMG) cells show modest improvements in salivary secretory function, but there is limited evidence of salivary gland regeneration. We have recently shown that poly(ethylene glycol) (PEG) hydrogels can support the survival and proliferation of SMG cells as multicellular spheres in vitro. To further develop this approach for cell-based salivary gland regeneration, we have investigated how different modes of PEG hydrogel degradation affect the proliferation, cell-specific gene expression, and epithelial morphology within encapsulated salivary gland spheres. Comparison of non-degradable, hydrolytically-degradable, matrix metalloproteinase (MMP)-degradable, and mixed mode-degradable hydrogels showed that hydrogel degradation by any mechanism is required for significant proliferation of encapsulated cells. The expression of acinar phenotypic markers Aqp5 and Nkcc1 was increased in hydrogels that are MMP-degradable compared with other hydrogel compositions. However, expression of secretory acinar proteins Mist1 and Pip was not maintained to the same extent as phenotypic markers, suggesting changes in cell function upon encapsulation. Nevertheless, MMP- and mixed mode-degradability promoted organization of polarized cell types forming tight junctions and expression of the basement membrane proteins laminin and collagen IV within encapsulated SMG spheres. This work demonstrates that cellularly remodeled hydrogels can promote proliferation and gland-like organization by encapsulated salivary gland cells as well as maintenance of acinar cell characteristics required for regenerative approaches. Investigation is required to identify approaches to further enhance acinar secretory properties.

  19. Functional somatostatin receptors on a rat pancreatic acinar cell line

    SciTech Connect

    Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.; Clerc, P.; Logsdon, C.; Svoboda, M.; Susini, C.; Vaysse, N.; Ribet, A. Mount Zion Hospital and Medical Center, San Francisco, CA Universite Libre de Bruxelles, Brussels )

    1988-07-01

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of {sup 125}I-(Tyr{sup 11})Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 {plus minus} 20 fmol/10{sup 6} cells. Somatostatin receptor structure was analyzed by covalently cross-linking {sup 125}I-(Tyr{sup 11})somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N{sub i} to inhibit adenylate cyclase.

  20. Ca2+-activated K channels in parotid acinar cells

    PubMed Central

    Romanenko, Victor G; Thompson, Jill

    2010-01-01

    Fluid secretion relies on a close interplay between Ca2+-activated Cl and K channels. Salivary acinar cells contain both large conductance, BK, and intermediate conductance, IK1, K channels. Physiological fluid secretion occurs with only modest (<500 nM) increases in intracellular Ca2+ levels but BK channels in many cell types and in heterologous expression systems require very high concentrations for significant activation. We report here our efforts to understand this apparent contradiction. We determined the Ca2+ dependence of IK1 and BK channels in mouse parotid acinar cells. IK1 channels activated with an apparent Ca2+ affinity of about 350 nM and a hill coefficient near 3. Native parotid BK channels activated at similar Ca2+ levels unlike the BK channels in other cell types. Since the parotid BK channel is encoded by an uncommon splice variant, we examined this clone in a heterologous expression system. In contrast to the native parotid channel, activation of this expressed “parslo” channel required very high levels of Ca2+. In order to understand the functional basis for the special properties of the native channels, we analyzed the parotid BK channel in the context of the horrigan-Aldrich model of BK channel gating. We found that the shifted activation of parotid BK channels resulted from a hyperpolarizing shift of the voltage dependence of voltage sensor activation and channel opening and included a large change in the coupling of these two processes. PMID:20519930

  1. PNA lectin for purifying mouse acinar cells from the inflamed pancreas.

    PubMed

    Xiao, Xiangwei; Fischbach, Shane; Fusco, Joseph; Zimmerman, Ray; Song, Zewen; Nebres, Philip; Ricks, David Matthew; Prasadan, Krishna; Shiota, Chiyo; Husain, Sohail Z; Gittes, George K

    2016-02-17

    Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer.

  2. PNA lectin for purifying mouse acinar cells from the inflamed pancreas

    PubMed Central

    Xiao, Xiangwei; Fischbach, Shane; Fusco, Joseph; Zimmerman, Ray; Song, Zewen; Nebres, Philip; Ricks, David Matthew; Prasadan, Krishna; Shiota, Chiyo; Husain, Sohail Z.; Gittes, George K.

    2016-01-01

    Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer. PMID:26884345

  3. Acinar cell carcinoma of exocrine pancreas in two horses.

    PubMed

    de Brot, S; Junge, H; Hilbe, M

    2014-05-01

    Two horses were presented with non-specific clinical signs of several weeks' duration and were humanely destroyed due to a poor prognosis. At necropsy examination, both horses had multiple small, white nodules replacing pancreatic tissue and involving the serosal surface of the abdominal cavity, the liver and the lung. Microscopically, neoplastic cells were organized in acini and contained abundant (case 1) or sparse (horse 2) intracytoplasmic zymogen granules. Immunohistochemically, both tumours expressed amylase and pan-cytokeratin, but not insulin or neuron-specific enolase. In case 2, a low percentage of neoplastic cells expressed glucagon and synaptophysin. The presence of zymogen granules was confirmed in both cases by electron microscopy and occasional fibrillary or glucagon granules were observed in cases 1 and 2, respectively. A diagnosis of pancreatic acinar cell carcinoma was established in both horses.

  4. Pancreatic acinar cells: effects of micro-ionophoretic polypeptide application on membrane potential and resistance.

    PubMed

    Petersen, O H; Philpott, H G

    1979-05-01

    1. Acinar cell membrane potential and resistance were measured from superfused segments of mouse pancreas, in vitro, using intracellular glass micro-electrodes. One or two extracellular micropipettes containing caerulein, bombesin nonapeptide (Bn) or acetylcholine (ACh) were placed near to the surface of the impaled acinus. The secretagogues were ejected rapidly from the micropipettes by ionophoresis.2. Each secretagogue evoked a similar electrical response from the impaled acinar cell: membrane depolarization and a simultaneous reduction in input resistance. The duration of cell activation from caerulein ionophoresis was longer than that observed for ACh and Bn. The cell response to the peptide hormone applications could be repeated in the presence of atropine.3. The minimum interval before the onset of cell depolarization after caerulein ionophoresis was determined. Values ranged between 500 and 1000 msec. The minimum latencies after Bn ionophoresis were 500-1400 msec.4. With two electrodes inserted into electrically coupled acinar cells, direct measurements of the caerulein and Bn null potentials were made. At high negative membrane potentials an enhanced depolarization was evoked by caerulein ionophoresis. At low negative membrane potentials the caerulein stimulation produced a diminished depolarization, and at membrane potentials less than - 10 mV acinar cell hyperpolarizations were observed. A similar series of responses was obtained in experiments where Bn ionophoresis was used. The caerulein and the Bn null potentials were always contained within - 10 to - 15 mV.5. The results describe the almost identical electrical response of acinar cells to stimulation by ACh, caerulein and bombesin. All three secretagogues have similar null potentials and latencies of activation on acinar cells. The bombesin latency responses appear as short as those measured for caerulein and provide electro-physiological evidence that Bn acts directly on acinar cells. The findings

  5. Characterization of single potassium channels in mouse pancreatic acinar cells.

    PubMed Central

    Schmid, A; Schulz, I

    1995-01-01

    1. Single K(+)-selective channels with a conductance of about 48 pS (pipette, 145 mM KCl; bath, 140 mM NaCl + 4.7 mM KCl) were recorded in the patch-clamp whole-cell configuration in isolated mouse pancreatic acinar cells. 2. Neither application of the secretagogues acetylcholine (second messenger, inositol 1,4,5-trisphosphate) or secretin (second messenger, cAMP), nor addition of the catalytic subunit of protein kinase A to the pipette solution changed the activity of the 48 pS K+ channel. 3. Intracellular acidification with sodium propionate (20 mM) diminished activity of the 48 pS channel, whereas channel open probability was increased by cytosolic alkalization with 20 mM NH4Cl. 4. BaCl2 (5 mM), TEA (10 mM) or apamin (1 microM) added to the bath solution had no obvious effect on the kinetics of the 48 pS channel. Similarly, glibenclamide and diazoxide failed to influence the channel activity. 5. When extracellular NaCl was replaced by KCl, whole-cell recordings revealed an inwardly rectifying K+ current carried by a 17 pS K+ channel. 6. The inwardly rectifying K+ current was not pH dependent and could largely be blocked by Ba2+ but not by TEA. 7. Since the 48 pS K+ channel is neither Ca2+ nor cAMP regulated, we suggest that this channel could play a role in the maintenance of the negative cell resting potential. PMID:7623283

  6. Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells.

    PubMed

    Teichenne, Joan; Morró, Meritxell; Casellas, Alba; Jimenez, Veronica; Tellez, Noelia; Leger, Adrien; Bosch, Fatima; Ayuso, Eduard

    2015-01-01

    Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes.

  7. Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

    PubMed Central

    Teichenne, Joan; Morró, Meritxell; Casellas, Alba; Jimenez, Veronica; Tellez, Noelia; Leger, Adrien; Bosch, Fatima; Ayuso, Eduard

    2015-01-01

    Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes. PMID:26690959

  8. Differentiation of pancreatic acinar carcinoma cells cultured on rat testicular seminiferous tubular basement membranes

    SciTech Connect

    Watanabe, T.K.; Hansen, L.J.; Reddy, N.K.; Kanwar, Y.S.; Reddy, J.K.

    1984-11-01

    The use of rat testicular seminiferous tubular basement membrane (STBM) segments as a model substratum for the in vitro maintenance of tumor cells dissociated from a transplantable pancreatic acinar rat carcinoma is described. Ultrastructurally pure, hollow tubular segments of STBM were prepared by mechanical disaggregation, DNase digestion, and deoxycholate treatment. Dissociated pancreatic acinar carcinoma cells adhered readily to STBM segments within 1 to 6 hr, and these STBM-tumor cell aggregates were maintained for up to 7 days in serum-free chemically defined medium supplemented with hydrocortisone, insulin, vitamin C, and soybean trypsin inhibitor. The tumor cells formed acinar-like clusters and displayed intercellular junctions and polarization of secretory granules toward the center of these clusters. By 4 days, virtually all cells of this acinar carcinoma maintained on STBM in supplemented chemically defined medium contained numerous secretory granules. Cell replication, as determined by (/sup 3/H)thymidine autoradiography, ceased within 18 hr of attachment of neoplastic cells to STBM; however, all cells incorporated (/sup 3/H)leucine as evidenced by light and electron microscopic autoradiography. In addition, two-dimensional analysis and fluorography of newly synthesized secretory proteins discharged by these cells in response to carbamylcholine revealed the presence of Mr 24,000 protein and 19 other secretory proteins characteristic of this tumor. The culture system utilizing STBM and supplemented chemically defined medium should allow investigation of the effects of a variety of factors on morphogenesis, cytodifferentiation, and gene expression in pancreatic acinar tumors.

  9. A Computer-Based Automated Algorithm for Assessing Acinar Cell Loss after Experimental Pancreatitis

    PubMed Central

    Eisses, John F.; Davis, Amy W.; Tosun, Akif Burak; Dionise, Zachary R.; Chen, Cheng; Ozolek, John A.; Rohde, Gustavo K.; Husain, Sohail Z.

    2014-01-01

    The change in exocrine mass is an important parameter to follow in experimental models of pancreatic injury and regeneration. However, at present, the quantitative assessment of exocrine content by histology is tedious and operator-dependent, requiring manual assessment of acinar area on serial pancreatic sections. In this study, we utilized a novel computer-generated learning algorithm to construct an accurate and rapid method of quantifying acinar content. The algorithm works by learning differences in pixel characteristics from input examples provided by human experts. HE-stained pancreatic sections were obtained in mice recovering from a 2-day, hourly caerulein hyperstimulation model of experimental pancreatitis. For training data, a pathologist carefully outlined discrete regions of acinar and non-acinar tissue in 21 sections at various stages of pancreatic injury and recovery (termed the “ground truth”). After the expert defined the ground truth, the computer was able to develop a prediction rule that was then applied to a unique set of high-resolution images in order to validate the process. For baseline, non-injured pancreatic sections, the software demonstrated close agreement with the ground truth in identifying baseline acinar tissue area with only a difference of 1%±0.05% (p = 0.21). Within regions of injured tissue, the software reported a difference of 2.5%±0.04% in acinar area compared with the pathologist (p = 0.47). Surprisingly, on detailed morphological examination, the discrepancy was primarily because the software outlined acini and excluded inter-acinar and luminal white space with greater precision. The findings suggest that the software will be of great potential benefit to both clinicians and researchers in quantifying pancreatic acinar cell flux in the injured and recovering pancreas. PMID:25343460

  10. Massive acinar cell apoptosis with secondary necrosis, origin of ducts in atrophic lobules and failure to regenerate in cyanohydroxybutene pancreatopathy in rats

    PubMed Central

    Kelly, Lyndell; Reid, Lynne; Walker, Neal I

    1999-01-01

    Cyanohydroxybutene (CHB), a glycosinolate breakdown product, causes pancreatic injury when given to animals in large amounts. To determine the course of CHB-induced pancreatopathy, rats were given a single subcutaneous dose of CHB and the pancreas weighed and examined by light and electron microscopy and immunohistochemistry at intervals from 2 h to 28 days. The pancreatic lesion was unusual in that there was marked early oedema with limited inflammatory cell infiltration, rapid synchronous onset of acinar cell apoptosis and early advanced atrophy engendering only a limited regenerative response. Acinar cell apoptosis was atypical in that cell fragmentation was limited and phagocytosis delayed, resulting in extensive secondary necrosis. As ducts were unaffected by CHB, the crowded ducts making up the epithelial component of atrophic lobules could be clearly shown to derive from their condensation and proliferation, not the redifferentiation of pre-existing acinar cells, widely held to produce this lesion. Although the basis of CHB selectivity and toxicity for pancreatic acinar cells remains unknown, the potential therapeutic benefit of such an agent in patients with pancreatitis or pancreatic tumours warrants further investigation. PMID:10583631

  11. Effect of taurine on acinar cell apoptosis and pancreatic fibrosis in dibutyltin dichloride-induced chronic pancreatitis.

    PubMed

    Matsushita, Koki; Mizushima, Takaaki; Shirahige, Akinori; Tanioka, Hiroaki; Sawa, Kiminari; Ochi, Koji; Tanimoto, Mitsune; Koide, Norio

    2012-01-01

    The relationship between pancreatic fibrosis and apoptosis of pancreatic acinar cells has not been fully elucidated. We reported that taurine had an anti-fibrotic effect in a dibutyltin dichloride (DBTC)-chronic pancreatitis model. However, the effect of taurine on apoptosis of pancreatic acinar cells is still unclear. Therefore, we examined apoptosis in DBTC-chronic pancreatitis and in the AR42J pancreatic acinar cell line with/without taurine. Pancreatic fibrosis was induced by a single administration of DBTC. Rats were fed a taurine-containing diet or a normal diet and were sacrificed at day 5. The AR42J pancreatic acinar cell line was incubated with/without DBTC with taurine chloramines. Apoptosis was determined by using terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. The expression of Bad and Bcl-2 proteins in the AR42J cells lysates was detected by Western blot analysis. The apoptotic index of pancreatic acinar cells in DBTC-administered rats was significantly increased. Taurine treatment inhibited pancreatic fibrosis and apoptosis of acinar cells induced by DBTC. The number of TUNEL-positive cells in the AR42J pancreatic acinar cell lines was significantly increased by the addition of DBTC. Incubation with taurine chloramines ameliorated these changes. In conclusion, taurine inhibits apoptosis of pancreatic acinar cells and pancreatitis in experimental chronic pancreatitis.

  12. Pancreatic acinar cells-derived cyclophilin A promotes pancreatic damage by activating NF-κB pathway in experimental pancreatitis

    SciTech Connect

    Yu, Ge; Wan, Rong; Hu, Yanling; Ni, Jianbo; Yin, Guojian; Xing, Miao; Shen, Jie; Tang, Maochun; Chen, Congying; Fan, Yuting; Xiao, Wenqin; Zhao, Yan; Wang, Xingpeng; and others

    2014-01-31

    Highlights: • CypA is upregulated in experimental pancreatitis. • CCK induces expression and release of CypA in acinar cell in vitro. • rCypA aggravates CCK-induced acinar cell death and inflammatory cytokine production. • rCypA activates the NF-κB pathway in acinar cells in vitro. - Abstract: Inflammation triggered by necrotic acinar cells contributes to the pathophysiology of acute pancreatitis (AP), but its precise mechanism remains unclear. Recent studies have shown that Cyclophilin A (CypA) released from necrotic cells is involved in the pathogenesis of several inflammatory diseases. We therefore investigated the role of CypA in experimental AP induced by administration of sodium taurocholate (STC). CypA was markedly upregulated and widely expressed in disrupted acinar cells, infiltrated inflammatory cells, and tubular complexes. In vitro, it was released from damaged acinar cells by cholecystokinin (CCK) induction. rCypA (recombinant CypA) aggravated CCK-induced acinar cell necrosis, promoted nuclear factor (NF)-κB p65 activation, and increased cytokine production. In conclusion, CypA promotes pancreatic damage by upregulating expression of inflammatory cytokines of acinar cells via the NF-κB pathway.

  13. Protein kinase D1 drives pancreatic acinar cell reprogramming and progression to intraepithelial neoplasia

    NASA Astrophysics Data System (ADS)

    Liou, Geou-Yarh; Döppler, Heike; Braun, Ursula B.; Panayiotou, Richard; Scotti Buzhardt, Michele; Radisky, Derek C.; Crawford, Howard C.; Fields, Alan P.; Murray, Nicole R.; Wang, Q. Jane; Leitges, Michael; Storz, Peter

    2015-02-01

    The transdifferentiation of pancreatic acinar cells to a ductal phenotype (acinar-to-ductal metaplasia, ADM) occurs after injury or inflammation of the pancreas and is a reversible process. However, in the presence of activating Kras mutations or persistent epidermal growth factor receptor (EGF-R) signalling, cells that underwent ADM can progress to pancreatic intraepithelial neoplasia (PanIN) and eventually pancreatic cancer. In transgenic animal models, ADM and PanINs are initiated by high-affinity ligands for EGF-R or activating Kras mutations, but the underlying signalling mechanisms are not well understood. Here, using a conditional knockout approach, we show that protein kinase D1 (PKD1) is sufficient to drive the reprogramming process to a ductal phenotype and progression to PanINs. Moreover, using 3D explant culture of primary pancreatic acinar cells, we show that PKD1 acts downstream of TGFα and Kras, to mediate formation of ductal structures through activation of the Notch pathway.

  14. Aspirin Protects against Acinar Cells Necrosis in Severe Acute Pancreatitis in Mice

    PubMed Central

    Lu, Guotao; Tong, Zhihui; Ding, Yanbing; Liu, Jinjiao; Pan, Yiyuan; Gao, Lin; Tu, Jianfeng; Liu, George

    2016-01-01

    Aspirin has a clear anti-inflammatory effect and is used as an anti-inflammatory agent for both acute and long-term inflammation. Previous study has indicated that aspirin alleviated acute pancreatitis induced by caerulein in rat. However, the role of aspirin on severe acute pancreatitis (SAP) and the necrosis of pancreatic acinar cell are not yet clear. The aim of this study was to determine the effects of aspirin treatment on a SAP model induced by caerulein combined with Lipopolysaccharide. We found that aspirin reduced serum amylase and lipase levels, decreased the MPO activity, and alleviated the histopathological manifestations of pancreas and pancreatitis-associated lung injury. Proinflammatory cytokines were decreased and the expression of NF-κB p65 in acinar cell nuclei was suppressed after aspirin treatment. Furthermore, aspirin induced the apoptosis of acinar cells by TUNEL assay, and the expression of Bax and caspase 3 was increased and the expression of Bcl-2 was decreased. Intriguingly, the downregulation of critical necrosis associated proteins RIP1, RIP3, and p-MLKL was observed; what is more, we additionally found that aspirin reduced the COX level of pancreatic tissue. In conclusion, our data showed that aspirin could protect pancreatic acinar cell against necrosis and reduce the severity of SAP. Clinically, aspirin may potentially be a therapeutic intervention for SAP. PMID:28119929

  15. Acinar Cell Apoptosis in Serpini2-Deficient Mice Models Pancreatic Insufficiency

    PubMed Central

    Loftus, Stacie K; Cannons, Jennifer L; Incao, Arturo; Pak, Evgenia; Chen, Amy; Zerfas, Patricia M; Bryant, Mark A; Biesecker, Leslie G; Schwartzberg, Pamela L; Pavan, William J

    2005-01-01

    Pancreatic insufficiency (PI) when left untreated results in a state of malnutrition due to an inability to absorb nutrients. Frequently, PI is diagnosed as part of a larger clinical presentation in cystic fibrosis or Shwachman–Diamond syndrome. In this study, a mouse model for isolated exocrine PI was identified in a mouse line generated by a transgene insertion. The trait is inherited in an autosomal recessive pattern, and homozygous animals are growth retarded, have abnormal immunity, and have reduced life span. Mice with the disease locus, named pequeño (pq), exhibit progressive apoptosis of pancreatic acinar cells with severe exocrine acinar cell loss by 8 wk of age, while the islets and ductal tissue persist. The mutation in pq/pq mice results from a random transgene insertion. Molecular characterization of the transgene insertion site by fluorescent in situ hybridization and genomic deletion mapping identified an approximately 210-kb deletion on Chromosome 3, deleting two genes. One of these genes, Serpini2, encodes a protein that is a member of the serpin family of protease inhibitors. Reintroduction of only the Serpini2 gene by bacterial artificial chromosome transgenic complementation corrected the acinar cell defect as well as body weight and immune phenotypes, showing that deletion of Serpini2 causes the pequeño phenotype. Dietary supplementation of pancreatic enzymes also corrected body size, body weight, and immunodeficiency, and increased the life span of Serpini2-deficient mice, despite continued acinar cell loss. To our knowledge, this study describes the first characterized genetic animal model for isolated PI. Genetic complementation of the transgene insertion mutant demonstrates that Serpini2 deficiency directly results in the acinar cell apoptosis, malabsorption, and malnutrition observed in pq/pq mice. The rescue of growth retardation, immunodeficiency, and mortality by either Serpini2 bacterial artificial chromosome transgenic expression

  16. The effect of hyposmotic and isosmotic cell swelling on the intracellular [Ca2+] in lactating rat mammary acinar cells.

    PubMed

    Shennan, D B; Grant, A C G; Gow, I F

    2002-04-01

    The effect of hyposmotic and isosmotic cell swelling on the free intracellular calcium concentration ([Ca2+]i) in rat mammary acinar cells has been examined using the fura-2 dye technique. Ahyposmotic shock (40% reduction) increased the [Ca2+]i in rat mammary acinar cells in a fashion which was transient; the [Ca2+]i returned to a value similar to that found under isomotic conditions within 180 sec. The increase in the [Ca2+]i was dependent upon the extent of the osmotic shock. The hyposmotically-activated increase in the [Ca2+]i could not be attributed to a reduction in extracellular Na+ or a change in the ionic strength of the incubation medium. Thapsigargin (1 microM) enhanced the hyposmotically-activated increase in the [Ca2+]i. Isosmotic swelling of rat mammary acinar cells, using urea, had no significant effect on the [Ca2+]i. Similarly, a hyperosmotic shock did not affect the [Ca2+]i in rat mammary acinar cells. It appears that the effect of cell swelling on the [Ca2+]i in rat mammary acinar cells depends on how the cells are swollen (hyposmotic vs. isosmotic). This finding may have important physiological implications given that it is predicted that mammary cell volume will change in vivo under isomotic conditions.

  17. The MET Receptor Tyrosine Kinase Confers Repair of Murine Pancreatic Acinar Cells following Acute and Chronic Injury

    PubMed Central

    Gaziova, Ivana; Jackson, Daniel; Boor, Paul J.; Carter, Dwayne; Cruz-Monserrate, Zobeida; Elferink, Cornelis J.; Joshi, Aditya D.; Kaphalia, Bhupendra; Logsdon, Craig D.; Pereira de Castro, Karen; Soong, Lynn; Tao, Xinrong; Qiu, Suimin; Elferink, Lisa A.

    2016-01-01

    Acinar cells represent the primary target in necroinflammatory diseases of the pancreas, including pancreatitis. The signaling pathways guiding acinar cell repair and regeneration following injury remain poorly understood. The purpose of this study was to determine the importance of Hepatocyte Growth Factor Receptor/MET signaling as an intrinsic repair mechanism for acinar cells following acute damage and chronic alcohol-associated injury. Here, we generated mice with targeted deletion of MET in adult acinar cells (MET-/-). Acute and repetitive pancreatic injury was induced in MET-/- and control mice with cerulein, and chronic injury by feeding mice Lieber-DeCarli diets containing alcohol with or without enhancement of repetitive pancreatic injury. We examined the exocrine pancreas of these mice histologically for acinar death, edema, inflammation and collagen deposition and changes in the transcriptional program. We show that MET expression is relatively low in normal adult pancreas. However, MET levels were elevated in ductal and acinar cells in human pancreatitis specimens, consistent with a role for MET in an adaptive repair mechanism. We report that genetic deletion of MET in adult murine acinar cells was linked to increased acinar cell death, chronic inflammation and delayed recovery (regeneration) of pancreatic exocrine tissue. Notably, increased pancreatic collagen deposition was detected in MET knockout mice following repetitive injury as well alcohol-associated injury. Finally, we identified specific alterations of the pancreatic transcriptome associated with MET signaling during injury, involved in tissue repair, inflammation and endoplasmic reticulum stress. Together, these data demonstrate the importance of MET signaling for acinar repair and regeneration, a novel finding that could attenuate the symptomology of pancreatic injury. PMID:27798657

  18. Expression pattern of REIC/Dkk-3 in various cell types and the implications of the soluble form in prostatic acinar development.

    PubMed

    Zhang, Kai; Watanabe, Masami; Kashiwakura, Yuji; Li, Shun-Ai; Edamura, Kohei; Huang, Peng; Yamaguchi, Ken; Nasu, Yasutomo; Kobayashi, Yasuyuki; Sakaguchi, Masakiyo; Ochiai, Kazuhiko; Yamada, Hiroshi; Takei, Kohji; Ueki, Hideo; Huh, Nam-Ho; Li, Ming; Kaku, Haruki; Na, Yanqun; Kumon, Hiromi

    2010-12-01

    The tumor suppressor REIC/Dkk-3 is a secretory protein which was originally identified to be downregulated in human immortalized cells. In the present study, we investigated the expression pattern of REIC/Dkk-3 in various cell types to characterize its physiological functions. We first examined the expression level of REIC/Dkk-3 in a broad range of cancer cell types and confirmed that it was significantly downregulated in all of the cell types. We also examined the tissue distribution pattern in a variety of normal mouse organs. Ubiquitous REIC/Dkk-3 protein expression was observed in the organs. The expression was abundant in the liver, heart and brain tissue, but was absent in the spleen and peripheral blood mononuclear cells. The immunohistochemical analyses revealed that the subcellular localization of REIC/Dkk-3 had a punctate pattern around the nucleus, indicating its association with secretory vesicles. In cancer cells stably transfected with REIC/Dkk-3, the protein was predominantly localized to the endoplasmic reticulum (ER) under observation with confocal microscopy. Because REIC/Dkk-3 was found to be abundantly expressed in the acinar epithelial cells of the mouse prostate, we analyzed the effects of recombinant REIC/Dkk-3 protein on the acinar morphogenesis of RWPE-1 cells, which are derived from human normal prostate epithelium. Statistically significant acinar growth was observed in the culture condition with 10 µg/ml REIC/Dkk-3 protein, implicating the soluble form in prostatic acinar development. Current results suggest that REIC/Dkk-3 may play a role in regulating the morphological process of normal tissue architecture through an autocrine and/or paracrine manner.

  19. Intracellular mediators of Na -K pump activity in guinea pig pancreatic acinar cells

    SciTech Connect

    Hootman, S.R.; Ochs, D.L.; Williams, J.A.

    1985-10-01

    The involvement of CaS and cyclic nucleotides in neurohormonal regulation of Na -K -ATPase (Na -K pump) activity in guinea pig pancreatic acinar cells was investigated. Changes in Na+-K+ pump activity elicited by secretagogues were assessed by (3H)ouabain binding and by ouabain-sensitive YWRb uptake. Carbachol (CCh) and cholecystokinin octapeptide (CCK-8) each stimulated both ouabain-sensitive 86Rb+ uptake and equilibrium binding of (TH)ouabain by approximately 60%. Secretin increased both indicators of Na+-K+ pump activity by approximately 40% as did forskolin, 8-bromo- and dibutyryl cAMP, theophylline, and isobutylmethylxanthine. Incubation of acinar cells in CaS -free HEPES-buffered Ringer (HR) with 0.5 mM EGTA reduced the stimulatory effects of CCh and CCK-8 by up to 90% but caused only a small reduction in the effects of secretin, forskolin, and cAMP analogues. In addition, CCh, CCK-8, secretin, and forskolin each stimulated ouabain-insensitive 86Rb+ uptake by acinar cells. The increase elicited by CCh and CCK-8 was greatly reduced in the absence of extracellular CaS , while that caused by the latter two agents was not substantially altered. The effects of secretagogues on free CaS levels in pancreatic acinar cells also were investigated with quin-2, a fluorescent CaS chelator. Basal intracellular CaS concentration ((CaS )i) was 161 nM in resting cells and increased to 713 and 803 nM within 15 s after addition of 100 microM CCh or 10 nM CCK-8, respectively.

  20. Sudden disappearance of the blood flow in a case of pancreatic acinar cell carcinoma.

    PubMed

    Kanno, Atsushi; Masamune, Atsushi; Hamada, Shin; Kikuta, Kazuhiro; Kume, Kiyoshi; Hirota, Morihisa; Shima, Kentaro; Okada, Takaho; Motoi, Fuyuhiko; Fujishima, Fumiyoshi; Ishida, Kazuyuki; Unno, Michiaki; Shimosegawa, Tooru

    2014-01-01

    A 55-year-old man was referred to our hospital for a further examination of a pancreatic cystic tumor with a solid component exhibiting vascularity. A few days later, the patient was admitted with a complaint of sudden severe epigastric pain. Enhanced CT showed the loss of vascularity in the tumor. In particular, contrast-enhanced endoscopic ultrasonography (EUS) clearly demonstrated the disappearance of the blood flow, and a histological examination revealed acinar cell carcinoma with central necrosis. To our knowledge, this is the first case in the literature of acinar cell carcinoma associated with the sudden disappearance of vascularity. In this case, contrast-enhanced harmonic EUS was especially useful for assessing the degree of vascularity.

  1. Loss of Ifnar1 in Pancreatic Acinar Cells Ameliorates the Disease Course of Acute Pancreatitis

    PubMed Central

    Miller, Katharina J.; Raulefs, Susanne; Kong, Bo; Steiger, Katja; Regel, Ivonne; Gewies, Andreas; Kleeff, Jörg; Michalski, Christoph W.

    2015-01-01

    Type I interferon constitutes an essential component of the combinational therapy against viral disease. Acute pancreatitis is one side effect of type I interferon-based therapy, implying that activation of type I interferon signaling affects the homeostasis and integrity of pancreatic acinar cells. Here, we investigated the role of type I interferon signaling in pancreatic acinar cells using a caerulein-induced murine model of acute pancreatitis. Pancreas-specific ablation of interferon (alpha and beta) receptor 1 (Ifnar1) partially protected animals from caerulein-induced pancreatitis, as demonstrated by reduced tissue damage. Profiling of infiltrating immune cells revealed that this dampened tissue damage response correlated with the number of macrophages in the pancreas. Pharmacologic depletion of macrophages reversed the protective effect of Ifnar1 deficiency. Furthermore, expression of chemokine (C-C motif) ligand 2 (Ccl2), a potent factor for macrophage recruitment, was significantly increased in the Ifnar1-deficient pancreas. Thus, type I interferon signaling in pancreatic acinar cells controls pancreatic homeostasis by affecting the macrophage-mediated inflammatory response in the pancreas. PMID:26618925

  2. Altered Gene Expression in Cerulein-Stimulated Pancreatic Acinar Cells: Pathologic Mechanism of Acute Pancreatitis

    PubMed Central

    Yu, Ji Hoon; Lim, Joo Weon

    2009-01-01

    Acute pancreatitis is a multifactorial disease associated with the premature activation of digestive enzymes. The genes expressed in pancreatic acinar cells determine the severity of the disease. The present study determined the differentially expressed genes in pancreatic acinar cells treated with cerulein as an in vitro model of acute pancreatitis. Pancreatic acinar AR42J cells were stimulated with 10-8 M cerulein for 4 h, and genes with altered expression were identified using a cDNA microarray for 4,000 rat genes and validated by real-time PCR. These genes showed a 2.5-fold or higher increase with cerulein: lithostatin, guanylate cyclase, myosin light chain kinase 2, cathepsin C, progestin-induced protein, and pancreatic trypsin 2. Stathin 1 and ribosomal protein S13 showed a 2.5-fold or higher decreases in expression. Real-time PCR analysis showed time-dependent alterations of these genes. Using commercially available antibodies specific for guanylate cyclase, myosin light chain kinase 2, and cathepsin C, a time-dependent increase in these proteins were observed by Western blotting. Thus, disturbances in proliferation, differentiation, cytoskeleton arrangement, enzyme activity, and secretion may be underlying mechanisms of acute pancreatitis. PMID:20054485

  3. Early acinar cell changes in caerulein-induced interstitial acute pancreatitis in the rat.

    PubMed

    Grönroos, J M; Aho, H J; Hietaranta, A J; Nevalainen, T J

    1991-01-01

    Early ultrastructural and immunohistochemical changes caused by supramaximal secretory stimulation with caerulein were studied in the rat pancreas. The morphological basis for the earlier reported decrease of pancreatic juice secretion after supramaximal caerulein was the appearance of swollen and irregular zymogen-like material containing structures with short segments of budding bristle-coated membranes in the apical parts of acinar cells. Images of exocytosis of zymogen granules were only few. Later, marked vacuolization and signs of autophagocytosis are seen in the basal cytoplasm. Immunohistochemistry showed that the large zymogen containing structures were intensively labelled for trypsin at the early stages of the experiment (4-30 min). Later (1-2 h), the vacuoles were empty or contained occasional, small-labelled granules only. The pancreozymin-receptor antagonist proglumide as well as cycloleucine that inhibits protein synthesis by inhibiting the synthesis of S-adenosylmethionine, effectively prevented the caerulein induced acinar cell changes. The irregular zymogen containing structures with coated pits on their surface indicate disturbed zymogen granule formation leading to the accumulation of large lakes of zymogen material and finally to marked autophagocytosis in acinar cells. The effects of caerulein are receptor-mediated and depend on the process of methylation in the formation of zymogen granules.

  4. Membrane Proteome Analysis of Cerulein-Stimulated Pancreatic Acinar Cells: Implication for Early Event of Acute Pancreatitis

    PubMed Central

    Lee, Jangwon; Seo, Ji Hye; Lim, Joo Weon

    2010-01-01

    Background/Aims Cerulein pancreatitis is similar to human edematous pancreatitis with dysregulation of the production and secretion of digestive enzymes, edema formation, cytoplasmic vacuolization and the death of acinar cells. We hypothesized that membrane proteins may be altered as the early event during the induction of acute pancreatitis. Present study aims to determine the differentially expressed proteins in the membranes of cerulein-treated pancreatic acinar cells. Methods Pancreatic acinar AR42J cells were treated with 10-8 M cerulein for 1 hour. Membrane proteins were isolated from the cells and separated by two-dimensional electrophoresis using pH gradients of 5-8. Membrane proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. The differentially expressed proteins, whose expression levels were more or less than three-fold in cerulein-treated cells, were analyzed. Results Two differentially expressed proteins (mannan-binding lectin-associated serine protease-2, heat shock protein 60) were up-regulated while four proteins (protein disulfide isomerase, γ-actin, isocitrate dehydrogenase 3, seven in absentia homolog 1A) were down-regulated by cerulein treatment in pancreatic acinar cells. These proteins are related to cell signaling, oxidative stress, and cytoskeleton arrangement. Conclusions Oxidative stress may induce cerulein-induced cell injury and disturbances in defense mechanism in pancreatic acinar cells. PMID:20479917

  5. Transplantable pancreatic acinar carcinoma

    SciTech Connect

    Warren, J.R.; Reddy, J.K.

    1981-03-15

    Fragments of the nafenopin-induced pancreatic acinar cell carcinoma of rat have been examined in vitro for patterns of intracellular protein transport and carbamylcholine-induced protein discharge. Continuous incubation of the fragments with (3H)-leucine for 60 minutes resulted in labeling of rough endoplasmic reticulum, Golgi cisternae, and mature zymogen granules, revealed by electron microscope autoradiography. This result indicates transport of newly synthesized protein from the rough endoplasmic reticulum to mature zymogen granules in approximately 60 minutes. The secretagogue carbamylcholine induced the discharge of radioactive protein by carcinoma fragments pulse-chase labeled with (3H)-leucine. A maximal effective carbamylcholine concentration of 10(-5) M was determined. The acinar carcinoma resembles normal exocrine pancreas in the observed rate of intracellular protein transport and effective secretagogue concentration. However, the acinar carcinoma fragments demonstrated an apparent low rate of carbamylcholine-induced radioactive protein discharge as compared with normal pancreatic lobules or acinar cells. It is suggested that the apparent low rate of radioactive protein discharge reflects functional immaturity of the acinar carcinoma. Possible relationships of functional differentiation to the heterogeneous cytodifferentiation of the pancreatic acinar carcinoma are discussed.

  6. Insulin Protects Pancreatic Acinar Cells from Palmitoleic Acid-induced Cellular Injury*

    PubMed Central

    Samad, Aysha; James, Andrew; Wong, James; Mankad, Parini; Whitehouse, John; Patel, Waseema; Alves-Simoes, Marta; Siriwardena, Ajith K.; Bruce, Jason I. E.

    2014-01-01

    Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca2+ ([Ca2+]i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca2+]i are linked critically by the ATP-driven plasma membrane Ca2+-ATPase (PMCA) important for maintaining low resting [Ca2+]i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca2+]i was measured by fura-2 imaging. An in situ [Ca2+]i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca2+ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca2+ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca2+ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca2+ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis. PMID:24993827

  7. Hydrogen peroxide attenuates refilling of intracellular calcium store in mouse pancreatic acinar cells

    PubMed Central

    Yoon, Mi Na; Kim, Dong Kwan; Kim, Se Hoon

    2017-01-01

    Intracellular calcium (Ca2+) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H2O2) on intracellular Ca2+ accumulation in mouse pancreatic acinar cells. Perfusion of H2O2 at 300 µM resulted in additional elevation of intracellular Ca2+ levels and termination of oscillatory Ca2+ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca2+. Antioxidants, catalase or DTT, completely prevented H2O2-induced additional Ca2+ increase and termination of Ca2+ oscillation. In Ca2+-free medium, H2O2 still enhanced CCh-induced intracellular Ca2+ levels and thapsigargin (TG) mimicked H2O2-induced cytosolic Ca2+ increase. Furthermore, H2O2-induced elevation of intracellular Ca2+ levels was abolished under sarco/endoplasmic reticulum Ca2+ ATPase-inactivated condition by TG pretreatment with CCh. H2O2 at 300 µM failed to affect store-operated Ca2+ entry or Ca2+ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca2+ uniporter blocker, failed to attenuate H2O2-induced intracellular Ca2+ elevation. These results provide evidence that excessive generation of H2O2 in pathological conditions could accumulate intracellular Ca2+ by attenuating refilling of internal Ca2+ stores rather than by inhibiting Ca2+ extrusion to extracellular fluid or enhancing Ca2+ mobilization from extracellular medium in mouse pancreatic acinar cells. PMID:28280417

  8. Pancreatic panniculitis as a paraneoplastic phenomenon of a pancreatic acinar cell carcinoma.

    PubMed

    Naeyaert, Charlotte; de Clerck, Frederik; De Wilde, Vincent

    2016-12-01

    We present the case of a 59-year-old patient admitted with extreme painful erythematous subcutaneous nodules of the lower extremities in association with arthritis and peripheral eosinophilia. Upon skin biopsy, the diagnosis of pancreatic panniculitis was made. On further investigation, an underlying acinar cell type pancreas carcinoma was revealed. This clinical case does illustrate how a seemingly innocuous skin condition may herald an underlying malignant disease. The presence of pancreatic panniculitis should trigger clinicians to undertake further thorough diagnostic investigation of the pancreas.

  9. Glycyrrhizin down-regulates CCL2 and CXCL2 expression in cerulein-stimulated pancreatic acinar cells

    PubMed Central

    Panahi, Yaser; Fakhari, Shohreh; Mohammadi, Mehdi; Rahmani, Mohammad Reza; Hakhamaneshi, Mohammad Saeid; Jalili, Ali

    2015-01-01

    Many inflammatory chemokines release from leukocytes and pancreatic acinar cells which play important roles in pathophysiology of acute pancreatitis (AP). Of interests, CXCL2 and CCL2 have been shown elevated in the plasma of patients with AP. We have recently found that Glycyrrhizin (GZ) attenuates AP in mice model. In this study, we aimed to investigate the direct effect of GZ on expression levels of CCL2 and CXCl2 in isolated pancreatic acinar cells. Isolated acinar cells were isolated from the pancreas of healthy C57BL/6 mice, stimulated with cerulein (10-7 M) and then treated with either PBS or different doses of GZ. The levels of CCL2 and CXCL2 expression at mRNA were assessed by qRT-PCR. Conditioned media from supernatants of each cells culture condition were collected for detection of CCL2 and CXCL2 levels by ELISA. First, we observed that cerulein significantly upregulates both cytokines expression in acinar cells. Moreover, we treated the acinar cells with GZ and found that GZ significantly downregulates CCL2 and CXCL2 expression at mRNA levels in a dose-dependent manner. Consistently, the conditioned media of GZ-treated cells contained a significant lower levels of CCL2 and CXCL2 (p<0.05). In conclusion, our data demonstrate for the first time that GZ directly downregulates CCL2 and CXCL2 levels in cerulein-stimulated acinar cells which may explain the mechanism of therapeutic effects of GZ in cerulein-induced AP in mice. PMID:26155433

  10. The role of Ca2+ influx in endocytic vacuole formation in pancreatic acinar cells

    PubMed Central

    Voronina, Svetlana; Collier, David; Chvanov, Michael; Middlehurst, Ben; Beckett, Alison J.; Prior, Ian A.; Criddle, David N.; Begg, Malcolm; Mikoshiba, Katsuhiko; Sutton, Robert; Tepikin, Alexei V.

    2014-01-01

    The inducers of acute pancreatitis trigger a prolonged increase in the cytosolic Ca2+ concentration ([Ca2+]c), which is responsible for the damage to and eventual death of pancreatic acinar cells. Vacuolization is an important indicator of pancreatic acinar cell damage. Furthermore, activation of trypsinogen occurs in the endocytic vacuoles; therefore the vacuoles can be considered as ‘initiating’ organelles in the development of the cell injury. In the present study, we investigated the relationship between the formation of endocytic vacuoles and Ca2+ influx developed in response to the inducers of acute pancreatitis [bile acid taurolithocholic acid 3-sulfate (TLC-S) and supramaximal concentration of cholecystokinin-8 (CCK)]. We found that the inhibitor of STIM (stromal interaction molecule)/Orai channels, GSK-7975A, effectively suppressed both the Ca2+ influx (stimulated by inducers of pancreatitis) and the formation of endocytic vacuoles. Cell death induced by TLC-S or CCK was also inhibited by GSK-7975A. We documented the formation of endocytic vacuoles in response to store-operated Ca2+ entry (SOCE) induced by thapsigargin [TG; inhibitor of sarcoplasmic/endoplasmic reticulum (ER) Ca2+ pumps] and observed strong inhibition of TG-induced vacuole formation by GSK-7975A. Finally, we found that structurally-unrelated inhibitors of calpain suppress formation of endocytic vacuoles, suggesting that this Ca2+-dependent protease is a mediator between Ca2+ elevation and endocytic vacuole formation. PMID:25370603

  11. Salivary gland acinar cells regenerate functional glandular structures in modified hydrogels

    NASA Astrophysics Data System (ADS)

    Pradhan, Swati

    Xerostomia, a condition resulting from irradiation of the head and neck, affects over 40,000 cancer patients each year in the United States. Direct radiation damage of the acinar cells that secrete fluid and protein results in salivary gland hypofunction. Present medical management for xerostomia for patients treated for upper respiratory cancer is largely ineffective. Patients who have survived their terminal diagnosis are often left with a diminished quality of life and are unable to enjoy the simple pleasures of eating and drinking. This project aims to ultimately reduce human suffering by developing a functional implantable artificial salivary gland. The goal was to create an extracellular matrix (ECM) modified hyaluronic acid (HA) based hydrogel culture system that allows for the growth and differentiation of salivary acinar cells into functional acini-like structures capable of secreting large amounts of protein and fluid unidirectionally and to ultimately engineer a functional artificial salivary gland that can be implanted into an animal model. A tissue collection protocol was established and salivary gland tissue was obtained from patients undergoing head and neck surgery. The tissue specimen was assessed by histology and immunohistochemistry to establish the phenotype of normal salivary gland cells including the native basement membranes. Hematoxylin and eosin staining confirmed normal glandular tissue structures including intercalated ducts, striated ducts and acini. alpha-Amylase and periodic acid schiff stain, used for structures with a high proportion of carbohydrate macromolecules, preferentially stained acinar cells in the tissue. Intercalated and striated duct structures were identified using cytokeratins 19 and 7 staining. Myoepithelial cells positive for cytokeratin 14 were found wrapped around the serous and mucous acini. Tight junction components including ZO-1 and E-cadherin were present between both ductal and acinar cells. Ductal and acinar

  12. The econobiology of pancreatic acinar cells granule inventory and the stealthy nano-machine behind it.

    PubMed

    Hammel, Ilan; Meilijson, Isaac

    2016-03-01

    The pancreatic gland secretes most of the enzymes and many other macromolecules needed for food digestion in the gastrointestinal tract. These molecules play an important role in digestion, host defense and lubrication. The secretion of pancreatic proteins ensures the availability of the correct mix of proteins when needed. This review describes model systems available for the study of the econobiology of secretory granule content. The secretory pancreatic molecules are stored in large dense-core secretory granules that may undergo either constitutive or evoked secretion, and constitute the granule inventory of the cell. It is proposed that the Golgi complex functions as a distribution center for secretory proteins in pancreatic acinar cells, packing the newly formed secretory molecules into maturing secretory granules, also known functionally as condensing vacuoles. Mathematical modelling brings forward a process underlying granule inventory maintenance at various physiological states of condensation and aggregation by homotypic fusion. These models suggest unique but simple mechanisms accountable for inventory buildup and size, as well as for the distribution of secretory molecules into different secretory pathways in pancreatic acinar cells.

  13. Hepcidin knockout mice spontaneously develop chronic pancreatitis owing to cytoplasmic iron overload in acinar cells.

    PubMed

    Lunova, Mariia; Schwarz, Peggy; Nuraldeen, Renwar; Levada, Kateryna; Kuscuoglu, Deniz; Stützle, Michael; Vujić Spasić, Maja; Haybaeck, Johannes; Ruchala, Piotr; Jirsa, Milan; Deschemin, Jean-Christophe; Vaulont, Sophie; Trautwein, Christian; Strnad, Pavel

    2017-01-01

    Iron is both an essential and a potentially toxic element, and its systemic homeostasis is controlled by the iron hormone hepcidin. Hepcidin binds to the cellular iron exporter ferroportin, causes its degradation, and thereby diminishes iron uptake from the intestine and the release of iron from macrophages. Given that hepcidin-resistant ferroportin mutant mice show exocrine pancreas dysfunction, we analysed pancreata of aging hepcidin knockout (KO) mice. Hepcidin and Hfe KO mice were compared with wild-type (WT) mice kept on standard or iron-rich diets. Twelve-month-old hepcidin KO mice were subjected to daily minihepcidin PR73 treatment for 1 week. Six-month-old hepcidin KO mice showed cytoplasmic acinar iron overload and mild pancreatitis, together with elevated expression of the iron uptake mediators DMT1 and Zip14. Acinar atrophy, massive macrophage infiltration, fatty changes and pancreas fibrosis were noted in 1-year-old hepcidin KO mice. As an underlying mechanism, 6-month-old hepcidin KO mice showed increased pancreatic oxidative stress, with elevated DNA damage, apoptosis and activated nuclear factor-κB (NF-κB) signalling. Neither iron overload nor pancreatic damage was observed in WT mice fed iron-rich diet or in Hfe KO mice. Minihepcidin application to hepcidin KO mice led to an improvement in general health status and to iron redistribution from acinar cells to macrophages. It also resulted in decreased NF-κB activation and reduced DNA damage. In conclusion, loss of hepcidin signalling in mice leads to iron overload-induced chronic pancreatitis that is not seen in situations with less severe iron accumulation. The observed tissue injury can be reversed by hepcidin supplementation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  14. Label retaining cells (LRCs) with myoepithelial characteristic from the proximal acinar region define stem cells in the sweat gland.

    PubMed

    Leung, Yvonne; Kandyba, Eve; Chen, Yi-Bu; Ruffins, Seth; Kobielak, Krzysztof

    2013-01-01

    Slow cycling is a common feature shared among several stem cells (SCs) identified in adult tissues including hair follicle and cornea. Recently, existence of unipotent SCs in basal and lumenal layers of sweat gland (SG) has been described and label retaining cells (LRCs) have also been localized in SGs; however, whether these LRCs possess SCs characteristic has not been investigated further. Here, we used a H2BGFP LRCs system for in vivo detection of infrequently dividing cells. This system allowed us to specifically localize and isolate SCs with label-retention and myoepithelial characteristics restricted to the SG proximal acinar region. Using an alternative genetic approach, we demonstrated that SG LRCs expressed keratin 15 (K15) in the acinar region and lineage tracing determined that K15 labeled cells contributed long term to the SG structure but not to epidermal homeostasis. Surprisingly, wound healing experiments did not activate proximal acinar SG cells to participate in epidermal healing. Instead, predominantly non-LRCs in the SG duct actively divided, whereas the majority of SG LRCs remained quiescent. However, when we further challenged the system under more favorable isolated wound healing conditions, we were able to trigger normally quiescent acinar LRCs to trans-differentiate into the epidermis and adopt its long term fate. In addition, dissociated SG cells were able to regenerate SGs and, surprisingly, hair follicles demonstrating their in vivo plasticity. By determining the gene expression profile of isolated SG LRCs and non-LRCs in vivo, we identified several Bone Morphogenetic Protein (BMP) pathway genes to be up-regulated and confirmed a functional requirement for BMP receptor 1A (BMPR1A)-mediated signaling in SG formation. Our data highlight the existence of SG stem cells (SGSCs) and their primary importance in SG homeostasis. It also emphasizes SGSCs as an alternative source of cells in wound healing and their plasticity for regenerating

  15. Bromoenol lactone enhances the permeabilization of rat submandibular acinar cells by P2X7 agonists

    PubMed Central

    Chaïb, N; Kabré, E; Alzola, E; Pochet, S; Dehaye, J P

    2000-01-01

    The permeabilizing effect of P2X7 agonists was tested in rat submandibular acinar cells using the uptake of ethidium bromide as an index. The uptake of ethidium bromide by acini incubated at 37°C in the presence of 1 mM ATP increased with time and reached after 5 min about 10% of maximal uptake measured in the presence of digitonin. The response to ATP was dose-dependent (half-maximal concentration around 40 μM) and it was decreased when the temperature was lowered to 25°C. Benzoyl-ATP reproduced the response to ATP (half-maximal concentration around 10 μM). UTP or 2-methylthioATP had no effect. The permeabilization in response to ATP was blocked by oxidized ATP and by magnesium and inhibited by Coomassie blue. ATP increased the activity of a calcium-insensitive phospholipase A2 (iPLA2). Bromoenol lactone (BEL) inhibited the iPLA2 stimulated by ATP but potentiated the uptake of ethidium bromide in response to the purinergic agonist. From these results it is concluded that the activation of P2X7 receptors permeabilizes rat submandibular acinar cells. The pore-forming activity of the receptor might be negatively regulated by the concomitant activation of the iPLA2 by the receptor. PMID:10683195

  16. Rhein Induces a Necrosis-Apoptosis Switch in Pancreatic Acinar Cells

    PubMed Central

    Zhao, Xianlin; Li, Juan; Zhu, Shifeng; Liu, Yiling; Zhao, Jianlei; Wan, Meihua; Tang, Wenfu

    2014-01-01

    Objectives. The Chinese herbal medicine Da-Cheng-Qi decoction can regulate a necrosis-apoptosis switch in injured pancreatic acinar cells. This study investigated the effects of rhein, a component of this medicine, on a necrosis-apoptosis switch in pancreatic rat AR42J cells. Methods. Cerulein-treated AR42J cells were used. After pretreatment with 479, 119.8, or 29.9 μg/L rhein, cells were cocultured with rhein and cerulein (10−8 M) for 4, 8, or 16 h. Apoptosis and necrosis were examined using annexin V and propidium iodide costaining. Mitochondria-dependent apoptosis-associated proteins were examined using enzyme-linked immunosorbent assays and western blotting. Results. Few cells died in untreated samples. The number was significantly higher in 16-h-cerulein-treated samples and treatment with 479 μg/L rhein most effectively increased the apoptotic-to-necrotic cell ratio (P < 0.05). In cerulein-treated cells, rhein increased the concentrations of p53, cytochrome C, and caspase-3, and increased the Bax/Bcl-2 ratio in a time- and dose-dependent manner, with the maximum effect in cells treated with 479 μg/L rhein for 16 h (P < 0.05). Conclusions. Rhein induces the necrosis-apoptosis switch in injured pancreatic acinar cells in a time- and dose-dependent manner. Mitochondria-dependent apoptosis signaling pathways might play an important role in this effect. PMID:24959186

  17. Ethanol exerts dual effects on calcium homeostasis in CCK-8-stimulated mouse pancreatic acinar cells

    PubMed Central

    Fernández-Sánchez, Marcela; del Castillo-Vaquero, Angel; Salido, Ginés M; González, Antonio

    2009-01-01

    Background A significant percentage of patients with pancreatitis often presents a history of excessive alcohol consumption. Nevertheless, the patho-physiological effect of ethanol on pancreatitis remains poorly understood. In the present study, we have investigated the early effects of acute ethanol exposure on CCK-8-evoked Ca2+ signals in mouse pancreatic acinar cells. Changes in [Ca2+]i and ROS production were analyzed employing fluorescence techniques after loading cells with fura-2 or CM-H2DCFDA, respectively. Results Ethanol, in the concentration range from 1 to 50 mM, evoked an oscillatory pattern in [Ca2+]i. In addition, ethanol evoked reactive oxygen species generation (ROS) production. Stimulation of cells with 1 nM or 20 pM CCK-8, respectively led to a transient change and oscillations in [Ca2+]i. In the presence of ethanol a transformation of 20 pM CCK-8-evoked physiological oscillations into a single transient increase in [Ca2+]i in the majority of cells was observed. Whereas, in response to 1 nM CCK-8, the total Ca2+ mobilization was significantly increased by ethanol pre-treatment. Preincubation of cells with 1 mM 4-MP, an inhibitor of alcohol dehydrogenase, or 10 μM of the antioxidant cinnamtannin B-1, reverted the effect of ethanol on total Ca2+ mobilization evoked by 1 nM CCK-8. Cinnamtannin B-1 blocked ethanol-evoked ROS production. Conclusion ethanol may lead, either directly or through ROS generation, to an over stimulation of pancreatic acinar cells in response to CCK-8, resulting in a higher Ca2+ mobilization compared to normal conditions. The actions of ethanol on CCK-8-stimulation of cells create a situation potentially leading to Ca2+ overload, which is a common pathological precursor that mediates pancreatitis. PMID:19878551

  18. Polyethylenimine-mediated expression of transgenes in the acinar cells of rats salivary glands in vivo

    PubMed Central

    Sramkova, Monika; Parente, Laura; Wigand, Timothy; Aye, Myo-Pale'; Shitara, Akiko; Weigert, Roberto

    2015-01-01

    Non viral-mediated transfection of plasmid DNA provides a fast and reliable way to express various transgenes in selected cell populations in live animals. Here, we show an improvement of a previously published method that is based on injecting plasmid DNA into the ductal system of the salivary glands in live rats. Specifically, using complexes between plasmid DNA and polyethyleneimine (PEI) we show that the expression of the transgenes is directed selectively to the salivary acinar cells. PEI does not affect the ability of cells to undergo regulated exocytosis, which was one of the main drawbacks of the previous methods. Moreover PEI does not affect the proper localization and targeting of transfected proteins, as shown for the apical plasma membrane water channel aquaporin 5 (AQP5). Overall, this approach, coupled with the use of intravital microscopy, permits to conduct localization and functional studies under physiological conditions, in a rapid, reliable, and affordable fashion. PMID:25621283

  19. Effect of glucagon on digestive enzyme synthesis, transport and secretion in mouse pancreatic acinar cells.

    PubMed Central

    Singh, M

    1980-01-01

    1. Effect of glucagon on amylase secretion and lactic dehydrogenase (LDH) release from functionally intact dissociated pancreatic acinar cells and acini was studied. 2. In dissociated rat pancreatic acinar cells, the rate of amylase secretion was increased by 70% with bethanechol (maximally effective concentration, 10(-4) M) and 125% with A23187 (10(-5) M), but the response to cholecystokinin-pancreozymin (CCK-PZ) was inconsistent. In dissociated cells from mouse pancreas, the increases amounted to 78% with bethanechol (10(-4) M), 134% with A23187 (10(-5) M) and 82% with CCK-PZ (maximally effective concentration, 0 . 01 u. ml.-1). Glucagon in concentrations ranging from 10(-7) to 10(-4) M increased amylase secretion by 3, 26, 67 and 80%, whereas secretin (10(-8)--10(-5) M) increased amylase secretion by 8, 39, 88 and 138%. LDH release was increased with A23187 in concentrations greater than 10(-6) M. 3. CCK-PZ, bethanechol and A23187 used in maximal concentrations potentiated the effect of a submaximal dose of glucagon whereas secretin did not have an additive or a potentiating effect. 4. Pancreatic acini were approximately 3 times more responsive to secretagogues than cells. The dose--response curves to bethanechol, glucagon and CCK-PZ for increase in amylase secretion were similar. LDH release was not increased by these agents. Cytochalasin B (5 microgram ml.-1) which is known to disrupt the integrity of luminal membrane inhibited the amylase secretion stimulated by glucagon, bethanechol and CCK-PZ. 5. Glucagon inhibited incorporation of a mixture of fifteen 14C-labelled amino acids (algal profile, Schwarz Mann) into perchloric acid precipitable proteins in dissociated mouse pancreatic acini within 30 min. 6. In 'pulse-chase' experiments, glucagon decreased the specific activity of zymogen granules isolated by differential centrifugation, from pancreatic lobules (120 min) and increased the specific activity of radiolabelled proteins in the medium (60 and 120 min

  20. The relation between apoptosis of acinar cells and nitric oxide during acute rejection of pancreas transplantation in rats.

    PubMed

    Xiaoguang, Ni; Zhong, Liu; Hailong, Chen; Ping, Zhao; Xiaofeng, Bai; Fenglin, Guan

    2003-01-01

    Apoptosis is an important mechanism of immune-mediated graft damage. Nitric oxide (NO) generated by inducible NO synthase (iNOS) has been demonstrated to induce apoptosis. This study investigated whether apoptosis occurs during pancreas allograft rejection and examined the relationship of apoptosis of acinar cells and NO. The rats were divided into three groups: untreated isograft group, untreated allograft group and aminoguanidine (AG)-treated group. The pancreatic grafts were harvested on the post-transplantation day 3, 5 and 7 and were used to detect the histopathological rejection grade, the expression of iNOS and the apoptotic index (AI) of the graft. iNOS presented faint positive in the acinar cells of untreated isografts and did not change greatly after transplantation (P>0.05), the level of iNOS in the untreated allografts increased progressively (P<0.01) and at the same time point was significantly higher than that of untreated isograft group and AG-treated group (P<0.01). The transferase-mediated dUTP nick end labeling showed that the apoptotic cells were mainly acinar cells. A significant correlation between AI and iNOS was noted (P<0.01, r=0.611). Therefore, NO-mediated apoptosis of acinar cells plays an important role in acute rejection of pancreas transplantation, AG can mitigate the damage of pancreas allografts.

  1. A Systems Biology Approach Identifies a Regulatory Network in Parotid Acinar Cell Terminal Differentiation

    PubMed Central

    Metzler, Melissa A.; Venkatesh, Srirangapatnam G.; Lakshmanan, Jaganathan; Carenbauer, Anne L.; Perez, Sara M.; Andres, Sarah A.; Appana, Savitri; Brock, Guy N.; Wittliff, James L.; Darling, Douglas S.

    2015-01-01

    Objective The transcription factor networks that drive parotid salivary gland progenitor cells to terminally differentiate, remain largely unknown and are vital to understanding the regeneration process. Methodology A systems biology approach was taken to measure mRNA and microRNA expression in vivo across acinar cell terminal differentiation in the rat parotid salivary gland. Laser capture microdissection (LCM) was used to specifically isolate acinar cell RNA at times spanning the month-long period of parotid differentiation. Results Clustering of microarray measurements suggests that expression occurs in four stages. mRNA expression patterns suggest a novel role for Pparg which is transiently increased during mid postnatal differentiation in concert with several target gene mRNAs. 79 microRNAs are significantly differentially expressed across time. Profiles of statistically significant changes of mRNA expression, combined with reciprocal correlations of microRNAs and their target mRNAs, suggest a putative network involving Klf4, a differentiation inhibiting transcription factor, which decreases as several targeting microRNAs increase late in differentiation. The network suggests a molecular switch (involving Prdm1, Sox11, Pax5, miR-200a, and miR-30a) progressively decreases repression of Xbp1 gene transcription, in concert with decreased translational repression by miR-214. The transcription factor Xbp1 mRNA is initially low, increases progressively, and may be maintained by a positive feedback loop with Atf6. Transfection studies show that Xbp1Mist1 promoter. In addition, Xbp1 and Mist1 each activate the parotid secretory protein (Psp) gene, which encodes an abundant salivary protein, and is a marker of terminal differentiation. Conclusion This study identifies novel expression patterns of Pparg, Klf4, and Sox11 during parotid acinar cell differentiation, as well as numerous differentially expressed microRNAs. Network analysis identifies a novel stemness arm, a

  2. Variations in the expression and distribution pattern of AQP5 in acinar cells of patients with sialadenosis.

    PubMed

    Teymoortash, Afshin; Wiegand, Susanne; Borkeloh, Martin; Bette, Michael; Ramaswamy, Annette; Steinbach-Hundt, Silke; Neff, Andreas; Werner, Jochen A; Mandic, Robert

    2012-01-01

    Previously, we pointed out on a possible role of aquaporin 5 (AQP5) in the development of sialadenosis. The goal of the present study was to further assess the association of AQP5 in the development of this salivary gland disease. The acinar diameter and mean surface area appeared elevated in sialadenosis tissues, which is a typical observation in this disease. AQP5 expression was evaluated by immunohistochemistry using tissue samples derived from salivary glands of patients with confirmed sialadenosis either as a primary diagnosis or as a secondary diagnosis within the framework of other salivary gland diseases. Normal salivary gland tissue served as a control. In sialadenosis tissues, the AQP5 signal at the apical plasma membrane of acinar cells frequently appeared stronger compared with that in normal salivary glands. In addition, the distribution of AQP5 at the apical region seemed to differ between normal and sialadenosis tissues, where AQP5 frequently was diffusely distributed near or at the apical plasma membrane of the acinar cells in contrast to normal controls where the AQP5 signal was strictly confined to the apical plasma membrane. These observations suggest that sialadenosis is associated with a different AQP5 expression and distribution pattern in salivary acinar cells.

  3. Sulforaphane Protects Pancreatic Acinar Cell Injury by Modulating Nrf2-Mediated Oxidative Stress and NLRP3 Inflammatory Pathway

    PubMed Central

    Dong, Zhaojun; Shang, Haixiao; Chen, Yong Q.; Pan, Li-Long

    2016-01-01

    Acute pancreatitis (AP) is characterized by early activation of intra-acinar proteases followed by acinar cell death and inflammation. Cellular oxidative stress is a key mechanism underlying these pathological events. Sulforaphane (SFN) is a natural organosulfur antioxidant with undescribed effects on AP. Here we investigated modulatory effects of SFN on cellular oxidation and inflammation in AP. AP was induced by cerulean hyperstimulation in BALB/c mice. Treatment group received a single dose of 5 mg/kg SFN for 3 consecutive days before AP. We found that SFN administration attenuated pancreatic injury as evidenced by serum amylase, pancreatic edema, and myeloperoxidase, as well as by histological examination. SFN administration reverted AP-associated dysregulation of oxidative stress markers including pancreatic malondialdehyde and redox enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx). In acinar cells, SFN treatment upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression and Nrf2-regulated redox genes including quinoneoxidoreductase-1, heme oxidase-1, SOD1, and GPx1. In addition, SFN selectively suppressed cerulein-induced activation of the nucleotide-binding domain leucine-rich repeat containing family, pyrin domain-containing 3 (NLRP3) inflammasome, in parallel with reduced nuclear factor- (NF-) κB activation and modulated NF-κB-responsive cytokine expression. Together, our data suggested that SFN modulates Nrf2-mediated oxidative stress and NLRP3/NF-κB inflammatory pathways in acinar cells, thereby protecting against AP. PMID:27847555

  4. Chronic alcohol exposure inhibits biotin uptake by pancreatic acinar cells: possible involvement of epigenetic mechanisms.

    PubMed

    Srinivasan, Padmanabhan; Kapadia, Rubina; Biswas, Arundhati; Said, Hamid M

    2014-11-01

    Chronic exposure to alcohol affects different physiological aspects of pancreatic acinar cells (PAC), but its effect on the uptake process of biotin is not known. We addressed this issue using mouse-derived pancreatic acinar 266-6 cells chronically exposed to alcohol and wild-type and transgenic mice (carrying the human SLC5A6 5'-promoter) fed alcohol chronically. First we established that biotin uptake by PAC is Na(+) dependent and carrier mediated and involves sodium-dependent multivitamin transporter (SMVT). Chronic exposure of 266-6 cells to alcohol led to a significant inhibition in biotin uptake, expression of SMVT protein, and mRNA as well as in the activity of the SLC5A6 promoter. Similarly, chronic alcohol feeding of wild-type and transgenic mice carrying the SLC5A6 promoter led to a significant inhibition in biotin uptake by PAC, as well as in the expression of SMVT protein and mRNA and the activity of the SLC5A6 promoters expressed in the transgenic mice. We also found that chronic alcohol feeding of mice is associated with a significant increase in the methylation status of CpG islands predicted to be in the mouse Slc5a6 promoters and a decrease in the level of expression of transcription factor KLF-4, which plays an important role in regulating SLC5A6 promoter activity. These results demonstrate, for the first time, that chronic alcohol exposure negatively impacts biotin uptake in PAC and that this effect is exerted (at least in part) at the level of transcription of the SLC5A6 gene and may involve epigenetic/molecular mechanisms.

  5. Cannabinoid receptor subtype 2 (CB2R) agonist, GW405833 reduces agonist-induced Ca2+ oscillations in mouse pancreatic acinar cells

    PubMed Central

    Huang, Zebing; Wang, Haiyan; Wang, Jingke; Zhao, Mengqin; Sun, Nana; Sun, Fangfang; Shen, Jianxin; Zhang, Haiying; Xia, Kunkun; Chen, Dejie; Gao, Ming; Hammer, Ronald P.; Liu, Qingrong; Xi, Zhengxiong; Fan, Xuegong; Wu, Jie

    2016-01-01

    Emerging evidence demonstrates that the blockade of intracellular Ca2+ signals may protect pancreatic acinar cells against Ca2+ overload, intracellular protease activation, and necrosis. The activation of cannabinoid receptor subtype 2 (CB2R) prevents acinar cell pathogenesis in animal models of acute pancreatitis. However, whether CB2Rs modulate intracellular Ca2+ signals in pancreatic acinar cells is largely unknown. We evaluated the roles of CB2R agonist, GW405833 (GW) in agonist-induced Ca2+ oscillations in pancreatic acinar cells using multiple experimental approaches with acute dissociated pancreatic acinar cells prepared from wild type, CB1R-knockout (KO), and CB2R-KO mice. Immunohistochemical labeling revealed that CB2R protein was expressed in mouse pancreatic acinar cells. Electrophysiological experiments showed that activation of CB2Rs by GW reduced acetylcholine (ACh)-, but not cholecystokinin (CCK)-induced Ca2+ oscillations in a concentration-dependent manner; this inhibition was prevented by a selective CB2R antagonist, AM630, or was absent in CB2R-KO but not CB1R-KO mice. In addition, GW eliminated L-arginine-induced enhancement of Ca2+ oscillations, pancreatic amylase, and pulmonary myeloperoxidase. Collectively, we provide novel evidence that activation of CB2Rs eliminates ACh-induced Ca2+ oscillations and L-arginine-induced enhancement of Ca2+ signaling in mouse pancreatic acinar cells, which suggests a potential cellular mechanism of CB2R-mediated protection in acute pancreatitis. PMID:27432473

  6. FK506 induces biphasic Ca2+ release from microsomal vesicles of rat pancreatic acinar cells.

    PubMed

    Ozawa, Terutaka

    2006-07-01

    The effect of the immunosuppressant drug FK506 on microsomal Ca2+ release was investigated in rat pancreatic acinar cells. When FK506 (0.1-200 microM) was added to the microsomal vesicles at a steady state of ATP-dependent 45Ca2+ uptake, FK506 caused a dose-dependent and a biphasic release of 45Ca2+. Almost 10% of total 45Ca2+ uptake was released at FK506 concentrations up to 10 microM (Km=0.47 microM), and 60% of total 45Ca2+ uptake was released at FK506 concentrations over 10 microM (Km=55 microM). Preincubation of the vesicles with cyclic ADP-ribose (cADPR, 0.5 microM) increased the FK506 (< or =10 microM)-induced 45Ca2+ release (Ozawa T, Biochim Biophys Acta 1693: 159-166, 2004). Preincubation with heparin (200 microg/ml) resulted in significant inhibition of the FK506 (30 microM)-induced 45Ca2+ release. Subsequent addition of inositol 1,4,5-trisphosphate (IP3, 5 microM) after FK506 (100 microM)-induced 45Ca2+ release did not cause any release of 45Ca2+. These results indicate that two types of FK506-induced Ca2+ release mechanism operate in the endoplasmic reticulum of rat pancreatic acinar cells: a high-affinity mechanism of Ca2+ release, which involves activation of the ryanodine receptor, and a low-affinity mechanism of Ca2+ release, which involves activation of the IP3 receptor.

  7. The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini

    PubMed Central

    Gaiko-Shcherbak, Aljona; Fabris, Gloria; Dreissen, Georg; Merkel, Rudolf; Hoffmann, Bernd; Noetzel, Erik

    2015-01-01

    The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function. PMID:26674091

  8. The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini.

    PubMed

    Gaiko-Shcherbak, Aljona; Fabris, Gloria; Dreissen, Georg; Merkel, Rudolf; Hoffmann, Bernd; Noetzel, Erik

    2015-01-01

    The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.

  9. Interaction of bombesin and litorin with specific membrane receptors on pancreatic acinar cells

    PubMed Central

    Jensen, R. T.; Moody, T.; Pert, C.; Rivier, J. E.; Gardner, J. D.

    1978-01-01

    We have prepared 125I-labeled [Tyr4]bombesin and have examined the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acini from guinea pig pancreas. Binding of 125I-labeled [Tyr4]-bombesin was saturable, temperature-dependent, and reversible and reflected interaction of the labeled peptide with a single class of binding sites on the plasma membrane of pancreatic acinar cells. Each acinar cell possessed approximately 5000 binding sites, and binding of the tracer to these sites could be inhibited by [Tyr4]bombesin [concentration for half-maximal effect (Kd), 2 nM], bombesin (Kd, 4 nM), or litorin (Kd, 40 nM) but not by eledoisin, physalemin, somatostatin, carbachol, atropine, secretin, vasocative intestinal peptide, neurotensin, or bovine pancreatic polypeptide. At high concentrations (>0.1 μM), cholecystokinin and caerulein each caused a small (15-20%) reduction in binding of lableled [Tyr4]bombesin. With bombesin, litorin, and [Tyr4]bombesin, there was a close correlation between the relative potency for inhibition of binding of labeled [Tyr4]bombesin and that for stimulation of amylase secretion. For a given peptide, however, a 10-fold higher concentration was required for half-maximal inhibition of binding than for half-maximal stimulation of amylase secretion, calcium outflux, or cyclic GMP accumulation. These results indicate that dispersed acini from guinea pig pancreas possess a single class of receptors that interact with [Tyr4]bombesin, bombesin, and litorin and that occupation of 25% of these receptors will cause a maximal biological response. PMID:216015

  10. Apoptotic Mechanisms of Peroxisome Proliferator–Activated Receptor-γ Activation in Acinar Cells During Acute Pancreatitis

    PubMed Central

    Xu, Ping; Lou, Xiao-Li; Chen, Cheng

    2016-01-01

    Objective The objective of this study was to determine the mechanism by which activation of peroxisome proliferator–activated receptor-γ promotes apoptosis of acinar cells in pancreatitis. Methods AR42j cells pretreated with the peroxisome proliferator–activated receptor-γ agonist pioglitazone were activated by cerulein as an in vitro model of acute pancreatitis. Inflammatory cytokines and amylase were detected by enzyme-linked immunosorbent assay. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was measured by flow cytometry and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining. Activity of caspases was determined. Bax and Bcl-2 levels were assayed by Western blot. Results Cytokines, amylase, and cellular proliferation decreased in pioglitazone-pretreated cells. Pioglitazone increased the activity of caspases 3, 8, and 9 in cerulein-activated AR42j cells as well as in the pancreas of rats 3 hours after induction of severe acute pancreatitis. Acinar cell apoptosis was induced by reducing the mitochondrial membrane potential in the pioglitazone group. Pioglitazone increased expression of proapoptotic Bax proteins and decreased antiapoptotic Bcl-2 in cerulein-induced AR42j cells and decreased Bcl-2 levels in pancreatic tissue of severe acute pancreatitis rats 1 and 3 hours after induction. Conclusion Pioglitazone may promote apoptosis of acinar cells through both intrinsic and extrinsic apoptotic pathways in acute pancreatitis. PMID:26495791

  11. Reversal of diabetes in rats using GLP-1-expressing adult pancreatic duct-like precursor cells transformed from acinar to ductal cells.

    PubMed

    Lee, Jieun; Wen, Jing; Park, Jeong Youp; Kim, Sun-A; Lee, Eun Jig; Song, Si Young

    2009-09-01

    Pancreatic injury induces replacement of exocrine acinar cells with ductal cells. These ductal cells have the potential to regenerate the pancreas, but their origin still remains unknown. It has been reported that adult pancreatic acinar cells have the potential to transdifferentiate to ductal progenitor cells. In this regards, we established novel adult pancreatic duct-like progenitor cell lines YGIC4 and YGIC5 and assessed the usefulness of these ductal progenitors in the cell therapy of diabetic rats. Acinar cells were cultured from pancreata of male Sprague Dawley rats and gradually attained ductal cell characteristics, such as expression of CK19 and CFTR with a concomitant down-regulation of amylase expression over time, suggesting transdifferentiation from acinar to ductal cells. During cell culture, the expression of Pdx-1, c-Kit, and vimentin peaked and then decreased, suggesting that transdifferentiation recapitulated embryogenesis. Overexpression of pancreas development regulatory genes and CK19, as well as the ability to differentiate into insulin-producing cells, suggests that the YGIC5 cells had characteristics of pancreatic progenitor cells. Finally, YGIC5 cells coexpressing Green fluorescent protein (GFP) and glucagon-like peptide (GLP)-1 under the activation of a zinc-inducible metallothionein promoter were intravenously infused to STZ-induced diabetic rats. Hyperglycemia was ameliorated with elevation of plasma insulin, and GFP-positive donor cells were colocalized in the acinar and islet areas of recipient pancreata following zinc treatment. In conclusion, after establishing pancreatic progenitor cell lines YGIC4 and YGIC5 under the concept of acinar to ductal transdifferentiation in vitro, we demonstrate how these adult pancreatic stem/progenitor cells can be used to regulate adult pancreatic differentiation toward developing therapy for pancreatic disease such as diabetes mellitus.

  12. TNF-α inhibits aquaporin 5 expression in human salivary gland acinar cells via suppression of histone H4 acetylation.

    PubMed

    Yamamura, Yoshiko; Motegi, Katsumi; Kani, Kouichi; Takano, Hideyuki; Momota, Yukihiro; Aota, Keiko; Yamanoi, Tomoko; Azuma, Masayuki

    2012-08-01

    Sjögren's syndrome is a systemic autoimmune disease characterized by reductions in salivary and lacrimal secretions. The mechanisms underlying these reductions remain unclear. We have previously shown that TNF-α plays an important role in the destruction of acinar structures. Here we examined TNF-α's function in the expression of aquaporin (AQP) 5 in human salivary gland acinar cells. Immortalized human salivary gland acinar (NS-SV-AC) cells were treated with TNF-α, and then the expression levels of AQP5 mRNA and protein were analysed. In addition, the mechanisms underlying the reduction of AQP5 expression by TNF-α treatment were investigated. TNF-α-treatment of NS-SV-AC cells significantly suppressed the expression levels of AQP5 mRNA and protein, and reduced the net fluid secretion rate. We examined the expression and activation levels of DNA methyltransferases (Dnmts) in NS-SV-AC cells treated with TNF-α. However, no significant changes were observed in the expression or activation levels of Dnmt1, Dnmt3a or Dnmt3b. Although we also investigated the role of NF-κB activity in the TNF-α-induced suppression of AQP5 expression in NS-SV-AC cells, we detected similar TNF-α suppression of AQP5 expression in non-transfected cells and in a super-repressor form of IκBα cDNA-transfected cell clones. However, interestingly, chromatin immunoprecipitation analysis demonstrated a remarkable decrease in levels of acetylated histone H4 associated with the AQP5 gene promoter after treatment with TNF-α in NS-SV-AC cells. Therefore, our results may indicate that TNF-α inhibition of AQP5 expression in human salivary gland acinar cells is due to the epigenetic mechanism by suppression of acetylation of histone H4.

  13. Alcohol oxidizing enzymes and ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells.

    PubMed

    Bhopale, Kamlesh K; Falzon, Miriam; Ansari, G A S; Kaphalia, Bhupendra S

    2014-04-01

    Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disease causing significant morbidity and mortality. Due to lack of a suitable animal model, the underlying mechanism of ACP is poorly understood. Chronic alcohol abuse inhibits alcohol dehydrogenase (ADH) and facilitates nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) in the pancreas frequently damaged during chronic ethanol abuse. Earlier, we reported a concentration-dependent formation of FAEEs and cytotoxicity in ethanol-treated rat pancreatic tumor (AR42J) cells, which express high FAEE synthase activity as compared to ADH and cytochrome P450 2E1. Therefore, the present study was undertaken to investigate the role of various ethanol oxidizing enzymes in ethanol-induced pancreatic acinar cell injury. Confluent AR42J cells were pre-treated with inhibitors of ADH class I and II [4-methylpyrazole (MP)] or class I, II, and III [1,10-phenanthroline (PT)], cytochrome P450 2E1 (trans-1,2-dichloroethylene) or catalase (sodium azide) followed by incubation with 800 mg% ethanol at 37°C for 6 h. Ethanol metabolism, cell viability, cytotoxicity (apoptosis and necrosis), cell proliferation status, and formation of FAEEs in AR42J cells were measured. The cell viability and cell proliferation rate were significantly reduced in cells pretreated with 1,10-PT + ethanol followed by those with 4-MP + ethanol. In situ formation of FAEEs was twofold greater in cells incubated with 1,10-PT + ethanol and ∼1.5-fold in those treated with 4-MP + ethanol vs. respective controls. However, cells treated with inhibitors of cytochrome P450 2E1 or catalase in combination of ethanol showed no significant changes either for FAEE formation, cell death or proliferation rate. Therefore, an impaired ADH class I-III catalyzed oxidation of ethanol appears to be a key contributing factor in ethanol-induced pancreatic injury via formation of nonoxidative metabolites of ethanol.

  14. Involvement of thrombopoietin in acinar cell necrosis in L-arginine-induced acute pancreatitis in mice.

    PubMed

    Shen, Jiaqing; Wan, Rong; Hu, Guoyong; Wang, Feng; Shen, Jie; Wang, Xingpeng

    2012-10-01

    Thrombopoietin (TPO) plays an important role in injuries of different tissues. However, the role of TPO in acute pancreatitis (AP) is not yet known. The aim of the study was to determine the involvement of TPO in AP. Serum TPO was assayed in necrotizing pancreatitis induced by L-arginine in mice. Recombinant TPO and anti-TPO antibody were given to mice with necrotizing pancreatitis. Amylase, lipase, lactate dehydrogenase, myeloperoxidase activity and pancreatic water content were assayed in serum and tissue samples. Pancreas and lung tissue samples were also collected for histological evaluation. Immunohistochemistry of amylase α and PCNA were applied for the study of acinar regeneration and TUNEL assay for the detection of apoptosis in the pancreas. Increased levels of serum TPO were found in necrotizing pancreatitis. After TPO administration, more severe acinar necrosis was found and blockade of TPO reduced the acinar necrosis in this AP model. Acinar regeneration and apoptosis in the pancreas were affected by TPO and antibody treatment in necrotizing pancreatitis. The severity of pancreatitis-associated lung injury was worsened after TPO treatment, but attenuated after Anti-TPO antibody treatment. In conclusion, serum TPO is up-regulated in the necrotizing pancreatitis induced by L-arginine in mice and may be a risk factor for the pancreatic acinar necrosis in AP. As a pro-necrotic factor, blockade of TPO can attenuate the acinar necrosis in AP and may be a possible therapeutic intervention for AP.

  15. Direct Imaging of RAB27B-Enriched Secretory Vesicle Biogenesis in Lacrimal Acinar Cells Reveals Origins on a Nascent Vesicle Budding Site

    PubMed Central

    Chiang, Lilian; Karvar, Serhan; Hamm-Alvarez, Sarah F.

    2012-01-01

    This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the “nascent vesicle site,” from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150Glued, a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules. PMID:22363735

  16. Long-term dexamethasone treatment alters the histomorphology of acinar cells in rat parotid and submandibular glands

    PubMed Central

    Bighetti, Bruna B; Assis, Gerson F d; Vieira, Danilo C; Violato, Natalia M; Cestari, Tania M; Taga, Rumio; Bosqueiro, José R; Rafacho, Alex

    2014-01-01

    Glucocorticoids (GCs) induce insulin resistance (IR), a condition known to alter oral homeostasis. This study investigated the effects of long-term dexamethasone administration on morphofunctional aspects of salivary glands. Male Wistar rats received daily injections of dexamethasone [0.1 mg/kg body weight (b.w.), intraperitoneally] for 10 days (DEX), whereas control rats received saline. Subsequently, glycaemia, insulinaemia, insulin secretion and salivary flow were analysed. The parotid and submandibular glands were collected for histomorphometric evaluation and Western blot experiments. The DEX rats were found to be normoglycaemic, hyperinsulinaemic, insulin resistant and glucose intolerant (P < 0.05). DEX rat islets secreted more insulin in response to glucose (P < 0.05). DEX rats had significant reductions in the masses of the parotid (29%) and submandibular (16%) glands (P < 0.05) that was associated with reduced salivary flux rate. The hypotrophy in both glands observed in the DEX group was associated with marked reduction in the volume of the acinar cells in these glands of 50% and 26% respectively (P < 0.05). The total number of acinar cells was increased in the submandibular glands of the DEX rats (P < 0.05) but not in the parotid glands. The levels of proteins related to insulin and survival signalling in both glands did not differ between the groups. In conclusion, the long-term administration of dexamethasone caused IR, which was associated with significant reductions in both mass and flux rate of the salivary glands. The parotid and submandibular glands exhibited reduced acinar cell volume; however, the submandibular glands displayed acinar hyperplasia, indicating a gland-specific response to GCs. Our data emphasize that GC-based therapies and insulin-resistant states have a negative impact on salivary gland homeostasis. PMID:25186305

  17. Integrins and epithelial cell polarity.

    PubMed

    Lee, Jessica L; Streuli, Charles H

    2014-08-01

    Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell-matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical-basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity.

  18. Pancreatic panniculitis associated with acinar cell carcinoma of the pancreas: a case report.

    PubMed

    Zheng, Zhen Jiang; Gong, Jun; Xiang, Guang Ming; Mai, Gang; Liu, Xu Bao

    2011-05-01

    Pancreatic panniculitis is a rare type of disorder associated with pancreatic diseases. We describe here a case of 54-year-old man who was admitted to the Department of Dermatology with the diagnosis of erythema nodosum. The patient presented with a 9-month history of painful erythematous nodules on the extremities, joint pain and swelling, and weight loss. A highly elevated level of pancreatic lipase was found on the laboratory examinations. The biopsy specimens from the skin lesions showed subcutaneous fat necrosis. Abdominal computed tomography (CT) revealed a large mass with central necrosis in the body and tail of the pancreas. Distal pancreatectomy, splenectomy and partial transverse colectomy were successfully performed on day 17 of the hospitalization. The histopathologic findings supported the diagnosis of acinar cell carcinoma of the pancreas (ACCP). Postoperatively, the level of serum lipase returned to normal, and the skin lesions and joint manifestations gradually regressed. However, the swelling did not significantly resolve in the left knee. In view of the non-specific clinical presentation of this disease, clinicians should be alert and have a high index of suspicion for pancreatic panniculitis.

  19. Pancreatic ducts as an important route of tumor extension for acinar cell carcinoma of the pancreas.

    PubMed

    Ban, Daisuke; Shimada, Kazuaki; Sekine, Shigeki; Sakamoto, Yoshihiro; Kosuge, Tomoo; Kanai, Yae; Hiraoka, Nobuyoshi

    2010-07-01

    Acinar cell carcinoma (ACC) of the pancreas is very rare, which usually grows expansively. Recently, a variant of ACC with predominant growth in the pancreatic ducts has been proposed, and is speculated to have potentially less aggressive behavior. The aim of this study was to investigate how the pancreatic duct system is related to the growth and extension of ACC. We reviewed the detailed gross and histologic features of 13 cases of ACC, of which 7 (54%) showed intraductal polypoid growth (IPG) of the tumor in the large pancreatic ducts with a mean IPG length of 24.8 mm. Tumors with IPG were found to spread characteristically along the pancreatic ducts as extending polypoid projections, filling the ducts and destroying the duct walls, although tumors did not tend to extend beyond the pancreatic parenchyma. Comparison of the clinicopathologic characteristics showed that ACC with IPG had less infiltrative features including lymphatic, venous, and neural invasion, formation of tumor thrombus in the portal vein, nodal metastasis, and invasion beyond the pancreas to the surrounding organs; death in only 1 case (14%) of ACC with IPG was the result of ACC itself. In contrast, ACC without IPG frequently showed more infiltrative growth, and was the cause of death in 50% of patients with this type of tumor. Intraductal dissemination of ACC in pancreatic ducts was proven in 1 case of ACC with IPG. These findings suggest that a significant proportion of ACC shows IPG, which is potentially linked to less aggressive clinicopathologic characteristics.

  20. [Protein malnutrition and response of pancreatic acinar cells to stimulation by cholecystokinin].

    PubMed

    Prost, J; Belleville, J

    1988-01-01

    Pancreatic lobules were isolated from 2 groups of male Wistar rats after 23 days of diet. A control group (C) fed on a 20% protein diet (16% gluten + 4% casein) and an experimental group (E) on a 5% protein diet (4% gluten + 1% casein). After isolation, lobules were preincubated 10 min with 10 muCi [3H]-leucine, washed, then incubate within Krebs Ringer bicarbonate Hepes. Basal secretion, then stimulated secretion (50 pM of cholecystokinin (CCK] of radioactive and non-radioactive protein and amylase outputs were measured. During basal secretion, in (E) group, lobules secreted more proteins than (C) one, the same outputs of amylase and radioactive protein were observed in both groups. The stimulated secretion by CCK increased the outputs of non-radioactive protein and amylase of lobules (T) (2-3 fold), but was without effect on lobule (E) outputs. Therefore, a low-protein diet involved a decrease of CCK sensibility on acinar cells, this fact might be mediated by a decreasing number and/or affinity of their CCK receptors.

  1. Ca²⁺ signaling and regulation of fluid secretion in salivary gland acinar cells.

    PubMed

    Ambudkar, Indu S

    2014-06-01

    Neurotransmitter stimulation of plasma membrane receptors stimulates salivary gland fluid secretion via a complex process that is determined by coordinated temporal and spatial regulation of several Ca(2+) signaling processes as well as ion flux systems. Studies over the past four decades have demonstrated that Ca(2+) is a critical factor in the control of salivary gland function. Importantly, critical components of this process have now been identified, including plasma membrane receptors, calcium channels, and regulatory proteins. The key event in activation of fluid secretion is an increase in intracellular [Ca(2+)] ([Ca(2+)]i) triggered by IP3-induced release of Ca(2+) from ER via the IP3R. This increase regulates the ion fluxes required to drive vectorial fluid secretion. IP3Rs determine the site of initiation and the pattern of [Ca(2+)]i signal in the cell. However, Ca(2+) entry into the cell is required to sustain the elevation of [Ca(2+)]i and fluid secretion. This Ca(2+) influx pathway, store-operated calcium influx pathway (SOCE), has been studied in great detail and the regulatory mechanisms as well as key molecular components have now been identified. Orai1, TRPC1, and STIM1 are critical components of SOCE and among these, Ca(2+) entry via TRPC1 is a major determinant of fluid secretion. The receptor-evoked Ca(2+) signal in salivary gland acinar cells is unique in that it starts at the apical pole and then rapidly increases across the cell. The basis for the polarized Ca(2+) signal can be ascribed to the polarized arrangement of the Ca(2+) channels, transporters, and signaling proteins. Distinct localization of these proteins in the cell suggests compartmentalization of Ca(2+) signals during regulation of fluid secretion. This chapter will discuss new concepts and findings regarding the polarization and control of Ca(2+) signals in the regulation of fluid secretion.

  2. Carbachol activates a K+ channel of very small conductance in the basolateral membrane of rat pancreatic acinar cells.

    PubMed

    Köttgen, M; Hoefer, A; Kim, S J; Beschorner, U; Schreiber, R; Hug, M J; Greger, R

    1999-10-01

    Secretion of Cl- requires the presence of a K+ conductance to hyperpolarize the cell, and to provide the driving force for Cl- exit via luminal Cl- channels. In the exocrine pancreas Cl- secretion is mediated by an increase in cytosolic Ca2+ ([Ca2+]i). Two types of Ca2+-activated K+ channels could be shown in pancreatic acinar cells of different species. However, there are no data on Ca2+-activated K+ channels in rat pancreatic acini. Here we examine the basolateral K+ conductance of freshly isolated rat pancreatic acinar cells in cell-attached and cell-excised patch-clamp experiments. Addition of carbachol (CCH, 1 micromol/l) to the bath led to the activation of very small conductance K+ channels in cell-attached patches (n=27), producing a noisy macroscopic outward current. The respective outward conductance increased significantly by a factor of 2.1+/-0.1 (n=27). Noise analysis revealed a Lorentzian noise component with a corner frequency (f(c)) of 30.3+/-3.5 Hz (n=19), consistent with channel activity in these patches. The estimated single-channel conductance was 1.5+/-0.4 pS (n=19). In cell-excised patches (inside out) from cells previously stimulated with CCH, channel activity was only observed in the presence of K+ in the bath solution. Under these conditions f(c) was 47.6+/-11.9 Hz (estimated single-channel conductance 1.1+/-0.2 pS, n=20). The current/voltage relationship of the noise showed weak inward rectification and the reversal potential shifted towards E(K+) when Na+ in the bath was replaced by K+. Channel activity in cell-excised patches was slightly reduced by 10 mmol/l Ba2+ (23.6+/-2.1% of the total outward current) and was completely absent when K+ in the bath was replaced by Na+. Reduction of the [Ca2+]i from 1 mmol/l to 1 micromol/l in cell-excised experiments decreased the current by 52.3+/-12.3% (n=5). Expression of K(v)LQT1, one of the possible candidates for a small-conductance K+ channel in rat pancreatic acinar cells, was shown by reverse

  3. Human bronchial epithelial cells differentiate to 3D glandular acini on basement membrane matrix.

    PubMed

    Wu, Xiaofang; Peters-Hall, Jennifer R; Bose, Sumit; Peña, Maria T; Rose, Mary C

    2011-06-01

    To create a model system that investigates mechanisms resulting in hyperplasia and hypertrophy of respiratory tract submucosal glands, we developed an in vitro three-dimensional (3D) system wherein normal human bronchial epithelial (HBE) cells differentiated into glandular acini when grown on a basement membrane matrix. The differentiation of primary HBE cells into glandular acini was monitored temporally by light microscopy. Apoptosis-induced lumen formation was observed by immunofluorescence analysis. The acinar cells expressed and secreted MUC5B mucin (marker for glandular mucous cells) and lysozyme, lactoferrin, and zinc-α2-glycoprotein (markers for glandular serous cells) at Day 22. β-Tubulin IV, a marker for ciliated cells, was not detected. Expression of mucous and serous cell markers in HBE glandular acini demonstrated that HBE cells grown on a basement membrane matrix differentiated into acini that exhibit molecular characteristics of respiratory tract glandular acinar cells. Inhibition studies with neutralizing antibodies resulted in a marked decrease in size of the spheroids at Day 7, demonstrating that laminin (a major component of the basement membrane matrix), the cell surface receptor integrin α6, and the cell junction marker E-cadherin have functional roles in HBE acinar morphogenesis. No significant variability was detected in the average size of glandular acini formed by HBE cells from two normal individuals. These results demonstrated that this in vitro model system is reproducible, stable, and potentially useful for studies of glandular differentiation and hyperplasia.

  4. Atp2c2 Is Transcribed From a Unique Transcriptional Start Site in Mouse Pancreatic Acinar Cells.

    PubMed

    Fenech, Melissa A; Sullivan, Caitlin M; Ferreira, Lucimar T; Mehmood, Rashid; MacDonald, William A; Stathopulos, Peter B; Pin, Christopher L

    2016-12-01

    Proper regulation of cytosolic Ca(2+) is critical for pancreatic acinar cell function. Disruptions in normal Ca(2+) concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca(2+) homeostasis by promoting rapid Ca(2+) movement. Determining how expression of Ca(2+) modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas-specific isoform for Secretory Pathway Ca(2+) ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA-seq), chromatin immunoprecipitation for epigenetic modifications and promoter-reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last four exons of the Atp2c2 gene (Atp2c2c). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2's 24th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca(2+) . RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca(2+) levels. Combined, these results suggest SPCA2C affects Ca(2+) homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca(2+) ATPases. J. Cell. Physiol. 231: 2768-2778, 2016. © 2016 Wiley Periodicals, Inc.

  5. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

    ABSTRACT Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell–matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical–basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: ‘ERM proteins at a glance’ by Andrea McClatchey (J. Cell Sci. 127, 3199–3204). ‘Establishment of epithelial polarity – GEF who's minding the GAP?’ by Siu Ngok et al. (J. Cell Sci. 127, 3205–3215). PMID:24994933

  6. Long-term persistence and development of induced pancreatic beta cells generated by lineage conversion of acinar cells.

    PubMed

    Li, Weida; Cavelti-Weder, Claudia; Zhang, Yingying; Zhang, Yinying; Clement, Kendell; Donovan, Scott; Gonzalez, Gabriel; Zhu, Jiang; Stemann, Marianne; Xu, Ke; Hashimoto, Tatsu; Yamada, Takatsugu; Nakanishi, Mio; Zhang, Yuemei; Zeng, Samuel; Gifford, David; Meissner, Alexander; Weir, Gordon; Zhou, Qiao

    2014-12-01

    Direct lineage conversion is a promising approach to generate therapeutically important cell types for disease modeling and tissue repair. However, the survival and function of lineage-reprogrammed cells in vivo over the long term has not been examined. Here, using an improved method for in vivo conversion of adult mouse pancreatic acinar cells toward beta cells, we show that induced beta cells persist for up to 13 months (the length of the experiment), form pancreatic islet-like structures and support normoglycemia in diabetic mice. Detailed molecular analyses of induced beta cells over 7 months reveal that global DNA methylation changes occur within 10 d, whereas the transcriptional network evolves over 2 months to resemble that of endogenous beta cells and remains stable thereafter. Progressive gain of beta-cell function occurs over 7 months, as measured by glucose-regulated insulin release and suppression of hyperglycemia. These studies demonstrate that lineage-reprogrammed cells persist for >1 year and undergo epigenetic, transcriptional, anatomical and functional development toward a beta-cell phenotype.

  7. ptf1a+, ela3l− cells are developmentally maintained progenitors for exocrine regeneration following extreme loss of acinar cells in zebrafish larvae

    PubMed Central

    Schmitner, Nicole; Kohno, Kenji

    2017-01-01

    ABSTRACT The exocrine pancreas displays a significant capacity for regeneration and renewal. In humans and mammalian model systems, the partial loss of exocrine tissue, such as after acute pancreatitis or partial pancreatectomy induces rapid recovery via expansion of surviving acinar cells. In mouse it was further found that an almost complete removal of acinar cells initiates regeneration from a currently not well-defined progenitor pool. Here, we used the zebrafish as an alternative model to study cellular mechanisms of exocrine regeneration following an almost complete removal of acinar cells. We introduced and validated two novel transgenic approaches for genetically encoded conditional cell ablation in the zebrafish, either by caspase-8-induced apoptosis or by rendering cells sensitive to diphtheria toxin. By using the ela3l promoter for exocrine-specific expression, we show that both approaches allowed cell-type-specific removal of >95% of acinar tissue in larval and adult zebrafish without causing any signs of unspecific side effects. We find that zebrafish larvae are able to recover from a virtually complete acinar tissue ablation within 2 weeks. Using short-term lineage-tracing experiments and EdU incorporation assays, we exclude duct-associated Notch-responsive cells as the source of regeneration. Rather, a rare population of slowly dividing ela3l-negative cells expressing ptf1a and CPA was identified as the origin of the newly forming exocrine cells. Cells are actively maintained, as revealed by a constant number of these cells at different larval stages and after repeated cell ablation. These cells establish ela3l expression about 4-6 days after ablation without signs of increased proliferation in between. With onset of ela3l expression, cells initiate rapid proliferation, leading to fast expansion of the ela3l-positive population. Finally, we show that this proliferation is blocked by overexpression of the Wnt-signaling antagonist dkk1b. In

  8. Calcium and pancreatic secretion-dynamics of subcellur calcium pools in resting and stimulated acinar cells.

    PubMed Central

    Clemente, F; Meldolesi, J

    1975-01-01

    1 Pulse-chase experiments were carried out on pancreatic tissue lobules incubated in vitro, with 45Ca as the tracer, in order to shed some light on the functional significance of the calcium pools associated with the various cell organelles of the acinar cell, especially in relation to stimulus-secretion coupling. 2 The kinetics of tracer uptake and release which were observed in the intact lobules suggest the existence of a number of intracellular pools, whose rate of exchange is slower than that across teh plasmalemma. 3 The various subcellular fractions accumulate the tracer in different amounts: some (rough microsomes and postmicrosomal supernatant) showed little radioactivity and some (smooth microsomes and zymogen granule membranes) were heavily labelled; mitochondria and zymogen granules showed intermediate values. 4 The fractions are heterogeneous also in relation to the time course of uptake and release of the tracer: in rough and smooth microsomes and, especially, in the postmicrosomal supernatant both rates were fast; zymogen granules and zymogen granule membranes showed slow rates of uptake and little release during chase; intermediate rates were found in mitochondria. 5 In agreement with previous findings we observed that in 45Ca preloaded lobules, stimulation of secretion (brought about by the secretagogue polypeptide caerulein) results in an increase of the tracer release which seems to be due primarily to the rise of the intracellular concentration of free Ca2+ and to the consequent increase of the transmembrane Ca2+ efflux. Among the cell fractions isolated from stimulated lobules only the mitochondria exhibited a significantly lower 45Ca level relative to the unstimulated controls. 6 It is concluded that, of the organelle-bound calcium pools, that associated with the mitochondria might be involved in the regulation of the calcium-dependent functions, including stimulus-secretion coupling; the calcium associated with the zymogen granule content

  9. Rap1 integrates tissue polarity, lumen formation, and tumorigenicpotential in human breast epithelial cells

    SciTech Connect

    Itoh, Masahiko; Nelson, Celeste M.; Myers, Connie A.; Bissell,Mina J.

    2006-09-29

    Maintenance of apico-basal polarity in normal breast epithelial acini requires a balance between cell proliferation, cell death, and proper cell-cell and cell-extracellular matrix signaling. Aberrations in any of these processes can disrupt tissue architecture and initiate tumor formation. Here we show that the small GTPase Rap1 is a crucial element in organizing acinar structure and inducing lumen formation. Rap1 activity in malignant HMT-3522 T4-2 cells is appreciably higher than in S1 cells, their non-malignant counterparts. Expression of dominant-negative Rap1 resulted in phenotypic reversion of T4-2 cells, led to formation of acinar structures with correct apico-basal polarity, and dramatically reduced tumor incidence despite the persistence of genomic abnormalities. The resulting acini contained prominent central lumina not observed when other reverting agents were used. Conversely, expression of dominant-active Rap1 in T4-2 cells inhibited phenotypic reversion and led to increased invasiveness and tumorigenicity. Thus, Rap1 acts as a central regulator of breast architecture, with normal levels of activation instructing apical polarity during acinar morphogenesis, and increased activation inducing tumor formation and progression to malignancy.

  10. Inhibitory effects of sho-seiryu-to on acetylcholine-induced responses in nasal gland acinar cells.

    PubMed

    Ikeda, K; Wu, D Z; Ishigaki, M; Sunose, H; Takasaka, T

    1994-01-01

    Sho-seiryu-to, a traditional Japanese herbal medicine, has been used extensively in the treatment of allergic rhinitis. The effects of Sho-seiryu-to on electrical responses induced by acetylcholine in dissociated nasal gland acinar cells were investigated using patch-clamp and microfluorimetric imaging techniques. The application of Sho-seiryu-to inhibited both K+ and Cl- currents augmented by acetylcholine. The elevation of intracellular Ca2+ and Na+ concentrations induced by acetylcholine was also inhibited by Sho-seriyu-to. These findings suggest that Sho-seiryu-to attenuated the secretion of water and electrolytes from the nasal glands through an anti-cholinergic effect.

  11. DNA quantification as prognostic factor in a case of acinar cell carcinoma of the parotid gland, diagnosed by FNA.

    PubMed

    Azúa-Romeo, Javier; Sánchez-Garnica, Juan Carlos; Azúa-Blanco, Javier; Tovar-Lázaro, Mayte

    2005-01-01

    Hereby we present a case of a 43-years-old male who complained of a three years history preauricular painful mass. Fine needle aspiration cytology was performed, diagnosing of compatible with acinar cell carcinoma, thus DNA quantification by image cytometry was carried out. Biological parameters studied (ploidy, S-phase, 5-c exceeding rate) showed that it is a low grade of malignancy lesion. Total parotidectomy conservative of facial nerve was recommended, without regional lymphadenectomy. Patient remains, one year later, asymptomatic and free of disease.

  12. Low-level (gallium-aluminum-arsenide) laser irradiation of Par-C10 cells and acinar cells of rat parotid gland.

    PubMed

    Onizawa, Katsuhiro; Muramatsu, Takashi; Matsuki, Miwako; Ohta, Kazumasa; Matsuzaka, Kenichi; Oda, Yutaka; Shimono, Masaki

    2009-03-01

    We investigated cell response, including cell proliferation and expression of heat stress protein and bcl-2, to clarify the influence of low-level [gallium-aluminum-arsenide (Ga-Al-As) diode] laser irradiation on Par-C10 cells derived from the acinar cells of rat parotid glands. Furthermore, we also investigated amylase release and cell death from irradiation in acinar cells from rat parotid glands. The number of Par-C10 cells in the laser-irradiated groups was higher than that in the non-irradiated group at days 5 and 7, and the difference was statistically significant (P < 0.01). Greater expression of heat shock protein (HSP)25 and bcl-2 was seen on days 1 and 3 in the irradiated group. Assay of the released amylase showed no significant difference statistically between the irradiated group and the non-irradiated group. Trypan blue exclusion assay revealed that there was no difference in the ratio of dead to live cells between the irradiated and the non-irradiated groups. These results suggest that low-level laser irradiation promotes cell proliferation and expression of anti-apoptosis proteins in Par-C10 cells, but it does not significantly affect amylase secretion and does not induce rapid cell death in isolated acinar cells from rat parotid glands.

  13. Beneficial effect of the bioflavonoid quercetin on cholecystokinin-induced mitochondrial dysfunction in isolated rat pancreatic acinar cells.

    PubMed

    Weber, Heike; Jonas, Ludwig; Wakileh, Michael; Krüger, Burkhard

    2014-03-01

    The pathogenesis of acute pancreatitis (AP) is still poorly understood. Thus, a reliable pharmacological therapy is currently lacking. In recent years, an impairment of the energy metabolism of pancreatic acinar cells, caused by Ca(2+)-mediated depolarization of the inner mitochondrial membrane and a decreased ATP supply, has been implicated as an important pathological event. In this study, we investigated whether quercetin exerts protection against mitochondrial dysfunction. Following treatment with or without quercetin, rat pancreatic acinar cells were stimulated with supramaximal cholecystokinin-8 (CCK). CCK caused a decrease in the mitochondrial membrane potential (MMP) and ATP concentration, whereas the mitochondrial dehydrogenase activity was significantly increased. Quercetin treatment before CCK application exerted no protection on MMP but increased ATP to a normal level, leading to a continuous decrease in the dehydrogenase activity. The protective effect of quercetin on mitochondrial function was accompanied by a reduction in CCK-induced changes to the cell membrane. Concerning the molecular mechanism underlying the protective effect of quercetin, an increased AMP/ATP ratio suggests that the AMP-activated protein kinase system may be activated. In addition, quercetin strongly inhibited CCK-induced trypsin activity. The results indicate that the use of quercetin may be a therapeutic strategy for reducing the severity of AP.

  14. Incorporation of (/sup 35/S)sulfate in normal and neoplastic rat pancreatic acinar cells in relationship to cytodifferentiation

    SciTech Connect

    Kanwar, Y.S.; Rao, M.S.; Longnecker, D.S.; Reddy, J.K.

    1984-11-01

    The rates of (/sup 35/S)sulfate incorporation in highly differentiated acinar cells from normal pancreas, moderately differentiated cells of nafenopin-induced transplantable pancreatic carcinoma, and poorly differentiated cells from azaserine-induced transplantable pancreatic carcinoma were examined in an attempt to determine if sulfation is a property of acinar cells with well-developed secretory granules. The cells were dissociated, pulsed with (/sup 35/S)sulfate (specific activity, approximately 1000 Ci/mmol) for 10 and 60 min, and chased with medium containing 100 X excess of cold inorganic sulfate for 0, 15, 60, and 120 min. The cells were then processed for determining their pool size and light and electron microscopic autoradiography. No significant differences among their pool sizes were observed. However, the light microscopic autoradiograms revealed the (/sup 35/S)sulfate incorporation as follows: azaserine-induced transplantable pancreatic carcinoma greater than nafenopin-induced transplantable pancreatic carcinoma greater than normal pancreas. Electron microscopic autoradiograms revealed similar trends. The grain densities (concentration of radiation) were highest in the Golgi regions immediately postpulse (0 min) and gradually shifted toward the secretory granules over a 120-min period. In addition, the grain density values of the secretory granule-rich cells of nafenopin-induced transplantable pancreatic carcinoma were relatively similar to the cells of normal pancreas, whereas the grain density values of secretory granule-deficient cells from this tumor were similar to those of poorly differentiated neoplastic cells of azaserine-induced transplantable pancreatic carcinoma. These results show that poorly differentiated neoplastic cells incorporate more (/sup 35/S)sulfate than do the well-differentiated cells, but the reasons for this unexpected differential incorporation are at present unknown.

  15. Ion Channels in Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Palmer, Lawrence G.

    Ion channels in epithelial cells serve to move ions, and in some cases fluid, between compartments of the body. This function of the transfer of material is fundamentally different from that of the transfer of information, which is the main job of most channels in excitable cells. Nevertheless the basic construction of the channels is similar in many respects in the two tissue types. This chapter reviews the nature of channels in epithelia and discusses how their functions have evolved to accomplish the basic tasks for which they are responsible. I will focus on three channel types: epithelial Na+ channels, inward-rectifier K+ channels, and CFTR Cl- channels.

  16. Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell

    PubMed Central

    1995-01-01

    Receptor desensitization is a key process for the protection of the cell from continuous or repeated exposure to high concentrations of an agonist. Well-established mechanisms for desensitization of guanine nucleotide-binding protein (G protein)-coupled receptors include phosphorylation, sequestration/internalization, and down-regulation. In this work, we have examined some mechanisms for desensitization of the cholecystokinin (CCK) receptor which is native to the pancreatic acinar cell, and have found the predominant mechanism to be distinct from these recognized processes. Upon fluorescent agonist occupancy of the native receptor, it becomes "insulated" from the effects of acid washing and becomes immobilized on the surface of the plasma membrane in a time- and temperature-dependent manner. This localization was assessed by ultrastructural studies using a colloidal gold conjugate of CCK, and lateral mobility of the receptor was assessed using fluorescence recovery after photobleaching. Of note, recent application of the same morphologic techniques to a CCK receptor-bearing Chinese hamster ovary cell line demonstrated prominent internalization via the clathrin-dependent endocytic pathway, as well as entry into caveolae (Roettger, B.F., R.U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J.M. Larkin, and L.J. Miller, 1995, J. Cell Biol. 128: 1029-1041). These organelles are not observed to represent prominent compartments for the same receptor to traverse in the acinar cell, although fluorescent insulin is clearly internalized in these cells via receptor-mediated endocytosis. In this work, the rate of lateral mobility of the CCK receptor is observed to be similar in both cell types (1-3 x 10(-10) cm2/s), while the fate of the agonist-occupied receptor is quite distinct in each cell. This supports the unique nature of desensitization processes which occur in a cell-specific manner. A plasmalemmal site of insulation of this important receptor on the pancreatic acinar cell

  17. Lycopene protects pancreatic acinar cells against severe acute pancreatitis by abating the oxidative stress through JNK pathway.

    PubMed

    Lv, J C; Wang, G; Pan, S H; Bai, X W; Sun, B

    2015-02-01

    This study investigated the anti-oxidative and anti-inflammatory effects of lycopene on severe acute pancreatitis (SAP) in both in vivo and in vitro models. Utilizing a rat model, we found that lycopene administration protected against SAP, as indicated by the decreased levels of serum amylase and C-reactive protein. Pathological changes were alleviated by pretreatment with lycopene. The serum levels of tumor necrosis factor-α, interleukin-6, macrophage inflammatory protein-1α, and monocyte chemotactic protein-1 were decreased by lycopene. The decreased reactive oxygen species (ROS) content in the pancreatic tissues of the lycopene-treated group were indirectly evaluated by measuring the levels of myeloperoxidase, lipid peroxidase, and superoxide dismutase. Lycopene protected acinar cells against necrosis and apoptosis by relieving the mitochondrial and endoplasmic stress caused by ROS which was shown in electron microscopy and immunohistochemistry staining of active nuclear factor-κB p65. The protective effect was also observed in a simulated SAP model in a rat acinar cell line. ROS and apoptotic staining were compared between groups. Lycopene exerts protective effects against SAP in rats that may be related to its anti-inflammatory property through inhibiting the expression of damage-associated molecular patterns, and anti-oxidative property which can thus maintain cellular homeostasis and prevent the phosphorylation of JNK pathway.

  18. Postnatal Pancreas of Mice Contains Tripotent Progenitors Capable of Giving Rise to Duct, Acinar, and Endocrine Cells In Vitro.

    PubMed

    Ghazalli, Nadiah; Mahdavi, Alborz; Feng, Tao; Jin, Liang; Kozlowski, Mark T; Hsu, Jasper; Riggs, Arthur D; Tirrell, David A; Ku, H Teresa

    2015-09-01

    Postnatal pancreas is a potential source for progenitor cells to generate endocrine β-cells for treating type 1 diabetes. However, it remains unclear whether young (1-week-old) pancreas harbors multipotent progenitors capable of differentiating into duct, acinar, and endocrine cells. Laminin is an extracellular matrix (ECM) protein important for β-cells' survival and function. We established an artificial extracellular matrix (aECM) protein that contains the functional IKVAV (Ile-Lys-Val-Ala-Val) sequence derived from laminin (designated aECM-lam). Whether IKVAV is necessary for endocrine differentiation in vitro is unknown. To answer these questions, we cultured single cells from 1-week-old pancreas in semi-solid media supplemented with aECM-lam, aECM-scr (which contains a scrambled sequence instead of IKVAV), or Matrigel. We found that colonies were generated in all materials. Individual colonies were examined by microfluidic reverse transcription-polymerase chain reaction, immunostaining, and electron microscopy analyses. The majority of the colonies expressed markers for endocrine, acinar, and ductal lineages, demonstrating tri-lineage potential of individual colony-forming progenitors. Colonies grown in aECM-lam expressed higher levels of endocrine markers Insulin1, Insulin2, and Glucagon compared with those grown in aECM-scr and Matrigel, indicating that the IKVAV sequence enhances endocrine differentiation. In contrast, Matrigel was inhibitory for endocrine gene expression. Colonies grown in aECM-lam displayed the hallmarks of functional β-cells: mature insulin granules and glucose-stimulated insulin secretion. Colony-forming progenitors were enriched in the CD133(high) fraction and among 230 micro-manipulated single CD133(high) cells, four gave rise to colonies that expressed tri-lineage markers. We conclude that young postnatal pancreas contains multipotent progenitor cells and that aECM-lam promotes differentiation of β-like cells in vitro.

  19. Distinct contributions by ionotropic purinoceptor subtypes to ATP-evoked calcium signals in mouse parotid acinar cells

    PubMed Central

    Bhattacharya, Sumit; Verrill, Douglas S; Carbone, Kristopher M; Brown, Stefanie; Yule, David I; Giovannucci, David R

    2012-01-01

    There is emerging consensus that P2X4 and P2X7 ionotropic purinoceptors (P2X4R and P2X7R) are critical players in regulating [Ca2+]i dynamics and fluid secretion in the salivary gland. In contrast, details regarding their compartmentalization and selective activation, contributions to the spatiotemporal properties of intracellular signals and roles in regulating protein exocytosis and ion channel activity have remained largely undefined. To address these concerns, we profiled mouse parotid acinar cells using live-cell imaging to follow the spatial and temporal features of ATP-evoked Ca2+ dynamics and exocytotic activity. Selective activation of P2X7Rs revealed an apical-to-basal [Ca2+]i signal that initiated at the sub-luminal border and propagated with a wave speed estimated at 17.3 ± 4.3 μm s−1 (n = 6). The evoked Ca2+ spike consisted of Ca2+ influx and Ca2+-induced Ca2+ release from intracellular Ca2+ channels. In contrast, selective activation of P2X4Rs induced a Ca2+ signal that initiated basally and propagated toward the lumen with a wave speed of 4.3 ± 0.2 μm s−1 (n = 8) that was largely independent of intracellular Ca2+ channel blockade. Consistent with these observations, P2X7R expression was enriched in the sub-luminal regions of acinar cells while P2X4R appeared localized to basal areas. In addition, we showed that P2X4R and P2X7R activation evokes exocytosis in parotid acinar cells. Our studies also demonstrate that the P2X4R-mediated [Ca2+]i rise and subsequent protein exocytosis was enhanced by ivermectin (IVR). Thus, in addition to furthering our understanding of salivary gland physiology, this study identifies P2X4R as a potential target for treatment of salivary hypofunction diseases. PMID:22451435

  20. Agonist activation of arachidonate-regulated Ca2+-selective (ARC) channels in murine parotid and pancreatic acinar cells.

    PubMed

    Mignen, Olivier; Thompson, Jill L; Yule, David I; Shuttleworth, Trevor J

    2005-05-01

    ARC channels (arachidonate-regulated Ca(2+)-selective channels) are a novel type of highly Ca(2+)-selective channel that are specifically activated by low concentrations of agonist-induced arachidonic acid. This activation occurs in the absence of any depletion of internal Ca(2+) stores (i.e. they are 'non-capacitative'). Previous studies in HEK293 cells have shown that these channels provide the predominant pathway for the entry of Ca(2+) seen at low agonist concentrations where oscillatory [Ca(2+)](i) signals are typically produced. In contrast, activation of the more widely studied store-operated Ca(2+) channels (e.g. CRAC channels) is only seen at higher agonist concentrations where sustained 'plateau-type'[Ca(2+)](i) responses are observed. We have now demonstrated the presence of ARC channels in both parotid and pancreatic acinar cells and shown that, again, they are specifically activated by the low concentrations of appropriate agonists (carbachol in the parotid, and both carbachol and cholecystokinin in the pancreas) that are associated with oscillatory [Ca(2+)](i) signals in these cells. Uncoupling the receptor-mediated activation of cytosolic phospholipase A(2) (cPLA(2)) with isotetrandrine reduces the activation of the ARC channels by carbachol and, correspondingly, markedly inhibits the [Ca(2+)](i) signals induced by low carbachol concentrations, whilst those signals seen at high agonist concentrations are essentially unaffected. Interestingly, in the pancreatic acinar cells, activation by cholecystokinin induces a current through the ARC channels that is only approximately 60% of that seen with carbachol. This is consistent with previous reports indicating that carbachol-induced [Ca(2+)](i) signals in these cells are much more dependent on Ca(2+) entry than are the cholecystokinin-induced responses.

  1. An introduction to acinar pressures in BPH and prostate cancer.

    PubMed

    Wadhera, Panikar

    2013-06-01

    Intra-acinar and peri-acinar pressures in the prostate might be key factors in the evolution of its zonal morphology and the pathogenesis of BPH and cancer. Herein, I hypothesize that intra-acinar pressures lead to a decrease in apoptosis by distending or stretching acinar epithelium and its surrounding stroma. Increased prostatic smooth muscle content and tone might generate peri-acinar pressures, which could, in the long-term, counteract intra-acinar pressures and decrease epithelial stretch. Thus, it is proposed that BPH (characterized by increased prostatic smooth muscle and, therefore, raised peri-acinar pressures) might decrease the risk of prostate cancer progression by counteracting intra-acinar pressures. In the context of this theory, the transition zone might have evolved as a specialized region within the prostate that can mount a concerted stromal-epithelial response to increased urethral and intra-acinar pressures (BPH), and the urethral angulation, anterior stroma and the prostatic capsule have an adjunctive evolutionary role in this phenomenon.

  2. The effects of sigma ligands on protein release from lacrimal acinar cells: a potential agonist/antagonist assay.

    PubMed

    Schoenwald, R D; Barfknecht, C F; Shirolkar, S; Xia, E

    1995-03-03

    Sigma receptor antagonists have been proposed as leading clinical candidates for use in various psychotic disorders. Prior to clinical testing, it is imperative that a new agent be correctly identified as an antagonist and not an agonist since the latter may worsen the psychosis. For sigma-ligands many behavioral and pharmacological assays have been developed in an attempt to classify agonist/antagonist activity. These assays evaluate a response or a behavior in an animal model that can be related to clinical efficacy. However, is the action by the presumed antagonist a consequence of sigma-receptor activity? Previously we have identified sigma-receptors in acinar cells of the main lacrimal gland of the New Zealand white rabbit and have measured protein release after the addition of various N,N-disubstituted phenylalkylamine derivatives known to be sigma-ligands by receptor binding studies. Although protein release from acinar cells has been attributed to either muscarinic or alpha-adrenergic stimulation, protein release from sigma-receptor stimulation was also confirmed. In the reported studies here, we isolated and incubated acinar cells with varying concentrations of known sigma-ligands and measured protein concentration. A knowledge of the receptor profile for the disubstituted phenylalkylamines permitted experiments to be designed in which various alpha, muscarinic, serotonergic, and dopaminergic antagonists could be added in equimolar concentrations. Under the conditions of these experiments, statistically significant increases in protein release for sigma-ligands could be attributed to stimulation of sigma-receptors. Haloperidol, an apparent sigma-antagonist, caused a statistically significant decrease in protein release and also inhibited protein release when tested with a known sigma-ligand, AF2975 [N,N-dimethyl-2-phenylethylamine]. In this system, stimulation and inhibition of protein release were defined as agonist and antagonist behavior, respectively

  3. Collagen type IV stimulates an increase in intracellular Ca2+ in pancreatic acinar cells via activation of phospholipase C.

    PubMed Central

    Somogyi, L; Lasić, Z; Vukicević, S; Banfić, H

    1994-01-01

    Intracellular Ca2+ responses to extracellular matrix molecules were studied in suspensions of pancreatic acinar cells loaded with Fura-2. Collagen type I, laminin, fibrinogen and fibronectin were unable to raise cytosolic free Ca2+ concentration ([Ca2+]i), whereas collagen type IV, at concentrations from 5 to 50 micrograms/ml, significantly increased it. The effect of collagen type IV was not due to possible contamination with type-I transforming growth factor beta or plasminogen, as neither of these agents was able to increase [Ca2+]i. Using highly specific mass assays, concentrations of inositol lipids, 1,2-diacylglycerol (DAG) and Ins(1,4,5) P3 were measured in pancreatic acinar cells stimulated with collagen type IV. A decrease in the concentrations of PtdIns(4,5) P2 and PtdIns4 P with a concomitant increase in the concentrations of DAG and InsP3 mass were observed, showing that collagen type IV increases [Ca2+]i by activation of phospholipase C. The observed [Ca2+]i signals had two components, the first resulting from Ca2+ release from the intracellular stores, and the second resulting from Ca2+ flux from the extracellular medium through the verapamil-insensitive channels. A tyrosine kinase inhibitor (tyrphostine) was able to block inositol lipid signalling caused by collagen type IV, which together with the insensitivity of this pathway to cholera toxin and pertussis toxin or to preactivation of protein kinase C, the longer duration of the increase in [Ca2+]i and a longer lag period needed for observation of increases in DAG and InsP3 concentration with collagen type IV than with carbachol (50 mM) suggest that activation of phospholipase C by collagen type IV is caused by tyrosine kinase activation. Inositol lipid signalling and increases in [Ca2+]i were also observed with Arg-Gly-Asp (RGD)-containing peptide but not with Arg-Asp-Gly (RDG)-containing peptide. Collagen type IV and RGD-containing peptide, but not carbachol, competed in increasing [Ca2+]i and

  4. Progress Towards Drosophila Epithelial Cell Culture

    PubMed Central

    Simcox, Amanda

    2015-01-01

    Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens. PMID:23097097

  5. Role of protein kinase C in caerulein induced expression of substance P and neurokinin-1-receptors in murine pancreatic acinar cells

    PubMed Central

    Koh, Yung-Hua; Tamizhselvi, Ramasamy; Moochhala, Shabbir; Bian, Jin-Song; Bhatia, Madhav

    2011-01-01

    Substance P (SP) is involved in the pathophysiology of acute pancreatitis (AP) via binding to its high-affinity receptor, neurokinin-1-receptor (NK1R). An up-regulation of SP and NK1R expression was observed in experimental AP and in caerulein-stimulated pancreatic acinar cells. However, the mechanisms that lead to this up-regulation are not fully understood. In this study, we showed the role of protein kinase C (PKC) in caerulein-induced SP and NK1R production in isolated mouse pancreatic acinar cells. Caerulein (10−7 M) stimulation rapidly activated the conventional PKC-α and novel PKC-δ as observed by the phosphorylation of these molecules. Pre-treatment of pancreatic acinar cells with Gö6976 (1–10 nM) and rottlerin (1–10 μM) inhibited PKC-α and PKC-δ phosphorylation, respectively, but not the other way round. At these concentrations used, PKC-α and PKC-δ inhibition reversed the caerulein-induced up-regulation of SP and NK1R, indicating an important role of PKCs in the modulation of SP and NK1R expression. Further experiments looking into signalling mechanisms showed that treatment of pancreatic acinar cells with both Gö6976 and rottlerin inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Inhibition of PKC-α or PKC-δ also affected caerulein-induced transcription factor activation, as represented by nuclear factor-κB and AP-1 DNA-binding activity. The findings in this study suggested that PKC is upstream of the mitogen-activated protein kinases and transcription factors, which then lead to the up-regulation of SP/NK1R expression in caerulein-treated mouse pancreatic acinar cells. PMID:20973912

  6. Role of protein kinase C in caerulein induced expression of substance P and neurokinin-1-receptors in murine pancreatic acinar cells.

    PubMed

    Koh, Yung-Hua; Tamizhselvi, Ramasamy; Moochhala, Shabbir; Bian, Jin-Song; Bhatia, Madhav

    2011-10-01

    Substance P (SP) is involved in the pathophysiology of acute pancreatitis (AP) via binding to its high-affinity receptor, neurokinin-1-receptor (NK1R). An up-regulation of SP and NK1R expression was observed in experimental AP and in caerulein-stimulated pancreatic acinar cells. However, the mechanisms that lead to this up-regulation are not fully understood. In this study, we showed the role of protein kinase C (PKC) in caerulein-induced SP and NK1R production in isolated mouse pancreatic acinar cells. Caerulein (10(-7) M) stimulation rapidly activated the conventional PKC-α and novel PKC-δ as observed by the phosphorylation of these molecules. Pre-treatment of pancreatic acinar cells with Gö6976 (1-10 nM) and rottlerin (1-10 μM) inhibited PKC-α and PKC-δ phosphorylation, respectively, but not the other way round. At these concentrations used, PKC-α and PKC-δ inhibition reversed the caerulein-induced up-regulation of SP and NK1R, indicating an important role of PKCs in the modulation of SP and NK1R expression. Further experiments looking into signalling mechanisms showed that treatment of pancreatic acinar cells with both Gö6976 and rottlerin inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Inhibition of PKC-α or PKC-δ also affected caerulein-induced transcription factor activation, as represented by nuclear factor-κB and AP-1 DNA-binding activity. The findings in this study suggested that PKC is upstream of the mitogen-activated protein kinases and transcription factors, which then lead to the up-regulation of SP/NK1R expression in caerulein-treated mouse pancreatic acinar cells.

  7. Eosinophils promote epithelial to mesenchymal transition of bronchial epithelial cells.

    PubMed

    Yasukawa, Atsushi; Hosoki, Koa; Toda, Masaaki; Miyake, Yasushi; Matsushima, Yuki; Matsumoto, Takahiro; Boveda-Ruiz, Daniel; Gil-Bernabe, Paloma; Nagao, Mizuho; Sugimoto, Mayumi; Hiraguchi, Yukiko; Tokuda, Reiko; Naito, Masahiro; Takagi, Takehiro; D'Alessandro-Gabazza, Corina N; Suga, Shigeru; Kobayashi, Tetsu; Fujisawa, Takao; Taguchi, Osamu; Gabazza, Esteban C

    2013-01-01

    Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.

  8. Effects of Baicalin on inflammatory mediators and pancreatic acinar cell apoptosis in rats with sever acute pancreatitis

    PubMed Central

    Xiping, Zhang; Hua, Tian; Hanqing, Chen; Li, Chen; Binyan, Yu; Jing, Ma

    2009-01-01

    BACKGROUND: To investigate the effects of Baicalin and Octreotide on inflammatory mediators and pancreatic acinar cells apoptosis of rats with severe acute pancreatitis (SAP). METHODS: SD rats were randomly divided into sham operated group (I group), model control group (II group), Baicalin treated group (III group) and Octreotide treated group (IV group). Each group was also divided into subgroup of 3, 6 and 12 h (n = 15). The mortality rate, ascites/body weight ratio as well as the level of endotoxin, NO and ET-1 in blood were measured. The pathological severity score of pancreas, apoptotic indexes, and expression levels of Bax and Bcl-2 proteins in each group were investigated. RESULTS: The survival rate of III and IV group has a significant difference compared with II group (P12 h < 0.05). The ascites volume, contents of inflammatory mediators in blood and pathological severity score of pancreas of III and IV group declined at different degrees compared to II group (P < 0.05, P < 0.01 or P < 0.001). Apoptotic index in III group was significantly higher than that in II group at 3 and 6 h (P3, 6 h < 0.05). Apoptotic index in IV group was significantly higher than that in II group at pancreatic tail at 6 h (P6 h < 0.05). Expression level of Bax in III group was significantly higher than that in II group (pancreatic head P3 h,6 h < 0.01, pancreatic tail P3 h < 0.001). CONCLUSIONS: Compared with Octreotide in the treatment of SAP, the protective mechanisms of Baicalin include reducing the excessive inflammatory mediators’ release, inducing the pancreatic acinar cells apoptosis. PMID:21772857

  9. 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.

  10. Pancreatic (acinar) metaplasia of the gastric mucosa. Histology, ultrastructure, immunocytochemistry, and clinicopathologic correlations of 101 cases.

    PubMed

    Doglioni, C; Laurino, L; Dei Tos, A P; De Boni, M; Franzin, G; Braidotti, P; Viale, G

    1993-11-01

    The occasional finding within the gastric mucosa of unidentified epithelial cells with morphological features closely resembling those of pancreatic acinar cells has prompted us to investigate a retrospective series of 8,430 consecutive gastric biopsies and of 126 surgical specimens of gastric resection and total gastrectomy. The aims of the study were to morphologically and immunocytochemically characterize these cells, to define their actual prevalence in a large series of unselected cases, and to assess the clinicopathologic correlates of their occurrence. Pancreatic acinar-like cells characterized by abundant cytoplasm, which was acidophilic and finely granular in the apical and middle portions and basophilic in the basal compartment, have been identified in 101 cases (84 gastric biopsies and 17 gastrectomies). These cells, arranged in nests or in variably sized lobules among the gastric glands, were morphologically indistinguishable from pancreatic acinar cells, both by light and by electron microscopy. Furthermore, they were consistently immunoreactive for pancreatic lipase and trypsinogen and, in 75% of the cases, for pancreatic alpha-amylase. The appearance of these cells within the gastric mucosa was correlated significantly with chronic gastritis (p = 0.032) and with the simultaneous occurrence of intestinal and pyloric types of gastric metaplasia (p = 0.021). The findings indicate that this is a previously unrecognized pancreatic (acinar) metaplasia of the gastric mucosa, clinically and morphologically distinct from pancreatic heterotopia.

  11. Ionizing radiation induces heritable disruption of epithelial cell interactions

    PubMed Central

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

    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, β-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. PMID:12960393

  12. Metastatic pancreatic acinar cell carcinoma in a younger male with marked AFP production: A potential pitfall on fine needle aspiration biopsy.

    PubMed

    Valente, Kari; Yacoub, George; Cappellari, James O; Parks, Graham

    2017-02-01

    A 30-year-old male presented to his doctor with complaints of abdominal pain and was found to have retroperitoneal as well as multiple hepatic masses. A serum alpha-fetoprotein (AFP) level was significantly elevated (17,373 ng mL(-1) ), raising suspicions for a metastatic germ cell tumor. Fine needle aspiration biopsy of the pancreatic lesion revealed atypical epithelioid cells with round nuclei, large prominent nucleoli, and granular cytoplasm. The morphologic differential diagnosis included pancreatic neoplasm, metastatic germ cell tumor, other metastatic carcinoma, and melanoma. An extensive panel of immunohistochemical stains confirmed the diagnosis of acinar cell carcinoma. The diagnosis of acinar cell carcinoma could be confounded by the markedly increased AFP level, particularly in the setting of a retroperitoneal mass in a younger male. The increased AFP level in the setting of an acinar cell tumor is a potential pitfall to correct diagnosis by cytology. As the treatment for these two entities differs considerably, acute awareness of the phenomenon is important. We present a case of pancreatic ACC with an increased AFP level diagnosed on a cytology specimen. Diagn. Cytopathol. 2017;45:133-136. © 2016 Wiley Periodicals, Inc.

  13. Endoscopic ultrasound in the diagnosis of acinar cell carcinoma of the pancreas: contrast-enhanced endoscopic ultrasound, endoscopic ultrasound elastography, and pathological correlation.

    PubMed

    Chantarojanasiri, Tanyaporn; Hirooka, Yoshiki; Kawashima, Hiroki; Ohno, Eizaburo; Yamamura, Takeshi; Funasaka, Kohei; Nakamura, Masanao; Miyahara, Ryoji; Ishigami, Masatoshi; Watanabe, Osamu; Nakaguro, Masato; Shimoyama, Yoshie; Nakamura, Shigeo; Goto, Hidemi

    2016-11-01

    We report a case series of five patients with pancreatic acinar cell carcinoma who received surgical treatment and compared the preoperative contrast-enhanced endoscopic ultrasound (EUS) and EUS elastography patterns with the surgical specimens. The contrast-enhanced EUS indicated vascular tumors with gradual enhancement in four patients and a hypovascular tumor in one patient. The elastography indicated an elastic score of 3 (hard lesion with softer border) in two patients and a score of 5 (hard lesion, which included the surrounding area) in two patients. In tumors with an elastic score of 5, the pathology exhibited abundant hyalinizing fibrous stroma or massive tumor invasion to the surrounding tissue. We concluded that acinar cell carcinoma of the pancreas has various patterns of EUS contrast-enhancement and elastography, depending on the pathologic phenotype.

  14. Endoscopic ultrasound in the diagnosis of acinar cell carcinoma of the pancreas: contrast-enhanced endoscopic ultrasound, endoscopic ultrasound elastography, and pathological correlation

    PubMed Central

    Chantarojanasiri, Tanyaporn; Hirooka, Yoshiki; Kawashima, Hiroki; Ohno, Eizaburo; Yamamura, Takeshi; Funasaka, Kohei; Nakamura, Masanao; Miyahara, Ryoji; Ishigami, Masatoshi; Watanabe, Osamu; Nakaguro, Masato; Shimoyama, Yoshie; Nakamura, Shigeo; Goto, Hidemi

    2016-01-01

    We report a case series of five patients with pancreatic acinar cell carcinoma who received surgical treatment and compared the preoperative contrast-enhanced endoscopic ultrasound (EUS) and EUS elastography patterns with the surgical specimens. The contrast-enhanced EUS indicated vascular tumors with gradual enhancement in four patients and a hypovascular tumor in one patient. The elastography indicated an elastic score of 3 (hard lesion with softer border) in two patients and a score of 5 (hard lesion, which included the surrounding area) in two patients. In tumors with an elastic score of 5, the pathology exhibited abundant hyalinizing fibrous stroma or massive tumor invasion to the surrounding tissue. We concluded that acinar cell carcinoma of the pancreas has various patterns of EUS contrast-enhancement and elastography, depending on the pathologic phenotype. PMID:27853750

  15. Successful Salvage Chemotherapy with FOLFIRINOX for Recurrent Mixed Acinar Cell Carcinoma and Ductal Adenocarcinoma of the Pancreas in an Adolescent Patient.

    PubMed

    Pfrommer, Sarah; Weber, Achim; Dutkowski, Philipp; Schäfer, Niklaus G; Müllhaupt, Beat; Bourquin, Jean-Pierre; Breitenstein, Stefan; Pestalozzi, Bernhard C; Stenner, Frank; Renner, Christoph; D'Addario, Giannicola; Graf, Hans-Jörg; Knuth, Alexander; Clavien, Pierre-Alain; Samaras, Panagiotis

    2013-01-01

    Pancreatic tumors are rare in children and adolescents. Here, we report the case of a 15-year-old boy who presented with a mixed acinar cell carcinoma/ductal adenocarcinoma with blastomatous components. He received multimodal treatment including various chemotherapy regimens and multistep surgery including liver transplantation. Introduction of FOLFIRINOX after relapse repeatedly achieved a durable metabolic and clinical response with good quality of life.

  16. Successful Salvage Chemotherapy with FOLFIRINOX for Recurrent Mixed Acinar Cell Carcinoma and Ductal Adenocarcinoma of the Pancreas in an Adolescent Patient

    PubMed Central

    Pfrommer, Sarah; Weber, Achim; Dutkowski, Philipp; Schäfer, Niklaus G.; Müllhaupt, Beat; Bourquin, Jean-Pierre; Breitenstein, Stefan; Pestalozzi, Bernhard C.; Stenner, Frank; Renner, Christoph; D'Addario, Giannicola; Graf, Hans-Jörg; Knuth, Alexander; Clavien, Pierre-Alain; Samaras, Panagiotis

    2013-01-01

    Pancreatic tumors are rare in children and adolescents. Here, we report the case of a 15-year-old boy who presented with a mixed acinar cell carcinoma/ductal adenocarcinoma with blastomatous components. He received multimodal treatment including various chemotherapy regimens and multistep surgery including liver transplantation. Introduction of FOLFIRINOX after relapse repeatedly achieved a durable metabolic and clinical response with good quality of life. PMID:24163668

  17. E-cadherin-negative acinar cell carcinoma of the pancreas: report of a case showing a solid pseudopapillary growth pattern.

    PubMed

    Tajima, Shogo; Waki, Michihiko; Azuma, Masaki; Koda, Kenji; Ohata, Akihiko

    2016-09-01

    E-cadherin expression patterns in acinar cell carcinomas (ACCs) of the pancreas have not been well documented. Herein, we present a hitherto undescribed case of E-cadherin-negative ACC with a solid pseudopapillary growth pattern in a 65-year-old man. We used an antibody against the extracellular domain of E-cadherin. As a further unusual status in ACC, faint β-catenin expression was observed in the cytoplasm of carcinoma cells. Morphological distinction from a solid pseudopapillary neoplasm (SPN) of the pancreas might be problematic in such a case, because of their similarities concerned with the growth pattern and E-cadherin negativity. Without nuclear accumulation of β-catenin, a diagnosis of SPN was almost excluded. Immunoreactivity for trypsin and BCL10 made an accurate diagnosis of ACC to this case. The tumor recurred 10 months post-surgery as rapidly enlarging masses in the liver, presumably indicating the aggressiveness of the E-cadherin-negative phenotype among ACCs.

  18. Pancreatic Fat Accumulation, Fibrosis, and Acinar Cell Injury in the Zucker Diabetic Fatty Rat Fed a Chronic High-Fat Diet

    PubMed Central

    Matsuda, Akiko; Makino, Naohiko; Tozawa, Tomohiro; Shirahata, Nakao; Honda, Teiichiro; Ikeda, Yushi; Sato, Hideyuki; Ito, Miho; Kakizaki, Yasuharu; Akamatsu, Manabu; Ueno, Yoshiyuki; Kawata, Sumio

    2014-01-01

    Objective The histological alteration of the exocrine pancreas in obesity has not been clarified. In the present study, we investigated biochemical and histological changes in the exocrine pancreas of obese model rats. Methods Zucker lean rats were fed a standard diet, and Zucker diabetic fatty (ZDF) rats were divided into 2 groups fed a standard diet and a high-fat diet, respectively. These experimental groups were fed each of the diets from 6 weeks until 12, 18, 24 weeks of age. We performed blood biochemical assays and histological analysis of the pancreas. Results In the ZDF rats fed a high-fat diet, the ratio of accumulated pancreatic fat area relative to exocrine gland area was increased significantly at 18 weeks of age in comparison with the other 2 groups (P < 0.05), and lipid droplets were observed in acinar cells. Subsequently, at 24 weeks of age in this group, pancreatic fibrosis and the serum exocrine pancreatic enzyme levels were increased significantly relative to the other 2 groups (P < 0.01). Conclusions In ZDF rats fed a chronic high-fat diet, fat accumulates in pancreatic acinar cells, and this fatty change seems to be related to subsequent pancreatic fibrosis and acinar cell injury. PMID:24717823

  19. RAS inhibitors decrease apoptosis of acinar cells and increase elimination of pancreatic stellate cells after in the course of experimental chronic pancreatitis induced by dibutyltin dichloride.

    PubMed

    Madro, A; Korolczuk, A; Czechowska, G; Celiński, K; Słomka, M; Prozorow-Król, B; Korobowicz, E

    2008-08-01

    Chronic pancreatitis (CP) is a progressive disease, in which the exocrine function of the gland is gradually lost and fibrosis develops due to repeated episodes of acute pancreatitis. The aim of the study was to investigate the effects of RAS inhibitors on the apoptosis of acinar cells and pancreatic stellate cells (PSCs) elimination in experimental CP induced by dibutyltin dichloride (DBTC). CP was induced by administration of DBTC to the femoral vein. Simultaneously captopril, losartan, enalapril and lisinopril were administered intraperitoneally. The rats were decapitated after 60 days and tissue of pancreas was collected. In rats treated by DBTC the features of inflammatory infiltration, ductal lumen dilatation, fibrosis were found. Strong reactivity with caspase2(L) and clusterin-beta antibodies was observed in areas of fibrosis. In animals treated with RAS inhibitors inflammatory changes and fibrosis were less severe. In groups of rats treated with DBTC and RAS inhibitors immunoreactivity of caspase(2L) and clusterin-beta was weak. Positive immunostaining against smooth muscle actine and desmin was observed in the elongated cells (PSC-s). This reaction was weak in groups of rat treated with DBTC and RAS inhibitors. Treatment of CP rats with RAS inhibitors alleviate apoptosis of pancreatic acinar cells and induces PSCs elimination.

  20. TP53 alterations in pancreatic acinar cell carcinoma: new insights into the molecular pathology of this rare cancer.

    PubMed

    La Rosa, Stefano; Bernasconi, Barbara; Frattini, Milo; Tibiletti, Maria Grazia; Molinari, Francesca; Furlan, Daniela; Sahnane, Nora; Vanoli, Alessandro; Albarello, Luca; Zhang, Lizhi; Notohara, Kenji; Casnedi, Selenia; Chenard, Marie-Pierre; Adsay, Volkan; Asioli, Sofia; Capella, Carlo; Sessa, Fausto

    2016-03-01

    The molecular alterations of pancreatic acinar cell carcinomas (ACCs) are poorly understood and have been reported as being different from those in ductal adenocarcinomas. Loss of TP53 gene function in the pathogenesis of ACCs is controversial since contradictory findings have been published. A comprehensive analysis of the different possible genetic and epigenetic mechanisms leading to TP53 alteration in ACC has never been reported and hence the role of TP53 in the pathogenesis and/or progression of ACC remains unclear. We investigated TP53 alterations in 54 tumor samples from 44 patients, including primary and metastatic ACC, using sequencing analysis, methylation-specific multiplex ligation probe amplification, fluorescence in situ hybridization, and immunohistochemistry. TP53 mutations were found in 13 % of primary ACCs and in 31 % of metastases. Primary ACCs and metastases showed the same mutational profile, with the exception of one case, characterized by a wild-type sequence in the primary carcinoma and a mutation in the corresponding metastasis. FISH analysis revealed deletion of the TP53 region in 53 % of primary ACCs and in 50 % of metastases. Promoter hypermethylation was found in one case. The molecular alterations correlated well with the immunohistochemical findings. A statistically significant association was found between the combination of mutation of one allele and loss of the other allele of TP53 and worse survival.

  1. Increased ROS production in non-polarized mammary epithelial cells induces monocyte infiltration in 3D culture.

    PubMed

    Li, Linzhang; Chen, Jie; Xiong, Gaofeng; St Clair, Daret K; Xu, Wei; Xu, Ren

    2017-01-01

    Loss of epithelial cell polarity promotes cell invasion and cancer dissemination. Therefore, identification of factors that disrupt polarized acinar formation is crucial. Reactive oxygen species (ROS) drive cancer progression and promote inflammation. Here, we show that the non-polarized breast cancer cell line T4-2 generates significantly higher ROS levels than polarized S1 and T4R cells in three-dimensional (3D) culture, accompanied by induction of the nuclear factor κB (NF-κB) pathway and cytokine expression. Minimizing ROS in T4-2 cells with antioxidants reestablished basal polarity and inhibited cell proliferation. Introducing constitutively activated RAC1 disrupted cell polarity and increased ROS levels, indicating that RAC1 is a crucial regulator that links cell polarity and ROS generation. We also linked monocyte infiltration with disruption of polarized acinar structure using a 3D co-culture system. Gain- and loss-of-function experiments demonstrated that increased ROS in non-polarized cells is necessary and sufficient to enhance monocyte recruitment. ROS also induced cytokine expression and NF-κB activity. These results suggest that increased ROS production in mammary epithelial cell leads to disruption of cell polarity and promotes monocyte infiltration.

  2. Patterning Bacterial Communities on Epithelial Cells

    PubMed Central

    Dwidar, Mohammed; Leung, Brendan M.; Yaguchi, Toshiyuki; Takayama, Shuichi; Mitchell, Robert J.

    2013-01-01

    Micropatterning of bacteria using aqueous two phase system (ATPS) enables the localized culture and formation of physically separated bacterial communities on human epithelial cell sheets. This method was used to compare the effects of Escherichia coli strain MG1655 and an isogenic invasive counterpart that expresses the invasin (inv) gene from Yersinia pseudotuberculosis on the underlying epithelial cell layer. Large portions of the cell layer beneath the invasive strain were killed or detached while the non-invasive E. coli had no apparent effect on the epithelial cell layer over a 24 h observation period. In addition, simultaneous testing of the localized effects of three different bacterial species; E. coli MG1655, Shigella boydii KACC 10792 and Pseudomonas sp DSM 50906 on an epithelial cell layer is also demonstrated. The paper further shows the ability to use a bacterial predator, Bdellovibriobacteriovorus HD 100, to selectively remove the E. coli, S. boydii and P. sp communities from this bacteria-patterned epithelial cell layer. Importantly, predation and removal of the P. Sp was critical for maintaining viability of the underlying epithelial cells. Although this paper focuses on a few specific cell types, the technique should be broadly applicable to understand a variety of bacteria-epithelial cell interactions. PMID:23785519

  3. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

    SciTech Connect

    Lee, Jingu; Park, Sangkyu; Roh, Sangho

    2015-05-15

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy.

  4. Ionizing irradiation induces apoptotic damage of salivary gland acinar cells via NADPH oxidase 1-dependent superoxide generation

    SciTech Connect

    Tateishi, Yoshihisa Sasabe, Eri; Ueta, Eisaku; Yamamoto, Tetsuya

    2008-02-08

    Reactive oxygen species (ROS) have important roles in various physiological processes. Recently, several novel homologues of the phagocytic NADPH oxidase have been discovered and this protein family is now designated as the Nox family. We investigated the involvement of Nox family proteins in ionizing irradiation-induced ROS generation and impairment in immortalized salivary gland acinar cells (NS-SV-AC), which are radiosensitive, and immortalized ductal cells (NS-SV-DC), which are radioresistant. Nox1-mRNA was upregulated by {gamma}-ray irradiation in NS-SV-AC, and the ROS level in NS-SV-AC was increased to approximately threefold of the control level after 10 Gy irradiation. The increase of ROS level in NS-SV-AC was suppressed by Nox1-siRNA-transfection. In parallel with the suppression of ROS generation and Nox1-mRNA expression by Nox1-siRNA, ionizing irradiation-induced apoptosis was strongly decreased in Nox1-siRNA-transfected NS-SV-AC. There were no large differences in total SOD or catalase activities between NS-SV-AC and NS-SV-DC although the post-irradiation ROS level in NS-SV-AC was higher than that in NS-SV-DC. In conclusion, these results indicate that Nox1 plays a crucial role in irradiation-induced ROS generation and ROS-associated impairment of salivary gland cells and that Nox1 gene may be targeted for preservation of the salivary gland function from radiation-induced impairment.

  5. Bone marrow-derived lung epithelial cells.

    PubMed

    Krause, Diane S

    2008-08-15

    Bone marrow-derived cells can take on the phenotype of epithelial cells and express epithelial-specific genes in multiple organs. Here, we focus on recent data on the appearance of marrow-derived epithelial cells in the adult lung. These findings have garnered significant skepticism because in most cases marrow-derived epithelial cells are very rare, the marrow cell of origin is not known, the techniques for detection have needed improvement, and there seem to be multiple mechanisms by which this occurs. Recent studies have focused on these concerns. Once these important concerns are addressed, further studies on the function(s) of these cells will need to be performed to determine whether this engraftment has any clinical significance-either beneficial or detrimental.

  6. Ionizing radiation predisposes nonmalignant human mammary epithelial cells to undergo transforming growth factor beta induced epithelial to mesenchymal transition.

    PubMed

    Andarawewa, Kumari L; Erickson, Anna C; Chou, William S; Costes, Sylvain V; Gascard, Philippe; Mott, Joni D; Bissell, Mina J; Barcellos-Hoff, Mary Helen

    2007-09-15

    Transforming growth factor beta1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 S1, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFbeta (0.4 ng/mL) or double treated. All double-treated (IR + TGFbeta) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, beta-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT, although IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogen-activated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFbeta, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

  7. PKCθ activation in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors is needed for stimulation of numerous important cellular signaling cascades.

    PubMed

    Sancho, Veronica; Berna, Marc J; Thill, Michelle; Jensen, R T

    2011-12-01

    The novel PKCθ isoform is highly expressed in T-cells, brain and skeletal muscle and originally thought to have a restricted distribution. It has been extensively studied in T-cells and shown to be important for apoptosis, T-cell activation and proliferation. Recent studies showed its presence in other tissues and importance in insulin signaling, lung surfactant secretion, intestinal barrier permeability, platelet and mast-cell functions. However, little information is available for PKCθ activation by gastrointestinal (GI) hormones/neurotransmitters and growth factors. In the present study we used rat pancreatic acinar cells to explore their ability to activate PKCθ and the possible interactions with important cellular mediators of their actions. Particular attention was paid to cholecystokinin (CCK), a physiological regulator of pancreatic function and important in pathological processes affecting acinar function, like pancreatitis. PKCθ-protein/mRNA was present in the pancreatic acini, and T538-PKCθ phosphorylation/activation was stimulated only by hormones/neurotransmitters activating phospholipase C. PKCθ was activated in time- and dose-related manner by CCK, mediated 30% by high-affinity CCK(A)-receptor activation. CCK stimulated PKCθ translocation from cytosol to membrane. PKCθ inhibition (by pseudostrate-inhibitor or dominant negative) inhibited CCK- and TPA-stimulation of PKD, Src, RafC, PYK2, p125(FAK) and IKKα/β, but not basal/stimulated enzyme secretion. Also CCK- and TPA-induced PKCθ activation produced an increment in PKCθ's direct association with AKT, RafA, RafC and Lyn. These results show for the first time the PKCθ presence in pancreatic acinar cells, its activation by some GI hormones/neurotransmitters and involvement in important cell signaling pathways mediating physiological responses (enzyme secretion, proliferation, apoptosis, cytokine expression, and pathological responses like pancreatitis and cancer growth).

  8. PKCθ activation in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors is needed for stimulation of numerous important cellular signaling cascades

    PubMed Central

    Sancho, Veronica; Berna, Marc J.; Thill, Michelle; Jensen, R. T.

    2011-01-01

    The novel PKCθ isoform is highly expressed in T-cells, brain and skeletal muscle and originally thought to have a restricted distribution. It has been extensively studied in T-cells and shown to be important for apoptosis, T-cell activation and proliferation. Recent studies showed its presence in other tissues and importance in insulin signaling, lung surfactant secretion, intestinal barrier permeability, platelet and mast-cell functions. However, little information is available for PKCθ activation by gastrointestinal(GI) hormones/neurotransmitters and growth factors. In the present study we used rat pancreatic acinar cells to explore their ability to activate PKCθ and the possible interactions with important cellular mediators of their actions. Particular attention was paid to cholecystokinin(CCK), a physiological regulator of pancreatic function and important in pathological processes affecting acinar function, like pancreatitis. PKCθ-protein/mRNA were present in the pancreatic acini, and T538-PKCθ phosphorylation/activation was stimulated only by hormones/neurotransmitters activating phospholipase C. PKCθ was activated in time- and dose-related manner by CCK, mediated 30% by high-affinity CCKA-receptor activation. CCK stimulated PKCθ translocation from cytosol to membrane. PKCθ inhibition (by pseudostrate-inhibitor or dominant negative) inhibited CCK- and TPA-stimulation of PKD, Src, RafC, PYK2, p125FAK and IKKα/β, but not basal/stimulated enzyme secretion. Also CCK- and TPA-induced PKCθ activation produced an increment in PKCθ’s direct association with AKT, RafA, RafC and Lyn. These results show for the first time PKCθ presence in pancreatic acinar cells, its activation by some GI hormones/neurotransmitters and involvement in important cell signaling pathways mediating physiological responses (enzyme secretion, proliferation, apoptosis, cytokine expression, and pathological responses like pancreatitis and cancer growth). PMID:21810446

  9. Prostate epithelial cell of origin determines cancer differentiation state in an organoid transformation assay.

    PubMed

    Park, Jung Wook; Lee, John K; Phillips, John W; Huang, Patrick; Cheng, Donghui; Huang, Jiaoti; Witte, Owen N

    2016-04-19

    The cell of origin for prostate cancer remains a subject of debate. Genetically engineered mouse models have demonstrated that both basal and luminal cells can serve as cells of origin for prostate cancer. Using a human prostate regeneration and transformation assay, our group previously demonstrated that basal cells can serve as efficient targets for transformation. Recently, a subpopulation of multipotent human luminal cells defined by CD26 expression that retains progenitor activity in a defined organoid culture was identified. We transduced primary human prostate basal and luminal cells with lentiviruses expressing c-Myc and activated AKT1 (myristoylated AKT1 or myrAKT1) to mimic theMYCamplification andPTENloss commonly detected in human prostate cancer. These cells were propagated in organoid culture before being transplanted into immunodeficient mice. We found that c-Myc/myrAKT1-transduced luminal xenografts exhibited histological features of well-differentiated acinar adenocarcinoma, with strong androgen receptor (AR) and prostate-specific antigen (PSA) expression. In contrast, c-Myc/myrAKT1-transduced basal xenografts were histologically more aggressive, with a loss of acinar structures and low/absent AR and PSA expression. Our findings imply that distinct subtypes of prostate cancer may arise from luminal and basal epithelial cell types subjected to the same oncogenic insults. This study provides a platform for the functional evaluation of oncogenes in basal and luminal epithelial populations of the human prostate. Tumors derived in this fashion with defined genetics can be used in the preclinical development of targeted therapeutics.

  10. Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells.

    PubMed

    Srinivasan, Padmanabhan; Thrower, Edwin C; Loganathan, Gopalakrishnan; Balamurugan, A N; Subramanian, Veedamali S; Gorelick, Fred S; Said, Hamid M

    2015-01-01

    Thiamin (vitamin B1), a member of the water-soluble family of vitamins, is essential for normal cellular functions; its deficiency results in oxidative stress and mitochondrial dysfunction. Pancreatic acinar cells (PAC) obtain thiamin from the circulation using a specific carrier-mediated process mediated by both thiamin transporters -1 and -2 (THTR-1 and THTR-2; encoded by the SLC19A2 and SLC19A3 genes, respectively). The aim of the current study was to examine the effect of chronic exposure of mouse PAC in vivo and human PAC in vitro to nicotine (a major component of cigarette smoke that has been implicated in pancreatic diseases) on thiamin uptake and to delineate the mechanism involved. The results showed that chronic exposure of mice to nicotine significantly inhibits thiamin uptake in murine PAC, and that this inhibition is associated with a marked decrease in expression of THTR-1 and THTR-2 at the protein, mRNA and hnRNAs level. Furthermore, expression of the important thiamin-metabolizing enzyme, thiamin pyrophosphokinase (TPKase), was significantly reduced in PAC of mice exposed to nicotine. Similarly, chronic exposure of cultured human PAC to nicotine (0.5 μM, 48 h) significantly inhibited thiamin uptake, which was also associated with a decrease in expression of THTR-1 and THTR-2 proteins and mRNAs. This study demonstrates that chronic exposure of PAC to nicotine impairs the physiology and the molecular biology of the thiamin uptake process. Furthermore, the study suggests that the effect is, in part, mediated through transcriptional mechanism(s) affecting the SLC19A2 and SLC19A3 genes.

  11. Chronic alcohol exposure affects pancreatic acinar mitochondrial thiamin pyrophosphate uptake: studies with mouse 266-6 cell line and primary cells

    PubMed Central

    Srinivasan, Padmanabhan; Nabokina, Svetlana

    2015-01-01

    Thiamin is essential for normal metabolic activity of all mammalian cells, including those of the pancreas. Cells obtain thiamin from their surroundings and enzymatically convert it into thiamin pyrophosphate (TPP) in the cytoplasm; TPP is then taken up by mitochondria via a specific carrier the mitochondrial TPP transporter (MTPPT; product of the SLC25A19 gene). Chronic alcohol exposure negatively impacts the health of pancreatic acinar cells (PAC), but its effect on physiological/molecular parameters of MTPPT is not known. We addressed this issue using mouse pancreatic acinar tumor cell line 266-6 and primary PAC of wild-type and transgenic mice carrying the SLC25A19 promoter that were fed alcohol chronically. Chronic alcohol exposure of 266-6 cells (but not to its nonoxidative metabolites ethyl palmitate and ethyl oleate) led to a significant inhibition in mitochondrial TPP uptake, which was associated with a decreased expression of MTPPT protein, mRNA, and activity of the SLC25A19 promoter. Similarly, chronic alcohol feeding of mice led to a significant inhibition in expression of MTPPT protein, mRNA, heterogeneous nuclear RNA, as well as in activity of SLC25A19 promoter in PAC. While chronic alcohol exposure did not affect DNA methylation of the Slc25a19 promoter, a significant decrease in histone H3 euchromatin markers and an increase in H3 heterochromatin marker were observed. These findings show, for the first time, that chronic alcohol exposure negatively impacts pancreatic MTPPT, and that this effect is exerted, at least in part, at the level of Slc25a19 transcription and appears to involve epigenetic mechanism(s). PMID:26316591

  12. In situ lineage tracking of human prostatic epithelial stem cell fate reveals a common clonal origin for basal and luminal cells.

    PubMed

    Blackwood, John K; Williamson, Stuart C; Greaves, Laura C; Wilson, Laura; Rigas, Anastasia C; Sandher, Raveen; Pickard, Robert S; Robson, Craig N; Turnbull, Douglass M; Taylor, Robert W; Heer, Rakesh

    2011-10-01

    Stem cells accumulate mitochondrial DNA (mtDNA) mutations resulting in an observable respiratory chain defect in their progeny, allowing the mapping of stem cell fate. There is considerable uncertainty in prostate epithelial biology where both basal and luminal stem cells have been described, and in this study the clonal relationships within the human prostate epithelial cell layers were explored by tracing stem cell fate. Fresh-frozen and formalin-fixed histologically-benign prostate samples from 35 patients were studied using sequential cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) enzyme histochemistry and COX subunit I immunofluorescence to identify areas of respiratory chain deficiency; mtDNA mutations were identified by whole mitochondrial genome sequencing of laser-captured areas. We demonstrated that cells with respiratory chain defects due to somatic mtDNA point mutations were present in prostate epithelia and clonally expand in acini. Lineage tracing revealed distinct patterning of stem cell fate with mtDNA mutations spreading throughout the whole acinus or, more commonly, present as mosaic acinar defects. This suggests that individual acini are typically generated from multiple stem cells, and the presence of whole COX-deficient acini suggests that a single stem cell can also generate an entire branching acinar subunit of the gland. Significantly, a common clonal origin for basal, luminal and neuroendocrine cells is demonstrated, helping to resolve a key area of debate in human prostate stem cell biology.

  13. Cell Division Drives Epithelial Cell Rearrangements during Gastrulation in Chick.

    PubMed

    Firmino, Joao; Rocancourt, Didier; Saadaoui, Mehdi; Moreau, Chloe; Gros, Jerome

    2016-02-08

    During early embryonic development, cells are organized as cohesive epithelial sheets that are continuously growing and remodeled without losing their integrity, giving rise to a wide array of tissue shapes. Here, using live imaging in chick embryo, we investigate how epithelial cells rearrange during gastrulation. We find that cell division is a major rearrangement driver that powers dramatic epithelial cell intercalation events. We show that these cell division-mediated intercalations, which represent the majority of epithelial rearrangements within the early embryo, are absolutely necessary for the spatial patterning of gastrulation movements. Furthermore, we demonstrate that these intercalation events result from overall low cortical actomyosin accumulation within the epithelial cells of the embryo, which enables dividing cells to remodel junctions in their vicinity. These findings uncover a role for cell division as coordinator of epithelial growth and remodeling that might underlie various developmental, homeostatic, or pathological processes in amniotes.

  14. 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.

  15. Epiplakin deficiency aggravates murine caerulein-induced acute pancreatitis and favors the formation of acinar keratin granules.

    PubMed

    Wögenstein, Karl L; Szabo, Sandra; Lunova, Mariia; Wiche, Gerhard; Haybaeck, Johannes; Strnad, Pavel; Boor, Peter; Wagner, Martin; Fuchs, Peter

    2014-01-01

    Epiplakin, a member of the plakin protein family, is exclusively expressed in epithelial tissues and was shown to bind to keratins. Epiplakin-deficient (EPPK-/-) mice showed no obvious spontaneous phenotype, however, EPPK-/- keratinocytes displayed faster keratin network breakdown in response to stress. The role of epiplakin in pancreas, a tissue with abundant keratin expression, was not yet known. We analyzed epiplakin's expression in healthy and inflamed pancreatic tissue and compared wild-type and EPPK-/- mice during caerulein-induced acute pancreatitis. We found that epiplakin was expressed primarily in ductal cells of the pancreas and colocalized with apicolateral keratin bundles in murine pancreatic acinar cells. Epiplakin's diffuse subcellular localization in keratin filament-free acini of K8-deficient mice indicated that its filament-associated localization in acinar cells completely depends on its binding partner keratin. During acute pancreatitis, epiplakin was upregulated in acinar cells and its redistribution closely paralleled keratin reorganization. EPPK-/- mice suffered from aggravated pancreatitis but showed no obvious regeneration phenotype. At the most severe stage of the disease, EPPK-/- acinar cells displayed more keratin aggregates than those of wild-type mice. Our data propose epiplakin to be a protective protein during acute pancreatitis, and that its loss causes impaired disease-associated keratin reorganization.

  16. Autophagy in pancreatic acinar cells in caerulein-treated mice: immunolocalization of related proteins and their potential as markers of pancreatitis.

    PubMed

    Zhang, Leshuai; Zhang, Jun; Shea, Katherine; Xu, Lin; Tobin, Grainne; Knapton, Alan; Sharron, Stewart; Rouse, Rodney

    2014-01-01

    Drug-induced pancreatitis (DIP) is an underdiagnosed condition that lacks sensitive and specific biomarkers. To better understand the mechanisms of DIP and to identify potential tissue biomarkers, we studied experimental pancreatitis induced in male C57BL/6 mice by intraperitoneal injection of caerulein (10 or 50 μg/kg) at 1-hr intervals for a total of 7 injections. Pancreata from caerulein-treated mice exhibited consistent acinar cell autophagy and apoptosis with infrequent necrosis. Kinetic assays for serum amylase and lipase also showed a dose-dependent increase. Terminal deoxynucleotidyl transferase-mediated biotin-dNTP nick labeling (TUNEL) detected dose-dependent acinar cell apoptosis. By light microscopy, autophagy was characterized by the formation of autophagosomes and autolysosomes (ALs) within the cytoplasm of acinar cells. Immunohistochemical studies with specific antibodies for proteins related to autophagy and pancreatic stress were conducted to evaluate these proteins as potential biomarkers of pancreatitis. Western blots were used to confirm immunohistochemical results using pancreatic lysates from control and treated animals. Autophagy was identified as a contributing process in caerulein-induced pancreatitis and proteins previously associated with autophagy were upregulated following caerulein treatment. Autophagosomes and ALs were found to be a common pathway, in which cathepsins, lysosome-associated membrane protein 2, vacuole membrane protein 1, microtubule-associated protein 1 light chain 3 (LC3), autophagy-related protein 9, Beclin1, and pancreatitis-associated proteins were simultaneously involved in response to caerulein stimulus. Regenerating islet-derived 3 gamma (Reg3γ), a pancreatic acute response protein, was dose-dependently induced in caerulein-treated mice and colocalized with the autophagosomal marker, LC3. This finding supports Reg3γ as a candidate biomarker for pancreatic injury.

  17. Control of lens epithelial cell survival

    PubMed Central

    1993-01-01

    We have studied the survival requirements of developing lens epithelial cells to test the hypothesis that most cells are programmed to kill themselves unless they are continuously signaled by other cells not to do so. The lens cells survived for weeks in both explant cultures and high-density dissociated cell cultures in the absence of other cells or added serum or protein, suggesting that they do not require signals from other cell types to survive. When cultured at low density, however, they died by apoptosis, suggesting that they depend on other lens epithelial cells for their survival. Lens epithelial cells cultured at high density in agarose gels also survived for weeks, even though they were not in direct contact with one another, suggesting that they can promote one another's survival in the absence of cell- cell contact. Conditioned medium from high density cultures promoted the survival of cells cultured at low density, suggesting that lens epithelial cells support one another's survival by secreting survival factors. We show for the first time that normal cell death occurs within the anterior epithelium in the mature lens, but this death is strictly confined to the region of the anterior suture. PMID:8491781

  18. Growth requirements of human mammary epithelial cells in culture.

    PubMed

    Taylor-Papadimitriou, J; Shearer, M; Stoker, M G

    1977-12-15

    Colony-forming epithelial cells can be separated from the non-dividing "foam cells" in human milk by differential adhesion to glass and freezing. The growth of such partially purified mammary epithelial cells is stimulated by co-culture with non-dividing feeder cells. Foam cells, mitomycin-treated mouse fibroblast lines and human mammary fibroblasts and calf lens epithelial cells are all effective in promoting mammary epithelial cell growth. Contact between epithelial cells and feeders is not required for the growth-promoting effect. The mitogenic effect of epidermal growth factor on mammary epithelial cells also requires feeder cell activity.

  19. Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology.

    PubMed

    Marciniak, Anja; Cohrs, Christian M; Tsata, Vasiliki; Chouinard, Julie A; Selck, Claudia; Stertmann, Julia; Reichelt, Saskia; Rose, Tobias; Ehehalt, Florian; Weitz, Jürgen; Solimena, Michele; Slak Rupnik, Marjan; Speier, Stephan

    2014-12-01

    Studies on the cellular function of the pancreas are typically performed in vitro on its isolated functional units, the endocrine islets of Langerhans and the exocrine acini. However, these approaches are hampered by preparation-induced changes of cell physiology and the lack of an intact surrounding. We present here a detailed protocol for the preparation of pancreas tissue slices. This procedure is less damaging to the tissue and faster than alternative approaches, and it enables the in situ study of pancreatic endocrine and exocrine cell physiology in a conserved environment. Pancreas tissue slices facilitate the investigation of cellular mechanisms underlying the function, pathology and interaction of the endocrine and exocrine components of the pancreas. We provide examples for several experimental applications of pancreas tissue slices to study various aspects of pancreas cell biology. Furthermore, we describe the preparation of human and porcine pancreas tissue slices for the validation and translation of research findings obtained in the mouse model. Preparation of pancreas tissue slices according to the protocol described here takes less than 45 min from tissue preparation to receipt of the first slices.

  20. Evidence for epithelial-mesenchymal transitions in adult liver cells.

    PubMed

    Sicklick, Jason K; Choi, Steve S; Bustamante, Marcia; McCall, Shannon J; Pérez, Elizabeth Hernández; Huang, Jiawen; Li, Yin-Xiong; Rojkind, Marcos; Diehl, Anna Mae

    2006-10-01

    Both myofibroblastic hepatic stellate cells (HSC) and hepatic epithelial progenitors accumulate in damaged livers. In some injured organs, the ability to distinguish between fibroblastic and epithelial cells is sometimes difficult because cells undergo epithelial-mesenchymal transitions (EMT). During EMT, cells coexpress epithelial and mesenchymal cell markers. To determine whether EMT occurs in adult liver cells, we analyzed the expression profile of primary HSC, two HSC lines, and hepatic epithelial progenitors. As expected, all HSC expressed HSC markers. Surprisingly, these markers were also expressed by epithelial progenitors. In addition, one HSC line expressed typical epithelial progenitor mRNAs, and these epithelial markers were inducible in the second HSC line. In normal and damaged livers, small ductular-type cells stained positive for an HSC marker. In conclusion, HSC and hepatic epithelial progenitors both coexpress epithelial and mesenchymal markers, providing evidence that EMT occurs in adult liver cells.

  1. Epithelial stem cells and intestinal cancer.

    PubMed

    Tan, Shawna; Barker, Nick

    2015-06-01

    The mammalian intestine is comprised of an epithelial layer that serves multiple functions in order to maintain digestive activity as well as intestinal homeostasis. This epithelial layer contains highly proliferative stem cells which facilitate its characteristic rapid regeneration. How these stem cells contribute to tissue repair and normal homeostasis are actively studied, and while we have a greater understanding of the molecular mechanisms and cellular locations that underlie stem cell regulation in this tissue, much still remains undiscovered. This review describes epithelial stem cells in both intestinal and non-intestinal tissues, as well as the strategies that have been used to further characterize the cells. Through a discussion of the current understanding of intestinal self-renewal and tissue regeneration in response to injury, we focus on how dysregulation of critical signaling pathways results in potentially oncogenic aberrations, and highlight issues that should be addressed in order for effective intestinal cancer therapies to be devised.

  2. Epithelial cell extrusion: Pathways and pathologies.

    PubMed

    Gudipaty, Swapna Aravind; Rosenblatt, Jody

    2016-05-19

    To remove dying or unwanted cells from an epithelium while preserving the barrier function of the layer, epithelia use a unique process called cell extrusion. To extrude, the cell fated to die emits the lipid Sphingosine 1 Phosphate (S1P), which binds the G-protein-coupled receptor Sphingosine 1 Phosphate receptor 2 (S1P2) in the neighboring cells that activates Rho-mediated contraction of an actomyosin ring circumferentially and basally. This contraction acts to squeeze the cell out apically while drawing together neighboring cells and preventing any gaps to the epithelial barrier. Epithelia can extrude out cells targeted to die by apoptotic stimuli to repair the barrier in the face of death or extrude live cells to promote cell death when epithelial cells become too crowded. Indeed, because epithelial cells naturally turn over by cell death and division at some of the highest rates in the body, epithelia depend on crowding-induced live cell extrusion to preserve constant cell numbers. If extrusion is defective, epithelial cells rapidly lose contact inhibition and form masses. Additionally, because epithelia act as the first line of defense in innate immunity, preservation of this barrier is critical for preventing pathogens from invading the body. Given its role in controlling constant cell numbers and maintaining barrier function, a number of different pathologies can result when extrusion is disrupted. Here, we review mechanisms and signaling pathways that control epithelial extrusion and discuss how defects in these mechanisms can lead to multiple diseases. We also discuss tactics pathogens have devised to hijack the extrusion process to infect and colonize epithelia.

  3. Quantitative characterization of the protein contents of the exocrine pancreatic acinar cell by soft x-ray microscopy and advanced digital imaging methods

    SciTech Connect

    Loo, Jr., Billy W.

    2000-06-01

    The study of the exocrine pancreatic acinar cell has been central to the development of models of many cellular processes, especially of protein transport and secretion. Traditional methods used to examine this system have provided a wealth of qualitative information from which mechanistic models have been inferred. However they have lacked the ability to make quantitative measurements, particularly of the distribution of protein in the cell, information critical for grounding of models in terms of magnitude and relative significance. This dissertation describes the development and application of new tools that were used to measure the protein content of the major intracellular compartments in the acinar cell, particularly the zymogen granule. Soft x-ray microscopy permits image formation with high resolution and contrast determined by the underlying protein content of tissue rather than staining avidity. A sample preparation method compatible with x-ray microscopy was developed and its properties evaluated. Automatic computerized methods were developed to acquire, calibrate, and analyze large volumes of x-ray microscopic images of exocrine pancreatic tissue sections. Statistics were compiled on the protein density of several organelles, and on the protein density, size, and spatial distribution of tens of thousands of zymogen granules. The results of these measurements, and how they compare to predictions of different models of protein transport, are discussed.

  4. Tauroursodeoxycholic acid reduces endoplasmic reticulum stress, acinar cell damage, and systemic inflammation in acute pancreatitis.

    PubMed

    Seyhun, Ersin; Malo, Antje; Schäfer, Claus; Moskaluk, Christopher A; Hoffmann, Ralf-Thorsten; Göke, Burkhard; Kubisch, Constanze H

    2011-11-01

    In acute pancreatitis, endoplasmic reticulum (ER) stress prompts an accumulation of malfolded proteins inside the ER, initiating the unfolded protein response (UPR). Because the ER chaperone tauroursodeoxycholic acid (TUDCA) is known to inhibit the UPR in vitro, this study examined the in vivo effects of TUDCA in an acute experimental pancreatitis model. Acute pancreatitis was induced in Wistar rats using caerulein, with or without prior TUDCA treatment. UPR components were analyzed, including chaperone binding protein (BiP), phosphorylated protein kinase-like ER kinase (pPERK), X-box binding protein (XBP)-1, phosphorylated c-Jun NH(2)-terminal kinase (pJNK), CCAAT/enhancer binding protein homologues protein, and caspase 12 and 3 activation. In addition, pancreatitis biomarkers were measured, such as serum amylase, trypsin activation, edema formation, histology, and the inflammatory reaction in pancreatic and lung tissue. TUDCA treatment reduced intracellular trypsin activation, edema formation, and cell damage, while leaving amylase levels unaltered. The activation of myeloperoxidase was clearly reduced in pancreas and lung. Furthermore, TUDCA prevented caerulein-induced BiP upregulation, reduced XBP-1 splicing, and caspase 12 and 3 activation. It accelerated the downregulation of pJNK. In controls without pancreatitis, TUDCA showed cytoprotective effects including pPERK signaling and activation of downstream targets. We concluded that ER stress responses activated in acute pancreatitis are grossly attenuated by TUDCA. The chaperone reduced the UPR and inhibited ER stress-associated proapoptotic pathways. TUDCA has a cytoprotective potential in the exocrine pancreas. These data hint at new perspectives for an employment of chemical chaperones, such as TUDCA, in prevention of acute pancreatitis.

  5. Epithelial cell-extracellular matrix interactions and stem cells in airway epithelial regeneration.

    PubMed

    Coraux, Christelle; Roux, Jacqueline; Jolly, Thomas; Birembaut, Philippe

    2008-08-15

    In healthy subjects, the respiratory epithelium forms a continuous lining to the airways and to the environment, and plays a unique role as a barrier against external deleterious agents to protect the airways from the insults. In respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), chronic bronchitis, or asthma, the airway epithelium is frequently remodeled and injured, leading to the impairment of its defense functions. The rapid restoration of the epithelial barrier is crucial for these patients. The complete regeneration of the airway epithelium is a complex phenomenon, including not only the epithelial wound repair but also the epithelial differentiation to reconstitute a fully well differentiated and functional epithelium. The regeneration implies two partners: the epithelial stem/progenitor cells and factors able to regulate this process. Among these factors, epithelial cells-extracellular matrix (ECM) interactions play a crucial role. The secretion of a provisional ECM, the cell-ECM relationships through epithelial receptors, and the remodeling of the ECM by proteases (mainly matrix metalloproteinases) contribute not only to airway epithelial repair by modulating epithelial cell migration and proliferation, but also to the differentiation of repairing cells leading to the complete restoration of the wounded epithelium. A better characterization of resident stem cells and of effectors of the regeneration process is an essential prerequisite to propose new regenerative therapeutics to patients suffering from infectious/inflammatory respiratory diseases.

  6. Stimulus-secretion coupling in pancreatic acinar cells: inhibitory effects of calcium removal and manganese addition on pancreozymin-induced amylase release.

    PubMed Central

    Kanno, T; Nishimura, O

    1976-01-01

    The role of Ca ions in stimulus-secretion coupling has been analysed in the isolated and perfused rat pancreas. 2. The omission of [Ca2+]O diminished but did not abolish the release of amylase in response to continuous stimulation with 5 m-u. pancreozymin (Pz)/ml. The addition of Mn2+ (1-0 mM) to this Ca-deficient environment abolished the residual release of amylase. This was followed by a complete recovery of amylase output when the control [Ca2+]O was reestablished. 3. The addition of Mn2+ (1-0 mM) to the extracellular environment containing 2-5 mM-Ca2+ reversibly inhibited the Pz-induced release of amylase. 4. A kinetic scheme based on competition of Ca and Mn at a carrier in the acinar cell membrane could quantitatively explain the effects of Ca and Mn upon the Pz-induced amylase release. 5. These results support the view that the Ca2+ influx into the acinar cells is the major contributor to the rise in [Ca2+]i which, in turn, mediates the processes in the stimulus-secretion coupling in the exocrine pancreas, and suggest that the mode of Ca influx is a facilitated diffusion. PMID:950596

  7. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    EPA Science Inventory

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  8. Bim Regulation of Lumen Formation in Cultured Mammary Epithelial Acini Is Targeted by Oncogenes

    PubMed Central

    Reginato, Mauricio J.; Mills, Kenna R.; Becker, Esther B. E.; Lynch, Danielle K.; Bonni, Azad; Muthuswamy, Senthil K.; Brugge, Joan S.

    2005-01-01

    Epithelial cells organize into cyst-like structures that contain a spherical monolayer of cells that enclose a central lumen. Using a three-dimensional basement membrane culture model in which mammary epithelial cells form hollow, acinus-like structures, we previously demonstrated that lumen formation is achieved, in part, through apoptosis of centrally localized cells. We demonstrate that the proapoptotic protein Bim may selectively trigger apoptosis of the centrally localized acinar cells, leading to temporally controlled lumen formation. Bim is not detectable during early stages of three-dimensional mammary acinar morphogenesis and is then highly upregulated in all cells of acini, coincident with detection of apoptosis in the centrally localized acinar cells. Inhibition of Bim expression by RNA interference transiently blocks luminal apoptosis and delays lumen formation. Oncogenes that induce acinar luminal filling, such as ErbB2 and v-Src, suppress expression of Bim through a pathway dependent on Erk-mitogen-activated protein kinase; however, HPV 16 E7, an oncogene that stimulates cell proliferation but not luminal filling, is unable to reduce Bim expression. Thus, Bim is a critical regulator of luminal apoptosis during mammary acinar morphogenesis in vitro and may be an important target of oncogenes that disrupt glandular epithelial architecture. PMID:15899862

  9. Minor Salivary Gland Changes in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma - A Histopathological Study

    PubMed Central

    Chitturi, Ravi Teja; Ragunathan, Yoithapprabhunath Thukanayakanpalayam; Lakshmi, Suman Jhansi; Nallusamy, Jaisanghar; Joseph, Isaac

    2016-01-01

    Introduction The most common etiology for Oral Squamous Cell Carcinoma (OSCC) is tobacco and tobacco related products which cause nuclear damage to the keratinocytes. The chemical carcinogens not only affect the lining of oral epithelium but also affect the lining epithelium of the excretory ducts of the salivary glands. Thus, there is a possibility of epithelial dysplasia of the salivary duct epithelium which may lead to potential malignant transformation. Aim The study was performed to see the changes in the minor salivary glands and excretory ducts in cases of oral epithelial dysplasia and OSCC. Materials and Methods A total of 278 archival cases of mild, moderate and severe epithelial dysplasia, carcinoma in situ, OSCC including verrucous carcinoma were histopathologically evaluated to observe changes in the excretory ducts and the minor salivary glands. Results In the study there were 56.5% males and 43.5% females. The age group that was most commonly affected in both the sexes was 50-60 yr old. Buccal mucosa was the most common site of involvement. Ductal changes observed in the excretory duct include simple hyperplasia, metaplastic changes such as mucous, oncocytic & squamous, and infiltration of inflammatory cells and malignant cells. Acinar changes observed were degeneration, squamous metaplasia, myoepithelial cell proliferation and inflammatory cell infiltration. Both the excretory ducts and ducts within the gland showed dysplasia. Conclusion According to observations in our study it is suggested that histopathological interpretation for oral mucosal lesions especially oral epithelial dysplasias and OSCC should also include changes related to salivary gland tissue to provide a better treatment plan and prevent recurrence of the malignant tumours. PMID:27630945

  10. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system.

    PubMed

    Lee, Jingu; Park, Sangkyu; Roh, Sangho

    2015-05-15

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage.

  11. Promoting effect of arachidonic acid supplementation on N-methyl-N-nitrosourea-induced pancreatic acinar cell hyperplasia in young Lewis rats.

    PubMed

    Yoshizawa, Katsuhiko; Uehara, Norihisa; Kimura, Ayako; Emoto, Yuko; Kinoshita, Yuichi; Yuri, Takashi; Takada, Hideho; Moriguchi, Toru; Hamazaki, Tomohito; Tsubura, Airo

    2013-01-01

    Arachidonic acid (AA) is naturally found in human breast milk. AA, together with docosahexaenoic acid, is commonly added as a functional food ingredient to commercial infant formula worldwide, in accordance with the international standard of Codex Alimentarius. However, few studies have been performed that are concerned with the possible carcinogenic effects of AA supplementation during neonatal life. The effect of dietary AA supplementation in dams, during gestation and lactation, was investigated in N-methyl-N-nitrosourea (MNU)-induced preneoplastic lesions in the exocrine pancreas of young Lewis rats. Dams were fed either an AA (2.0% AA) or a basal (<0.01% AA) diet. On postnatal day 0 (at birth), male and female pups received a single intraperitoneal injection of either 35 mg/kg MNU or vehicle. The morphology and proliferating activity of the exocrine pancreas were examined by proliferative cell nuclear antigen immunohistochemistry 7, 14, 21, 28 and/or 60 days post-MNU. Histopathologically, acinar cell hyperplasia (ACH) occurred in the MNU-treated groups 60 days after MNU injection, irrespecitive of whether the rats had been fed an AA diet. Morphometrically, the number and area of ACH per 1 mm(2) in MNU-treated rats increased significantly in the AA diet-fed rats, compared with basal diet-fed rats. The number of proliferative cell nuclear antigen-positive acinar cells in both the normal and hyperplastic areas of MNU-treated rats increased significantly in the AA diet-fed rats. In conclusion, providing dams with an AA-rich diet during gestation and lactation promotes MNU-induced pancreatic ACH in young Lewis rats.

  12. CCN1 induces a reversible epithelial-mesenchymal transition in gastric epithelial cells.

    PubMed

    Chai, Jianyuan; Norng, Manith; Modak, Cristina; Reavis, Kevin M; Mouazzen, Wasim; Pham, Jennifer

    2010-08-01

    CCN1 is a matricellular protein that activates many genes related to wound healing and tissue remodeling in fibroblasts, but its effect on epithelial cells remains unclear. This study examined the role of CCN1 in epithelial wound healing using rat gastric epithelial cells and rat stomach ulcer as in vitro and in vivo models, respectively. We found that CCN1 expression is highly upregulated in the epithelial cells adjacent to a wound and remains high until the wound is healed. Upregulation of CCN1 activates a transient epithelial-mesenchymal transition in the epithelial cells at the migrating front and drives wound closure. Once the wound is healed, these epithelial cells and their progeny can resume their original epithelial phenotype. We also found that CCN1-induced E-cadherin loss is not due to transcriptional regulation but rather protein degradation due to the collapse of adherens junctions, which is contributed by beta-catenin translocation. CCN1-activated integrin-linked kinase mediates this process. Finally, our in vivo study showed that locally neutralizing CCN1 drastically impairs wound closure, whereas local injection of recombinant CCN1 protein induces expression of vimentin and smooth muscle alpha-actin in normal gastric mucosal epithelial cells and accelerates re-epithelialization during ulcer healing. In conclusion, our study indicates that CCN1 can induce reversible epithelial-mesenchymal transition, and this feature may have great value for clinical wound healing.

  13. Adherence of skin bacteria to human epithelial cells.

    PubMed Central

    Romero-Steiner, S; Witek, T; Balish, E

    1990-01-01

    Aerobic and anaerobic bacteria isolated from human axillae were tested for their capacity to adhere to buccal epithelial cells, immortalized human epithelial (HEp-2) cells, and undifferentiated and differentiated human epithelial cells. In general, both aerobic and anaerobic diphtheroids adhered better to differentiated human epithelial cells than to HEp-2 and undifferentiated human epithelial cells (P less than 0.05). Mannose, galactose, fucose, N-acetyl-D-glucosamine, and fibronectin were also assayed for their capacity to inhibit the adherence of diphtheroids to human epithelial cells. A great deal of variability was observed in the capacity of the latter compounds to inhibit the attachment of aerobic diphtheroids to undifferentiated and differentiated epithelial cells. Overall, mannose appeared to be best at inhibiting the adherence of the aerobic diphtheroids to undifferentiated human epithelial cells. Galactose, fucose, N-acetyl-D-glucosamine, and fibronectin showed a greater capacity to inhibit attachment of aerobic diphtheroids to differentiated than to undifferentiated human epithelial cells. The inhibition of adherence to differentiated human epithelial cells varied with the microorganism and the compound tested; however, the highest and most consistent inhibition of adherence (76.1 to 88.6%) was observed with a 5% solution of N-acetyl-D-glucosamine. The in vitro adherence and adherence inhibition assays presented here demonstrate that a number of adhesins and receptors are involved in the adherence of skin bacteria to human epithelial cells and receptors on human epithelial cells are apparently altered during differentiation. PMID:2298877

  14. Effects of the type of dietary fat on acetylcholine-evoked amylase secretion and calcium mobilization in isolated rat pancreatic acinar cells.

    PubMed

    Yago, María D; Díaz, Ricardo J; Martínez, María A; Audi, Nama'a; Naranjo, José A; Martínez-Victoria, Emilio; Mañas, Mariano

    2006-04-01

    Olive oil is a major component of the Mediterranean diet, and its role in human health is being actively debated. This study aimed to clarify the mechanism of pancreatic adaptation to dietary fat. For this purpose, we examined whether dietary-induced modification of pancreatic membranes affects acinar cell function in response to the secretagogue acetylcholine (ACh). Weaning male Wistar rats were assigned to one of two experimental groups and fed for 8 weeks with a commercial chow (C) or a semisynthetic diet containing virgin olive oil as dietary fat (OO). The fatty acid composition of pancreatic plasma membranes was determined by gas-liquid chromatography. For assessment of secretory function, viable acini were incubated with ACh and amylase of supernatant was further assayed with a substrate reagent. Changes in cytosolic Ca(2+) concentration in response to ACh were measured by fura-2 AM fluorimetry. Compared to C rats, pancreatic cell membranes of OO rats had a higher level of monounsaturated fatty acids and a lower level of both saturated and polyunsaturated fatty acids, thus, reflecting the type of dietary fat given. Net amylase secretion in response to ACh was greatly enhanced after OO feeding, although this was not paralleled by enhancement of ACh-evoked Ca(2+) peak increases. In conclusion, chronic intake of diets that differ in the fat type influences not only the fatty acid composition of rat pancreatic membranes but also the responsiveness of acinar cells to ACh. This mechanism may be, at least in part, responsible for the adaptation of the exocrine pancreas to the type of fat available.

  15. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    SciTech Connect

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  16. Isolation by Size of Epithelial Tumor Cells

    PubMed Central

    Vona, Giovanna; Sabile, Abdelmajid; Louha, Malek; Sitruk, Veronique; Romana, Serge; Schütze, Karin; Capron, Frédérique; Franco, Dominique; Pazzagli, Mario; Vekemans, Michel; Lacour, Bernard; Bréchot, Christian; Paterlini-Bréchot, Patrizia

    2000-01-01

    We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine. PMID:10623654

  17. Force mapping in epithelial cell migration

    PubMed Central

    du Roure, Olivia; Saez, Alexandre; Buguin, Axel; Austin, Robert H.; Chavrier, Philippe; Siberzan, Pascal; Ladoux, Benoit

    2005-01-01

    We measure dynamic traction forces exerted by epithelial cells on a substrate. The force sensor is a high-density array of elastomeric microfabricated pillars that support the cells. Traction forces induced by cell migration are deduced from the measurement of the bending of these pillars and are correlated with actin localization by fluorescence microscopy. We use a multiple-particle tracking method to estimate the mechanical activity of cells in real time with a high-spatial resolution (down to 2 μm) imposed by the periodicity of the post array. For these experiments, we use differentiated Madin-Darby canine kidney (MDCK) epithelial cells. Our data provide definite information on mechanical forces exerted by a cellular assembly. The maximum intensity of the forces is localized on the edge of the epithelia. Hepatocyte growth factor promotes cell motility and induces strong scattering activity of MDCK cells. Thus, we compare forces generated by MDCK cells in subconfluent epithelia versus isolated cells after hepatocyte growth factor treatment. Maximal-traction stresses at the edge of a monolayer correspond to higher values than those measured for a single cell and may be due to a collective behavior. PMID:15695588

  18. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells.

    PubMed Central

    Simon, R H; DeHart, P D; Todd, R F

    1986-01-01

    The damage to pulmonary alveolar epithelial cells that occurs in many inflammatory conditions is thought to be caused in part by phagocytic neutrophils. To investigate this process, we exposed monolayers of purified rat alveolar epithelial cells to stimulated human neutrophils and measured cytotoxicity using a 51Cr-release assay. We found that stimulated neutrophils killed epithelial cells by a process that did not require neutrophil-generated reactive oxygen metabolites. Pretreatment of neutrophils with an antibody (anti-Mo1) that reduced neutrophil adherence to epithelial cells limited killing. Although a variety of serine protease inhibitors partially inhibited cytotoxicity, we found that neutrophil cytoplasts, neutrophil lysates, neutrophil-conditioned medium, purified azurophilic or specific granule contents, and purified human neutrophil elastase did not duplicate the injury. We conclude that stimulated neutrophils can kill alveolar epithelial cells in an oxygen metabolite-independent manner. Tight adherence of stimulated neutrophils to epithelial cell monolayers appears to promote epithelial cell killing. Images PMID:3771800

  19. Cell density determines epithelial migration in culture.

    PubMed Central

    Rosen, P; Misfeldt, D S

    1980-01-01

    The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approxmately 6.6 X 10(5) cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 day showed that the cells accelerated at a uniform rate of 0.24 micrometer . hr-2 and, by extrapolation, possessed an apparent initial velocity of 2.8 micrometer . hr-1 at the time of wounding. The apparent initial velocity was considered to be the result of a brief (< 10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 10(5) cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 micrometer . hr-2). Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet. Images PMID:6933523

  20. Interaction of E-cadherin and PTEN regulates morphogenesis and growth arrest in human mammary epithelial cells

    SciTech Connect

    Fournier, Marcia V.; Fata, Jimmie E.; Martin, Katherine J.; Yaswen, Paul; Bissell, Mina J.

    2009-06-03

    PTEN is a dual function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two non-malignant human mammary epithelial cell lines (HMECs) that form polarized, growth-arrested structures (acini) when cultured in 3-dimensional laminin-rich extracellular matrix gels (3D lrECM). As acini begin to form, PTEN accumulates in both the cytoplasm, and at cell-cell contacts where it colocalizes with E-cadherin/{beta}-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function-blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in SKBR3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in 3D lrECM indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus appears to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells.

  1. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2010-10-01

    breast is highly expressed by luminal epithelial cells and is less expressed by basal cells19,20. In contrast, CD49f (a6 integrin) has an inverse pattern...mouse stretched on its back. The hose and nose cone from the anesthetic vaporizer are securely attached to one side of the plate, and a heated pad is...the mouse by a nose cone. Check that the mouse has reached surgical anesthesia by loss of pedal withdrawal reflex . ! cautIon Institutional review

  2. Transcriptional Landscape of Glomerular Parietal Epithelial Cells

    PubMed Central

    Gharib, Sina A.; Pippin, Jeffrey W.; Ohse, Takamoto; Pickering, Scott G.; Krofft, Ronald D.; Shankland, Stuart J.

    2014-01-01

    Very little is known about the function of glomerular parietal epithelial cells (PECs). In this study, we performed genome-wide expression analysis on PEC-enriched capsulated vs. PEC-deprived decapsulated rat glomeruli to determine the transcriptional state of PECs under normal conditions. We identified hundreds of differentially expressed genes that mapped to distinct biologic modules including development, tight junction, ion transport, and metabolic processes. Since developmental programs were highly enriched in PECs, we characterized several of their candidate members at the protein level. Collectively, our findings confirm that PECs are multifaceted cells and help define their diverse functional repertoire. PMID:25127402

  3. Control of local immunity by airway epithelial cells.

    PubMed

    Weitnauer, M; Mijošek, V; Dalpke, A H

    2016-03-01

    The lung is ventilated by thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbial compounds, most of them harmless contaminants. Airway epithelial cells are known to have innate sensor functions, thus being able to detect microbial danger. To avoid chronic inflammation, the pulmonary system has developed specific means to control local immune responses. Even though airway epithelial cells can act as proinflammatory promoters, we propose that under homeostatic conditions airway epithelial cells are important modulators of immune responses in the lung. In this review, we discuss epithelial cell regulatory functions that control reactivity of professional immune cells within the microenvironment of the airways and how these mechanisms are altered in pulmonary diseases. Regulation by epithelial cells can be divided into two mechanisms: (1) mediators regulate epithelial cells' innate sensitivity in cis and (2) factors are produced that limit reactivity of immune cells in trans.

  4. Deoxycholic acid inhibited proliferation and induced apoptosis and necrosis by regulating the activity of transcription factors in rat pancreatic acinar cell line AR42J.

    PubMed

    Zhang, Guixin; Zhang, Jingwen; Shang, Dong; Qi, Bing; Chen, Hailong

    2015-09-01

    The objective of this study is to investigate the effect of deoxycholic acid (DCA) on rat pancreatic acinar cell line AR42J and the functional mechanisms of DCA on AR42J cells. AR42J cells were treated with various concentrations of DCA for 24 h and also treated with 0.4 mmol/L DCA for multiple times, and then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to detect the AR42J cell survival rate. Flow cytometric was used to detect the cell apoptosis and necrosis in AR42J cells treated with 0.4 mmol/L and 0.8 mmol/L DCA. The cells treated with phosphate buffer saline (PBS) were served as control. In addition, the DNA-binding activity assays of transcription factors (TFs) in nuclear proteins of cells treated with DCA were determined using Panomics Procarta Transcription Factor Assay Kit. The relative survival rates were markedly decreased (P < 0.05) in a dose- and time-dependent manner. Compared with control group, the cell apoptosis and necrosis ratio were both significantly elevated in 0.4 mmol/L DCA and 0.8 mmol/L DCA groups (P < 0.01). A significant increase (P < 0.05) in the activity of transcription factor 2 (ATF2), interferon-stimulated response element (ISRE), NKX-2.5, androgen receptor (AR), p53, and hypoxia-inducible factor-1 (HIF-1) was observed, and the activity of peroxisome proliferator-activated receptor (PPAR), activator protein 1 (AP1), and E2F1 was reduced (P < 0.05). In conclusion, DCA inhibited proliferation and induced apoptosis and necrosis in AR42J cells. The expression changes of related genes regulated by TFs might be the molecular mechanism of AR42J cell injury.

  5. Phenotypic plasticity in normal breast derived epithelial cells

    PubMed Central

    2014-01-01

    Background Normal, healthy human breast tissue from a variety of volunteer donors has become available for research thanks to the establishment of the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB). Multiple epithelial (K-HME) and stromal cells (K-HMS) were established from the donated tissue. Explant culture was utilized to isolate the cells from pieces of breast tissue. Selective media and trypsinization were employed to select either epithelial cells or stromal cells. The primary, non-transformed epithelial cells, the focus of this study, were characterized by immunohistochemistry, flow cytometry, and in vitro cell culture. Results All of the primary, non-transformed epithelial cells tested have the ability to differentiate in vitro into a variety of cell types when plated in or on biologic matrices. Cells identified include stratified squamous epithelial, osteoclasts, chondrocytes, adipocytes, neural progenitors/neurons, immature muscle and melanocytes. The cells also express markers of embryonic stem cells. Conclusions The cell culture conditions employed select an epithelial cell that is pluri/multipotent. The plasticity of the epithelial cells developed mimics that seen in metaplastic carcinoma of the breast (MCB), a subtype of triple negative breast cancer; and may provide clues to the origin of this particularly aggressive type of breast cancer. The KTB is a unique biorepository, and the normal breast epithelial cells isolated from donated tissue have significant potential as new research tools. PMID:24915897

  6. Scrib is required for epithelial cell identity and prevents epithelial to mesenchymal transition in the mouse.

    PubMed

    Yamben, Idella F; Rachel, Rivka A; Shatadal, Shalini; Copeland, Neal G; Jenkins, Nancy A; Warming, Soren; Griep, Anne E

    2013-12-01

    The integrity and function of epithelial tissues depend on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFβ signaling intermediates, accumulated in the nucleus and Snail, a TGFβ target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFβ signaling.

  7. Interaction with epithelial cells modifies airway macrophage response to ozone.

    PubMed

    Bauer, Rebecca N; Müller, Loretta; Brighton, Luisa E; Duncan, Kelly E; Jaspers, Ilona

    2015-03-01

    The initial innate immune response to ozone (O3) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell-Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell-derived signals are important determinants of Mac immunophenotype and response to O3.

  8. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2009-10-01

    Appendix……………………………………………………………………………… 11 Eirew,P., Stingl,J., Raouf,A., Turashvili,G., Aparicio ,S., Emerman,J.T., and Eaves,C.J. A method for... Aparicio , Joanne Emerman and Connie Eaves. A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability...Abstracts Peter Eirew, John Stingl, Afshin Raouf, Gulisa Turshvili, Sam Aparicio , Joanne Emerman and Connie Eaves, “Identification of Human Mammary

  9. Acinar neoplasms of the pancreas-A summary of 25 years of research.

    PubMed

    Klimstra, David S; Adsay, Volkan

    2016-09-01

    Our understanding about the family of acinar neoplasms of the pancreas has grown substantially over the past 25 years. The prototype is acinar cell carcinoma, an uncommon variant of pancreatic carcinoma that demonstrates production of pancreatic exocrine enzymes, verifiable using immunohistochemistry, and exhibits characteristic histologic features. Related neoplasms include mixed acinar carcinomas such as mixed acinar neuroendocrine carcinoma and mixed acinar ductal carcinoma. In the pediatric age group, pancreatoblastoma is also closely related. Cystic and extrapancreatic forms have been described. These neoplasms share molecular alterations that are distinct from the more common ductal and neuroendocrine neoplasms of the pancreas. Although there is a broad range of genetic findings, a number of potential therapeutic targets have emerged. This review explores the clinical and pathologic features of pancreatic acinar neoplasms along with their more common molecular phenotypes. The differential diagnosis with other pancreatic neoplasms is explored as well.

  10. Quercetin Blocks Airway Epithelial Cell Chemokine Expression

    PubMed Central

    Nanua, Suparna; Zick, Suzanna M.; Andrade, Juan E.; Sajjan, Umadevi S.; Burgess, John R.; Lukacs, Nicholas W.; Hershenson, Marc B.

    2006-01-01

    Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a dietary flavonoid, is an inhibitor of phosphatidylinositol (PI) 3-kinase and potent antioxidant. We hypothesized that quercetin blocks airway epithelial cell chemokine expression via PI 3-kinase–dependent mechanisms. Pretreatment with quercetin and the PI 3–kinase inhibitor LY294002 each reduced TNF-α–induced IL-8 and monocyte chemoattractant protein (MCP)-1 (also called CCL2) expression in cultured human airway epithelial cells. Quercetin also inhibited TNF-α–induced PI 3-kinase activity, Akt phosphorylation, intracellular H2O2 production, NF-κB transactivation, IL-8 promoter activity, and steady-state mRNA levels, consistent with the notion that quercetin inhibits chemokine expression by attenuating NF-κB transactivation via a PI 3-kinase/Akt-dependent pathway. Quercetin also reduced TNF-α–induced chemokine secretion in the presence of the transcriptional inhibitor actinomycin D, while inducing phosphorylation of eukaryotic translation initiation factor (eIF)-2α, suggesting that quercetin attenuates chemokine expression by post-transcriptional as well as transcriptional mechanisms. Finally, we tested the effects of quercetin in cockroach antigen–sensitized and –challenged mice. These mice show MCP-1–dependent airways hyperresponsiveness and inflammation. Quercetin significantly reduced lung MCP-1 and methacholine responsiveness. We conclude that quercetin blocks airway cell chemokine expression via transcriptional and post-transcriptional pathways. PMID:16794257

  11. Urokinase plasminogen activator released by alveolar epithelial cells modulates alveolar epithelial repair in vitro.

    PubMed

    Van Leer, Coretta; Stutz, Monika; Haeberli, André; Geiser, Thomas

    2005-12-01

    Intra-alveolar fibrin is formed following lung injury and inflammation and may contribute to the development of pulmonary fibrosis. Fibrin turnover is altered in patients with pulmonary fibrosis, resulting in intra-alveolar fibrin accumulation, mainly due to decreased fibrinolysis. Alveolar type II epithelial cells (AEC) repair the injured alveolar epithelium by migrating over the provisional fibrin matrix. We hypothesized that repairing alveolar epithelial cells modulate the underlying fibrin matrix by release of fibrinolytic activity, and that the degree of fibrinolysis modulates alveolar epithelial repair on fibrin. To test this hypothesis we studied alveolar epithelial wound repair in vitro using a modified epithelial wound repair model with human A549 alveolar epithelial cells cultured on a fibrin matrix. In presence of the inflammatory cytokine interleukin-1beta, wounds increase by 800% in 24 hours mainly due to detachment of the cells, whereas in serum-free medium wound areas decreases by 22.4 +/- 5.2% (p < 0.01). Increased levels of D-dimer, FDP and uPA in the cell supernatant of IL-1beta-stimulated A549 epithelial cells indicate activation of fibrinolysis by activation of the plasmin system. In presence of low concentrations of fibrinolysis inhibitors, including specific blocking anti-uPA antibodies, alveolar epithelial repair in vitro was improved, whereas in presence of high concentrations of fibrinolysis inhibitors, a decrease was observed mainly due to decreased spreading and migration of cells. These findings suggest the existence of a fibrinolytic optimum at which alveolar epithelial repair in vitro is most efficient. In conclusion, uPA released by AEC alters alveolar epithelial repair in vitro by modulating the underlying fibrin matrix.

  12. Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis

    PubMed Central

    Teoh-Fitzgerald, ML; Fitzgerald, MP; Zhong, W; Askeland, RW; Domann, FE

    2013-01-01

    Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging

  13. Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis.

    PubMed

    Teoh-Fitzgerald, M L; Fitzgerald, M P; Zhong, W; Askeland, R W; Domann, F E

    2014-01-16

    Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging

  14. Myosin 2 Maintains an Open Exocytic Fusion Pore in Secretory Epithelial Cells

    PubMed Central

    Bhat, Purnima

    2009-01-01

    Many studies have implicated F-actin and myosin 2 in the control of regulated secretion. Most recently, evidence suggests a role for the microfilament network in regulating the postfusion events of vesicle dynamics. This is of potential importance as postfusion behavior can influence the loss of vesicle content and may provide a new target for drug therapy. We have investigated the role of myosin 2 in regulating exocytosis in secretory epithelial cells by using novel assays to determine the behavior of the fusion pore in individual granules. We immunolocalize myosin 2A to the apical region of pancreatic acinar cells, suggesting it is this isoform that plays a role in granule exocytosis. We further show myosin 2 phosphorylation increased on cell stimulation, consistent with a regulatory role in secretion. Importantly, in a single-cell, single-granule secretion assay, neither the myosin 2 inhibitor (−)-blebbistatin nor the myosin light chain kinase inhibitor ML-9 had any effect on the numbers of granules stimulated to fuse after cell stimulation. These data indicate that myosin 2, if it has any action on secretion, must be targeting postfusion granule behavior. This interpretation is supported by direct study of fusion pore opening in which we show that (−)-blebbistatin and ML-9 promote fusion pore closure and decrease fusion pore lifetimes. Our work now adds to a growing body of evidence showing that myosin 2 is an essential regulator of postfusion granule behavior. In particular, in the case of the secretory epithelial cells, myosin 2 activity is necessary to maintain fusion pore opening. PMID:19158378

  15. Pancreatic Acinar Cells Employ miRNAs as Mediators of Intercellular Communication to Participate in the Regulation of Pancreatitis-Associated Macrophage Activation

    PubMed Central

    Zhao, Yong; Wang, Hao; Qiao, Xin; Sun, Bei

    2016-01-01

    Macrophage activation plays an important role in the inflammatory response in acute pancreatitis. In the present study, the activation of AR42J pancreatic acinar cells was induced by taurolithocholate treatment. The results showed that the culture medium from the activated AR42J cells significantly enhanced NFκB activation in the macrophages compared to that without taurolithocholate treatment. Additionally, the precipitates obtained from ultracentrifugation of the culture media that were rich in exosomes were markedly more potent in activating macrophages compared with the supernatant fraction lacking exosomes. The results indicated that the mediators carried by the exosomes played important roles in macrophage activation. Exosomal miRNAs were extracted and examined using microarrays. A total of 115 differentially expressed miRNAs were identified, and 30 showed upregulated expression, while 85 displayed downregulated expression. Target genes of the differentially expressed miRNAs were predicted using TargetScan, MiRanda, and PicTar software programs. The putative target genes were subjected to KEGG functional analysis. The functions of the target genes were primarily enriched in MAPK pathways. Specifically, the target genes regulated macrophage activation through the TRAF6-TAB2-TAK1-NIK/IKK-NFκB pathway. As the mediators of signal transduction, miRNAs and their predicted target mRNAs regulate every step in the MAPK pathway. PMID:27546996

  16. Glucocorticoid receptor in human respiratory epithelial cells.

    PubMed

    Pujolsa, Laura; Mullol, Joaquim; Picado, Cèsar

    2009-01-01

    Inhaled and intranasal glucocorticoids (GCs) are the most common and effective drugs for controlling symptoms and airway inflammation in respiratory diseases such as allergic rhinitis, chronic rhinosinusitis with/without nasal polyps, and asthma, and the respiratory epithelium is a primary target of GC anti-inflammatory actions. GC effects are mediated through the GC receptor (GR). In humans, one single GR gene gives rise to two main GR products, namely GRalpha and GRbeta, which are subject to translational and posttranslational modifications. GRalpha is expressed in virtually all human cells and tissues, including respiratory epithelial cells, and - at least in vitro - is downregulated by GC. GRalpha mediates the anti-inflammatory actions of GC by activating transcription of anti-inflammatory genes through binding of GRalpha to glucocorticoid response elements (GRE) located in the promoter region of target genes, repressing transcription of proinflammatory genes through direct interaction between GRalpha and proinflammatory transcription factors, such as AP-1 and NF-kappaB (transrepression), and also by destabilizing the mRNA of proinflammatory mediators. GRbeta acts as a dominant negative inhibitor of GRalpha-mediated transactivation and transrepression in certain in vitro studies with transfected cells. The GRbeta message is expressed at low levels in numerous tissues and its protein is mainly expressed in inflammatory cells, although it has also been detected in airway epithelial cells. Increased GRbeta expression has been reported in bronchial asthma and nasal polyposis, and after incubation of cells with certain proinflammatory stimuli. However, the role of GRbeta in modulating GC sensitivity in vivo has been highly debated and is as yet unclear.

  17. Henipavirus pathogenesis in human respiratory epithelial cells.

    PubMed

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz; Rockx, Barry

    2013-03-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection.

  18. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    SciTech Connect

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  19. Serotonin promotes acinar dedifferentiation following pancreatitis-induced regeneration in the adult pancreas.

    PubMed

    Saponara, Enrica; Grabliauskaite, Kamile; Bombardo, Marta; Buzzi, Raphael; Silva, Alberto B; Malagola, Ermanno; Tian, Yinghua; Hehl, Adrian B; Schraner, Elisabeth M; Seleznik, Gitta M; Zabel, Anja; Reding, Theresia; Sonda, Sabrina; Graf, Rolf

    2015-12-01

    The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help in developing therapeutic interventions. Serotonin has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy, and thyroid hormone-stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar dedifferentiation and cell cycle progression during the regenerative phase were investigated over the course of 2 weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect the clonal regeneration of mature acinar cells. Serotonin was, however, required for acinar dedifferentiation following inflammation-mediated tissue injury. Specifically, lack of serotonin resulted in delayed up-regulation of progenitor genes and delayed the formation of acinar-to-ductal metaplasia and defective acinar cell proliferation. We identified serotonin-dependent acinar secretion as a key step in progenitor-based regeneration, as it promoted acinar cell dedifferentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1-Ptfa axis in the uninjured adult pancreas, activated by zymogen secretion. Our findings indicated that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting the dedifferentiation of acinar cells.

  20. Epithelial Cell Innate Response to Candida albicans

    PubMed Central

    Naglik, J.R.; Moyes, D.

    2011-01-01

    With the advent of treatments and diseases such as AIDS resulting in increasing numbers of patients with suppressed immune systems, fungal diseases are an escalating problem. Candida albicans is the most common of these fungal pathogens, causing infections in many of these patients. It is therefore important to understand how immunity to this fungus is regulated and how it might be manipulated. Although work has been done to identify the receptors, fungal moieties, and responses involved in anti-Candida immunity, most studies have investigated interactions with myeloid or lymphoid cells. Given that the first site of contact of C. albicans with its host is the mucosal epithelial surface, recent studies have begun to focus on interactions of C. albicans with this site. The results are startling yet in retrospect obvious, indicating that epithelial cells play an important role in these interactions, initiating responses and even providing a level of protection. These findings have obvious implications, not just for fungal pathogens, but also for identifying how host organisms can distinguish between commensal and pathogenic microbes. This review highlights some of these recent findings and discusses their importance in the wider context of infection and immunity. PMID:21441481

  1. Nuclear microscopy of rat colon epithelial cells

    NASA Astrophysics Data System (ADS)

    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  2. Culture and differentiation of mouse tracheal epithelial cells.

    PubMed

    You, Yingjian; Brody, Steven L

    2013-01-01

    Airway epithelial cell biology has been greatly advanced by studies of genetically defined and modified mice; however it is often difficult to isolate, manipulate, and assay epithelial cell-specific responses in vivo. In vitro proliferation and differentiation of mouse airway epithelial cells are made possible by a high-fidelity system for primary culture of mouse tracheal epithelial cells described in this chapter. Using this method, epithelial cells purified from mouse tracheas proliferate in growth factor-enriched medium. Subsequent culture in defined medium and the use of the air-liquid interface condition result in the development of well-differentiated epithelia composed of ciliated and non-ciliated cells with characteristics of native airways. Methods are also provided for manipulation of differentiation and analysis of differentiation and gene expression. These approaches allow the assessment of global responses and those of specific cell subpopulations within the airway epithelium.

  3. 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

  4. Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction

    PubMed Central

    Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina

    2017-01-01

    Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1

  5. Documentation of angiotensin II receptors in glomerular epithelial cells

    NASA Technical Reports Server (NTRS)

    Sharma, M.; Sharma, R.; Greene, A. S.; McCarthy, E. T.; Savin, V. J.; Cowley, A. W. (Principal Investigator)

    1998-01-01

    Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial

  6. Klebsiella pneumoniae Is Able to Trigger Epithelial-Mesenchymal Transition Process in Cultured Airway Epithelial Cells

    PubMed Central

    Leone, Laura; Mazzetta, Francesca; Martinelli, Daniela; Valente, Sabatino; Alimandi, Maurizio; Raffa, Salvatore; Santino, Iolanda

    2016-01-01

    The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells. PMID:26812644

  7. Airway epithelial IL-15 transforms monocytes into dendritic cells.

    PubMed

    Regamey, Nicolas; Obregon, Carolina; Ferrari-Lacraz, Sylvie; van Leer, Coretta; Chanson, Marc; Nicod, Laurent P; Geiser, Thomas

    2007-07-01

    IL-15 has recently been shown to induce the differentiation of functional dendritic cells (DCs) from human peripheral blood monocytes. Since DCs lay in close proximity to epithelial cells in the airway mucosa, we investigated whether airway epithelial cells release IL-15 in response to inflammatory stimuli and thereby induce differentiation and maturation of DCs. Alveolar (A549) and bronchial (BEAS-2B) epithelial cells produced IL-15 spontaneously and in a time- and dose-dependent manner after stimulation with IL-1beta, IFN-gamma, or TNF-alpha. Airway epithelial cell supernatants induced an increase of IL-15Ralpha gene expression in ex vivo monocytes, and stimulated DCs enhanced their IL-15Ralpha gene expression up to 300-fold. Airway epithelial cell-conditioned media induced the differentiation of ex vivo monocytes into partially mature DCs (HLA-DR+, DC-SIGN+, CD14+, CD80-, CD83+, CD86+, CCR3+, CCR6(+), CCR7-). Based on their phenotypic (CD123+, BDCA2+, BDCA4+, BDCA1(-), CD1a-) and functional properties (limited maturation upon stimulation with LPS and limited capacity to induce T cell proliferation), these DCs resembled plasmacytoid DCs. The effects of airway epithelial cell supernatants were largely blocked by a neutralizing monoclonal antibody to IL-15. Thus, our results demonstrate that airway epithelial cell-conditioned media have the capacity to differentiate monocytes into functional DCs, a process substantially mediated by epithelial-derived IL-15.

  8. Mechanical stretch triggers rapid epithelial cell division through Piezo1.

    PubMed

    Gudipaty, S A; Lindblom, J; Loftus, P D; Redd, M J; Edes, K; Davey, C F; Krishnegowda, V; Rosenblatt, J

    2017-03-02

    Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.

  9. STUDIES ON AN EPITHELIAL (GLAND) CELL JUNCTION

    PubMed Central

    Loewenstein, Werner R.; Kanno, Yoshinobu

    1964-01-01

    Membrane permeability of an epithelial cell junction (Drosophila salivary gland) was examined with intracellular microelectrodes and with fluorescent tracers. In contrast to the non-junctional cell membrane surface, which has a low permeability to ions (10-4 mho/cm2), the junctional membrane surface is highly permeable. In fact, it introduces no substantial restriction to ion flow beyond that in the cytoplasm; the resistance through a chain of cells (150 Ω cm) is only slightly greater than in extruded cytoplasm (100 Ω cm). The diffusion resistance along the intercellular space to the exterior, on the other hand, is very high. Here, there exists an ion barrier of, at least, 104Ω cm2. As a result, small ions and fluorescein move rather freely from one cell to the next, but do not leak appreciably through the intercellular space to the exterior. The organ here, rather than the single cell, appears to be the unit of ion environment. The possible underlying structural aspects are discussed. PMID:14206423

  10. Sonic Hedgehog regulates thymic epithelial cell differentiation

    PubMed Central

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L.; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-01-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus. PMID

  11. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    PubMed

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-04-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.

  12. Multi-functionality and plasticity characterize epithelial cells in Hydra

    PubMed Central

    Buzgariu, W; Al Haddad, S; Tomczyk, S; Wenger, Y; Galliot, B

    2015-01-01

    Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra. PMID:26716072

  13. Technical note: Isolation and characterization of porcine mammary epithelial cells.

    PubMed

    Dahanayaka, S; Rezaei, R; Porter, W W; Johnson, G A; Burghardt, R C; Bazer, F W; Hou, Y Q; Wu, Z L; Wu, G

    2015-11-01

    Within the mammary gland, functional synthesis of milk is performed by its epithelial (alveolar) cells. The availability of a stable mammary epithelial cell line is essential for biochemical studies to elucidate cellular and molecular mechanisms responsible for nutritional regulation of lactation. Therefore, porcine mammary epithelial cells (PMEC) were isolated from mammary glands of a 9-mo-old nonpregnant and nonlactating gilt and cultured to establish a nonimmortalized cell line. These cells were characterized by expression of cytokeratin-18 (an intermediate filament specific for epithelial cells), β-casein (a specific marker for mammary epithelial cells), and α-lactalbumin. In culture, the PMEC doubled in number every 24 h and maintained a cobblestone morphology, typical for cultured epithelial cells, for at least 15 passages. Addition of 0.2 to 2 μg/mL prolactin to culture medium for 3 d induced the production of β-casein and α-lactalbumin by PMEC in a dose-dependent manner. Thus, we have successfully developed a useful PMEC line for future studies of cellular and molecular regulation of milk synthesis by mammary epithelial cells of the sow.

  14. p120 Catenin is required for normal tubulogenesis but not epithelial integrity in developing mouse pancreas.

    PubMed

    Hendley, Audrey M; Provost, Elayne; Bailey, Jennifer M; Wang, Yue J; Cleveland, Megan H; Blake, Danielle; Bittman, Ross W; Roeser, Jeffrey C; Maitra, Anirban; Reynolds, Albert B; Leach, Steven D

    2015-03-01

    The intracellular protein p120 catenin aids in maintenance of cell-cell adhesion by regulating E-cadherin stability in epithelial cells. In an effort to understand the biology of p120 catenin in pancreas development, we ablated p120 catenin in mouse pancreatic progenitor cells, which resulted in deletion of p120 catenin in all epithelial lineages of the developing mouse pancreas: islet, acinar, centroacinar, and ductal. Loss of p120 catenin resulted in formation of dilated epithelial tubules, expansion of ductal epithelia, loss of acinar cells, and the induction of pancreatic inflammation. Aberrant branching morphogenesis and tubulogenesis were also observed. Throughout development, the phenotype became more severe, ultimately resulting in an abnormal pancreas comprised primarily of duct-like epithelium expressing early progenitor markers. In pancreatic tissue lacking p120 catenin, overall epithelial architecture remained intact; however, actin cytoskeleton organization was disrupted, an observation associated with increased cytoplasmic PKCζ. Although we observed reduced expression of adherens junction proteins E-cadherin, β-catenin, and α-catenin, p120 catenin family members p0071, ARVCF, and δ-catenin remained present at cell membranes in homozygous p120(f/f) pancreases, potentially providing stability for maintenance of epithelial integrity during development. Adult mice homozygous for deletion of p120 catenin displayed dilated main pancreatic ducts, chronic pancreatitis, acinar to ductal metaplasia (ADM), and mucinous metaplasia that resembles PanIN1a. Taken together, our data demonstrate an essential role for p120 catenin in pancreas development.

  15. Novel Lipophilic Probe for Detecting Near-Membrane Reactive Oxygen Species Responses and Its Application for Studies of Pancreatic Acinar Cells: Effects of Pyocyanin and L-Ornithine

    PubMed Central

    Chvanov, Michael; Huang, Wei; Jin, Tao; Wen, Li; Armstrong, Jane; Elliot, Vicky; Alston, Ben; Burdyga, Alex; Criddle, David N.; Sutton, Robert

    2015-01-01

    Abstract Aims: The aim of this study was to develop a fluorescent reactive oxygen species (ROS) probe, which is preferentially localized in cellular membranes and displays a strong change in fluorescence upon oxidation. We also aimed to test the performance of this probe for detecting pathophysiologically relevant ROS responses in isolated cells. Results: We introduced a novel lipophilic ROS probe dihydrorhodamine B octadecyl ester (H2RB-C18). We then applied the new probe to characterize the ROS changes triggered by inducers of acute pancreatitis in pancreatic acinar cells. We resolved ROS changes produced by L-ornithine, L-arginine, cholecystokinin-8, acetylcholine, taurolithocholic acid 3-sulfate, palmitoleic acid ethyl ester, and the bacterial toxin pyocyanin. Particularly prominent ROS responses were induced by pyocyanin and L-ornithine. These ROS responses were accompanied by changes in cytosolic Ca2+concentration ([Ca2+]i), mitochondrial membrane potential (ΔΨ), and NAD(P)H concentration. Innovation: The study describes a novel sensitive lipophilic ROS probe. The probe is particularly suitable for detecting ROS in near-membrane regions and therefore for reporting the ROS environment of plasma membrane channels and pumps. Conclusions: In our experimental conditions, the novel probe was more sensitive than 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (CM-H2DCF) and dihydrorhodamine123 (H2R123) and allowed us to resolve ROS responses to secretagogues, pyocyanin, and L-ornithine. Changes in the fluorescence of the new probe were particularly prominent in the peripheral plasma membrane-associated regions. Our findings suggest that the new probe will be a useful tool in studies of the contribution of ROS to the pathophysiology of exocrine pancreas and other organs/tissues. Antioxid. Redox Signal. 22, 451–464. PMID:24635199

  16. Pathology and genetics of pancreatic neoplasms with acinar differentiation.

    PubMed

    Wood, Laura D; Klimstra, David S

    2014-11-01

    Pancreatic neoplasms with acinar differentiation, including acinar cell carcinoma, pancreatoblastoma, and carcinomas with mixed differentiation, are distinctive pancreatic neoplasms with a poor prognosis. These neoplasms are clinically, pathologically, and genetically unique when compared to other more common pancreatic neoplasms. Most occur in adults, although pancreatoblastomas usually affect children under 10 years old. All of these neoplasms exhibit characteristic histologic features including a solid or acinar growth pattern, dense neoplastic cellularity, uniform nuclei with prominent nucleoli, and granular eosinophilic cytoplasm. Exocrine enzymes are detectable by immunohistochemistry and, for carcinomas with mixed differentiation, neuroendocrine or ductal lineage markers are also expressed. The genetic alterations of this family of neoplasms largely differ from conventional ductal adenocarcinomas, with only rare mutations in TP53, KRAS, and p16, but no single gene or neoplastic pathway is consistently altered in acinar neoplasms. Instead, there is striking genomic instability, and a subset of cases has mutations in the APC/β-catenin pathway, mutations in SMAD4, RAF gene family fusions, or microsatellite instability. Therapeutically targetable mutations are often present. This review summarizes the clinical and pathologic features of acinar neoplasms and reviews the current molecular data on these uncommon tumors.

  17. Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells.

    PubMed

    Liu, Yuan; Zhang, Yueling; Gu, Zhaohui; Hao, Lina; Du, Juan; Yang, Qian; Li, Suping; Wang, Liying; Gong, Shilei

    2014-07-15

    Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite.

  18. Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells

    PubMed Central

    Liu, Yuan; Zhang, Yueling; Gu, Zhaohui; Hao, Lina; Du, Juan; Yang, Qian; Li, Suping; Wang, Liying; Gong, Shilei

    2014-01-01

    Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite. PMID:25221599

  19. Cell Chirality Induces Collective Cell Migration in Epithelial Sheets

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Shibata, Tatsuo

    2015-10-01

    During early development, epithelial cells form a monolayer sheet and migrate in a uniform direction. Here, we address how this collective migration can occur without breaking the cell-to-cell attachments. Repeated contraction and expansion of the cell-to-cell interfaces enables the cells to rearrange their positions autonomously within the sheet. We show that when the interface tension is strengthened in a direction that is tilted from the body axis, cell rearrangements occur in such a way that unidirectional movement is induced. We use a vertex model to demonstrate that such anisotropic tension can generate the unidirectional motion of cell sheets. Our results suggest that cell chirality facilitates collective cell migration during tissue morphogenesis.

  20. Epithelial cells as alternative human biomatrices for comet assay

    PubMed Central

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases. PMID:25506353

  1. Epithelial cells as alternative human biomatrices for comet assay.

    PubMed

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

  2. Clinical implications of epithelial cell plasticity in cancer progression.

    PubMed

    Aparicio, Luis A; Blanco, Moisés; Castosa, Raquel; Concha, Ángel; Valladares, Manuel; Calvo, Lourdes; Figueroa, Angélica

    2015-09-28

    In the last few years, the role of epithelial cell plasticity in cancer biology research has gained increasing attention. This concept refers to the ability of the epithelial cells to dynamically switch between different phenotypic cellular states. This programme is particularly relevant during the epithelial-to-mesenchymal transition (EMT) in cancer progression. During colonization, epithelial cells first activate the EMT programme to disseminate from a primary tumour to reach a distant tissue site. During this process, cells are transported into the circulation and are able to escape the immune system of the host. Then, a reverse process called mesenchymal-to-epithelial transition (MET) occurs on cells that settle in the distant organs. Although epithelial cell plasticity has an important impact on tumour biology, the clinical relevance of this concept remains to be recapitulated. In this review, we will update the current state of epithelial cell plasticity in cancer progression and its clinical implications for the design of therapeutic strategies, the acquisition of multidrug resistance, and future perspectives for the management of cancer patients.

  3. Gastrin stimulates MMP-1 expression in gastric epithelial cells: putative role in gastric epithelial cell migration.

    PubMed

    Kumar, J Dinesh; Steele, Islay; Moore, Andrew R; Murugesan, Senthil V; Rakonczay, Zoltan; Venglovecz, Viktoria; Pritchard, D Mark; Dimaline, Rodney; Tiszlavicz, Laszlo; Varro, Andrea; Dockray, Graham J

    2015-07-15

    The pyloric antral hormone gastrin plays a role in remodeling of the gastric epithelium, but the specific targets of gastrin that mediate these effects are poorly understood. Glandular epithelial cells of the gastric corpus express matrix metalloproteinase (MMP)-1, which is a potential determinant of tissue remodeling; some of these cells express the CCK-2 receptor at which gastrin acts. We have now examined the hypothesis that gastrin stimulates expression of MMP-1 in the stomach. We determined MMP-1 transcript abundance in gastric mucosal biopsies from Helicobacter pylori negative human subjects with normal gastric mucosal histology, who had a range of serum gastrin concentrations due in part to treatment with proton pump inhibitors (PPI). The effects of gastrin were studied on gastric epithelial AGS-GR cells using Western blot and migration assays. In human subjects with increased serum gastrin due to PPI usage, MMP-1 transcript abundance was increased 2-fold; there was also increased MMP-7 transcript abundance but not MMP-3. In Western blots, gastrin increased proMMP-1 abundance, as well that of a minor band corresponding to active MMP-1, in the media of AGS-GR cells, and the response was mediated by protein kinase C and p42/44 MAP kinase. There was also increased MMP-1 enzyme activity. Gastrin-stimulated AGS-GR cell migration in both scratch wound and Boyden chamber assays was inhibited by MMP-1 immunoneutralization. We conclude that MMP-1 expression is a target of gastrin implicated in mucosal remodeling.

  4. Genetics and epithelial cell dysfunction in cystic fibrosis

    SciTech Connect

    Riordan, J.R.; Buchwald, M.

    1987-01-01

    This book examines the advances being made in the study of the physiology, cell biology, and molecular genetics of cystic fibrosis. Emphasis is placed on various areas of research that involve epithelial cells (e.g., the CF-specific phenotypes exhibited by epithelial cells, abnormalities in epithelium ion transport, chloride channel regulation in CF epithelial.) Coverage is presented on the current status of CF, including data on the incidence of the disease, its mode of inheritance, chromosomal localization, genetic heterogeneity, and screening and management.

  5. Growth of corneal epithelial cells over in situ therapeutic contact lens after simple limbal epithelial transplantation (SLET).

    PubMed

    Bhalekar, Swapnil; Sangwan, Virender S; Basu, Sayan

    2013-06-27

    An 11-year-old boy underwent simple limbal epithelial transplantation (SLET) from the healthy right eye to his left eye for total limbal stem cell deficiency. One month later, corneal surface epithelialised and whitish plaques overlying the transplants were seen inferiorly. Those plaques were adherent to the surface of the contact lens and underlying corneal surface had smooth elevations. Similar findings were noted in a 23-year man following cyanoacrylate glue application for corneal perforation. On histological and immunohistochemical analysis, cells lining the contact lenses were identified as corneal epithelial cells. These cases illustrate epithelial cell growth on the contact lens and epithelial hyperplasia on corresponding surface of the cornea. Exorbitant proliferation of the epithelial cells may be owing to young age; therefore, early contact lens removal after SLET in young age, can possibly avoid epithelial hyperplasia. This also reiterates the possibility of using contact lens as a scaffold to grow epithelial cells.

  6. Polarized fibronectin secretion induced by adenosine regulates bacterial–epithelial interaction in human intestinal epithelial cells

    PubMed Central

    2004-01-01

    Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role

  7. Alignment of cell division axes in directed epithelial cell migration

    NASA Astrophysics Data System (ADS)

    Marel, Anna-Kristina; Podewitz, Nils; Zorn, Matthias; Oskar Rädler, Joachim; Elgeti, Jens

    2014-11-01

    Cell division is an essential dynamic event in tissue remodeling during wound healing, cancer and embryogenesis. In collective migration, tensile stresses affect cell shape and polarity, hence, the orientation of the cell division axis is expected to depend on cellular flow patterns. Here, we study the degree of orientation of cell division axes in migrating and resting epithelial cell sheets. We use microstructured channels to create a defined scenario of directed cell invasion and compare this situation to resting but proliferating cell monolayers. In experiments, we find a strong alignment of the axis due to directed flow while resting sheets show very weak global order, but local flow gradients still correlate strongly with the cell division axis. We compare experimental results with a previously published mesoscopic particle based simulation model. Most of the observed effects are reproduced by the simulations.

  8. Microfluidic approaches for epithelial cell layer culture and characterisation

    PubMed Central

    Thuenauer, Roland; Rodriguez-Boulan, Enrique; Römer, Winfried

    2014-01-01

    In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips, including methods to perform electrical impedance spectroscopy, methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry, techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress, and methods to carry out high-resolution imaging of vesicular trafficking with light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. PMID:24668405

  9. SWCNTs induced autophagic cell death in human bronchial epithelial cells.

    PubMed

    Park, Eun-Jung; Zahari, Nur Elida M; Lee, Eun-Woo; Song, Jaewhan; Lee, Jae-Hyeok; Cho, Myung-Haing; Kim, Jae-Ho

    2014-04-01

    Carbon nanotubes are being actively introduced in electronics, computer science, aerospace, and other industries. Thus, the urgent need for toxicological studies on CNTs is mounting. In this study, we investigated the alterations in cellular response with morphological changes induced by single-walled carbon nanotubes (SWCNTs) in BEAS-2B cells, a human bronchial epithelial cell line. At 24h after exposure, SWCNTs rapidly decreased ATP production and cell viability as well a slight increase in the number of cells in the subG1 and G1 phases. In addition, SWCNTs increased the expression of superoxide dismutase (SOD)-1, but not SOD-2, and the number of cells generating ROS. The concentration of Cu and Zn ions also increased in a dose-dependent manner in cells exposed to SWCNTs. SWCNTs significantly enhanced the release of nitric oxide, interleukin (IL)-6, and IL-8 and up-regulated the expression of chemokine- and cytokine-related genes. Furthermore, the levels of autophagy-related genes, especially the DRAM1 gene, and the autophagosome formation-related proteins, were clearly up-regulated together with an increase of autophagosome-like vacuoles. Based on these results, we suggest that SWCNTs induce autophagic cell death through mitochondrial dysfunction and cytosolic damage in human bronchial epithelial cells.

  10. Diversity of Epithelial Stem Cell Types in Adult Lung

    PubMed Central

    Li, Feng; He, Jinxi; Wei, Jun; Cho, William C.; Liu, Xiaoming

    2015-01-01

    Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays, in vitro and/or ex vivo models, and genetically engineered mice have suggested that these local epithelial stem/progenitor cells within spatially distinct regions along the pulmonary tree contribute to the injury repair of epithelium adjacent to their respective niches. This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer. PMID:25810726

  11. Stochastic Terminal Dynamics in Epithelial Cell Intercalation

    NASA Astrophysics Data System (ADS)

    Eule, Stephan; Metzger, Jakob; Reichl, Lars; Kong, Deqing; Zhang, Yujun; Grosshans, Joerg; Wolf, Fred

    2015-03-01

    We found that the constriction of epithelial cell contacts during intercalation in germ band extension in Drosophila embryos follows intriguingly simple quantitative laws. The mean contact length < L > follows < L > (t) ~(T - t) α , where T is the finite collapse time; the time dependent variance of contact length is proportional to the square of the mean; finally the time dependent probability density of the contact lengths remains close to Gaussian during the entire process. These observations suggest that the dynamics of contact collapse can be captured by a stochastic differential equation analytically tractable in small noise approximation. Here, we present such a model, providing an effective description of the non-equilibrium statistical mechanics of contact collapse. All model parameters are fixed by measurements of time dependent mean and variance of contact lengths. The model predicts the contact length covariance function that we obtain in closed form. The contact length covariance function closely matches experimental observations suggesting that the model well captures the dynamics of contact collapse.

  12. CUX1/Wnt signaling regulates Epithelial Mesenchymal Transition in EBV infected epithelial cells

    SciTech Connect

    Malizia, Andrea P.; Lacey, Noreen; Walls, Dermot; Egan, Jim J.; Doran, Peter P.

    2009-07-01

    Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix protein deposition. EBV, localised to alveolar epithelial cells of pulmonary fibrosis patients is associated with a poor prognosis. A strategy based on microarray-differential gene expression analysis to identify molecular drivers of EBV-associated lung fibrosis was utilized. Alveolar epithelial cells were infected with EBV to identify genes whose expression was altered following TGF{beta}1-mediated lytic phase. EBV lytic reactivation by TGF{beta}1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM{sub 1}81552) expression, inducing activation of non-canonical Wnt pathway mediators, implicating it in Epithelial Mesenchymal Transition (EMT), the molecular event underpinning scar production in tissue fibrosis. The role of EBV in EMT can be attenuated by antiviral strategies and inhibition of Wnt signaling by using All-Trans Retinoic Acids (ATRA). Activation of non-canonical Wnt signaling pathway by EBV in epithelial cells suggests a novel mechanism of EMT via CUX1 signaling. These data present a framework for further description of the link between infectious agents and fibrosis, a significant disease burden.

  13. The 18-kDa translocator protein (TSPO) disrupts mammary epithelial morphogenesis and promotes breast cancer cell migration.

    PubMed

    Wu, Xiaoting; Gallo, Kathleen A

    2013-01-01

    Mitochondria play important roles in cancer progression and have emerged as viable targets for cancer therapy. Increasing levels of the outer mitochondrial membrane protein, 18-kDa translocator protein (TSPO), are associated with advancing breast cancer stage. In particular, higher TSPO levels are found in estrogen receptor (ER)-negative breast tumors, compared with ER-positive tumors. In this study, we sought to define the roles of TSPO in the acquisition of breast cancer malignancy. Using a three-dimensional Matrigel culture system, we determined the impact of elevated TSPO levels on mammary epithelial morphogenesis. Our studies demonstrate that stable overexpression of TSPO in mammary epithelial MCF10A acini drives proliferation and provides partial resistance to luminal apoptosis, resulting in enlarged acinar structures with partially filled lumen that resemble early stage breast lesions leading to breast cancer. In breast cancer cell lines, TSPO silencing or TSPO overexpression significantly altered the migratory activity. In addition, we found that combination treatment with the TSPO ligands (PK 11195 or Ro5-4864) and lonidamine, a clinical phase II drug targeting mitochondria, decreased viability of ER-negative breast cancer cell lines. Taken together, these data demonstrate that increases in TSPO levels at different stages of breast cancer progression results in the acquisition of distinct properties associated with malignancy. Furthermore, targeting TSPO, particularly in combination with other mitochondria-targeting agents, may prove useful for the treatment of ER-negative breast cancer.

  14. The Src kinase Yes is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters, but not pancreatic growth factors, which stimulate its association with numerous other signaling molecules.

    PubMed

    Sancho, Veronica; Nuche-Berenguer, Bernardo; Jensen, R T

    2012-08-01

    For growth factors, cytokines, G-protein-coupled receptors and numerous other stimuli, the Src Family of kinases (SFK) play a central signaling role. SFKs also play an important role in pancreatic acinar cell function including metabolism, secretion, endocytosis, growth and cytoskeletal integrity, although the specific SFKs involved are not fully known. In the present study we used specific antibodies for the SFK, Yes, to determine its presence, activation by pancreatic secretagogues or growth factors, and interaction with cellular signaling cascades mediated by CCK in which Yes participates in to cause acinar cell responses. Yes was identified in acini and secretagogues known to activate phospholipase C (PLC) [CCK, carbachol, bombesin] as well as post-receptor stimulants activating PKC [TPA] or mobilizing cellular calcium [thapsigargin/calcium ionophore (A23187)] each activated Yes. Secretin, which activates adenylate cyclase did not stimulate Yes, nor did pancreatic growth factors. CCK activation of Yes required both high- and low-affinity CCK(1)-receptor states. TPA-/CCK-stimulated Yes activation was completely inhibited by thapsigargin and the PKC inhibitor, GF109203X. CCK/TPA stimulated the association of Yes with focal adhesion kinases (Pyk2, FAK) and its autophosphorylated forms (pY397FAK, pY402Pyk2). Moreover, CCK/TPA stimulated Yes interacted with a number of other signaling proteins, including Shc, PKD, p130(Cas), PI3K and PTEN. This study demonstrates that in rat pancreatic acini, the SFK member Yes is expressed and activated by CCK and other gastrointestinal hormones/neurotransmitters. Because its activation results in the direct activation of many cellular signaling cascades that have been shown to mediate CCK's effect in acinar cell function our results suggest that it is one of the important pancreatic SFKs mediating these effects.

  15. Characteristics and EGFP expression of porcine mammary gland epithelial cells.

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

    The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line.

  16. The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells.

    PubMed

    Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

    2015-01-01

    The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival.

  17. The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells

    PubMed Central

    Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

    2015-01-01

    The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival. PMID:25616580

  18. Ionizing radiation predisposes non-malignant human mammaryepithelial cells to undergo TGF beta-induced epithelial to mesenchymaltransition

    SciTech Connect

    Andarawewa, Kumari L.; Erickson, Anna C.; Chou, William S.; Costes, Sylvain; Gascard, Philippe; Mott, Joni D.; Bissell, Mina J.; Barcellos-Hoff, Mary Helen

    2007-04-06

    Transforming growth factor {beta}1 (TGF{beta}) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGF{beta}, activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGF{beta}-mediated epithelial to mesenchymal transition (EMT). Non-malignant HMEC (MCF10A, HMT3522 S1 and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture, or treated with a low concentration of TGF{beta} (0.4 ng/ml), or double-treated. All double-treated (IR+TGF{beta}) HMEC underwent a morphological shift from cuboidal to spindle-shaped. This phenotype was accompanied by decreased expression of epithelial markers E-cadherin, {beta}-catenin and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin and vimentin. Furthermore, double-treatment increased cell motility, promoted invasion and disrupted acinar morphogenesis of cells subsequently plated in Matrigel{trademark}. Neither radiation nor TGF{beta} alone elicited EMT, even though IR increased chronic TGF{beta} signaling and activity. Gene expression profiling revealed that double treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes non-malignant HMEC to undergo TGF{beta}-mediated EMT. Consistent with this, Erk phosphorylation were transiently induced by irradiation, persisted in irradiated cells treated with TGF{beta}, and treatment with U0126, a Mek inhibitor, blocked the EMT phenotype. Together, these data demonstrate that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

  19. Airway epithelial cell wound repair mediated by alpha-dystroglycan.

    PubMed

    White, S R; Wojcik, K R; Gruenert, D; Sun, S; Dorscheid, D R

    2001-02-01

    Dystroglycans (DGs) bind laminin matrix proteins in skeletal and cardiac muscle and are expressed in other nonmuscle tissues. However, their expression in airway epithelial cells has not been demonstrated. We examined expression of DGs in the human airway epithelial cell line 1HAEo(-), and in human primary airway epithelial cells. Expression of the common gene for alpha- and beta-DG was demonstrated by reverse transcriptase/ polymerase chain reaction in 1HAEo(-) cells. Protein expression of beta-DG was demonstrated by both Western blot and flow cytometry in cultured cells. Localization of alpha-DG, using both a monoclonal antibody and the alpha-DG binding lectin wheat-germ agglutinin (WGA), was to the cell membrane and nucleus. We then examined the function of DGs in modulating wound repair over laminin matrix. Blocking alpha-DG binding to laminin in 1HAEo(-) monolayers using either glycosyaminoglycans or WGA attenuated cell migration and spreading after mechanical injury. alpha-DG was not expressed in epithelial cells at the wound edge immediately after wound creation, but localized to the cell membrane in these cells within 12 h of injury. These data demonstrate the presence of DGs in airway epithelium. alpha-DG is dynamically expressed and serves as a lectin to bind laminin during airway epithelial cell repair.

  20. Ion pump sorting in polarized renal epithelial cells.

    PubMed

    Caplan, M J

    2001-08-01

    The plasma membranes of renal epithelial cells are divided into distinct apical and basolateral domains, which contain different inventories of ion transport proteins. Without this polarity vectorial ion and fluid transport would not be possible. Little is known of the signals and mechanisms that renal epithelial cells use to establish and maintain polarized distributions of their ion transport proteins. Analysis of ion pump sorting reveals that multiple complex signals participate in determining and regulating these proteins' subcellular localizations.

  1. Secretion of alpha 1-antitrypsin by alveolar epithelial cells.

    PubMed

    Venembre, P; Boutten, A; Seta, N; Dehoux, M S; Crestani, B; Aubier, M; Durand, G

    1994-06-13

    We have investigated the ability of alveolar epithelial cells (human A549 cell line and rat type-II pneumocytes) to produce alpha 1-antitrypsin (AAT). Northern blot analysis demonstrated the presence of an AAT-specific mRNA transcript in A549 cells. Unstimulated A549 cells secreted immunoreactive AAT at a rate of 0.51 +/- 0.04 ng/10(6) cells/h, with a modified glycosylation compared to serum AAT. AAT formed a complex with neutrophil elastase. Rat type-II pneumocytes secreted immunoreactive AAT. Our results suggest that alveolar epithelial cells could participate in antiprotease defense within the lung through local AAT production.

  2. Metformin inhibits the proliferation of benign prostatic epithelial cells

    PubMed Central

    Ge, Rongbin; Li, Jijun; Johnson, Cameron W.; Rassoulian, Cyrus; Olumi, Aria F.

    2017-01-01

    Objective Benign prostatic hyperplasia (BPH) is the most common proliferative abnormality of the prostate affecting elderly men throughout the world. Epidemiologic studies have shown that diabetes significantly increases the risk of developing BPH, although whether anti-diabetic medications preventing the development of BPH remains to be defined. We have previously found that stromally expressed insulin-like growth factor 1 (IGF-1) promotes benign prostatic epithelial cell proliferation through paracrine mechanisms. Here, we seek to understand if metformin, a first line medication for the treatment of type 2 diabetes, inhibits the proliferation of benign prostatic epithelial cells through reducing the expression of IGF-1 receptor (IGF-1R) and regulating cell cycle. Methods BPE cell lines BPH-1 and P69, murine fibroblasts3T3 and primary human prostatic fibroblasts were cultured and tested in this study. Cell proliferation and the cell cycle were analyzed by MTS assay and flow cytometry, respectively. The expression of IGF-1R was determined by western-blot and immunocytochemistry. The level of IGF-1 secretion in culture medium was measured by ELISA. Results Metformin (0.5-10mM, 6-48h) significantly inhibited the proliferation of BPH-1 and P69 cells in a dose-dependent and time-dependent manner. Treatment with metformin for 24 hours lowered the G2/M cell population by 43.24% in P69 cells and 24.22% in BPH-1 cells. On the other hand, IGF-1 (100ng/mL, 24h) stimulated the cell proliferation (increased by 28.81% in P69 cells and 20.95% in BPH-1 cells) and significantly enhanced the expression of IGF-1R in benign prostatic epithelial cells. Metformin (5mM) abrogated the proliferation of benign prostatic epithelial cells induced by IGF-1. In 3T3 cells, the secretion of IGF-1 was significantly inhibited by metformin from 574.31pg/ml to 197.61pg/ml. The conditioned media of 3T3 cells and human prostatic fibroblasts promoted the proliferation of epithelial cells and the

  3. Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

    SciTech Connect

    Hirai, H.; Lochter, A.; Galosy, S.; Koshida, S.; Niwa, S.; Bissell, M.J.

    1997-10-13

    Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

  4. Inhibition of corneal epithelial cell migration by cadmium and mercury

    SciTech Connect

    Ubels, J.L.; Osgood, T.B. Medical Coll. of Wisconsin, Milwaukee )

    1991-02-01

    In a previous comparative study of corneal healing in fish, the authors observed that corneal epithelial healing occurs very rapidly in vivo in the marine teleost Myoxocephalus octodecimspinosus (longhorn sculpin) with a 6-mm diameter wound on the mammalian cornea. This rapid healing which permits prompt restoration of the epithelial barrier is apparently an adaptation to the large ionic and osmotic gradients between the environment and the intraocular fluids of the fish. These observations suggested that epithelial healing in the sculpin cornea might be useful model in aquatic biomedical toxicology if an in vitro method for measurement of healing rates could be developed. In this report the authors demonstrate that sculpin eyes maintained in short-term organ culture have a rapid corneal epithelial healing response and that this model can be used to demonstrate the toxic effects of heavy metals on epithelial cell migration.

  5. Left-right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-12-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left-right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left-right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left-right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction.

  6. Characteristics and EGFP expression of goat mammary gland epithelial cells.

    PubMed

    Zheng, Y-M; He, X-Y; Zhang, Y

    2010-12-01

    The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing.

  7. Differentiation of Club Cells to Alveolar Epithelial Cells In Vitro

    PubMed Central

    Zheng, Dahai; Soh, Boon-Seng; Yin, Lu; Hu, Guangan; Chen, Qingfeng; Choi, Hyungwon; Han, Jongyoon; Chow, Vincent T. K.; Chen, Jianzhu

    2017-01-01

    Club cells are known to function as regional progenitor cells to repair the bronchiolar epithelium in response to lung damage. By lineage tracing in mice, we have shown recently that club cells also give rise to alveolar type 2 cells (AT2s) and alveolar type 1 cells (AT1s) during the repair of the damaged alveolar epithelium. Here, we show that when highly purified, anatomically and phenotypically confirmed club cells are seeded in 3-dimensional culture either in bulk or individually, they proliferate and differentiate into both AT2- and AT1-like cells and form alveolar-like structures. This differentiation was further confirmed by transcriptomic analysis of freshly isolated club cells and their cultured progeny. Freshly isolated club cells express Sca-1 and integrin α6, markers commonly used to characterize lung stem/progenitor cells. Together, current study for the first time isolated highly purified club cells for in vitro study and demonstrated club cells’ capacity to differentiate into alveolar epithelial cells at the single-cell level. PMID:28128362

  8. DA-6034 Induces [Ca(2+)]i Increase in Epithelial Cells.

    PubMed

    Yang, Yu-Mi; Park, Soonhong; Ji, Hyewon; Kim, Tae-Im; Kim, Eung Kweon; Kang, Kyung Koo; Shin, Dong Min

    2014-04-01

    DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca(2+) signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca(2+) signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca(2+)-activated Cl(-) channels (CaCCs) and increased intracellular calcium concentrations ([Ca(2+)]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca(2+)]i in mouse salivary gland cells and human corneal epithelial cells. [Ca(2+)]i increase of DA-6034 was dependent on the Ca(2+) entry from extracellular and Ca(2+) release from internal Ca(2+) stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca(2+) stores. These results suggest that DA-6034 induces Ca(2+) signaling via extracellular Ca(2+) entry and RyRs-sensitive Ca(2+) release from internal Ca(2+) stores in epithelial cells.

  9. Gastrin stimulates MMP-1 expression in gastric epithelial cells: putative role in gastric epithelial cell migration

    PubMed Central

    Kumar, J. Dinesh; Steele, Islay; Moore, Andrew R.; Murugesan, Senthil V.; Rakonczay, Zoltan; Venglovecz, Viktoria; Pritchard, D. Mark; Dimaline, Rodney; Tiszlavicz, Laszlo; Varro, Andrea

    2015-01-01

    The pyloric antral hormone gastrin plays a role in remodeling of the gastric epithelium, but the specific targets of gastrin that mediate these effects are poorly understood. Glandular epithelial cells of the gastric corpus express matrix metalloproteinase (MMP)-1, which is a potential determinant of tissue remodeling; some of these cells express the CCK-2 receptor at which gastrin acts. We have now examined the hypothesis that gastrin stimulates expression of MMP-1 in the stomach. We determined MMP-1 transcript abundance in gastric mucosal biopsies from Helicobacter pylori negative human subjects with normal gastric mucosal histology, who had a range of serum gastrin concentrations due in part to treatment with proton pump inhibitors (PPI). The effects of gastrin were studied on gastric epithelial AGS-GR cells using Western blot and migration assays. In human subjects with increased serum gastrin due to PPI usage, MMP-1 transcript abundance was increased 2-fold; there was also increased MMP-7 transcript abundance but not MMP-3. In Western blots, gastrin increased proMMP-1 abundance, as well that of a minor band corresponding to active MMP-1, in the media of AGS-GR cells, and the response was mediated by protein kinase C and p42/44 MAP kinase. There was also increased MMP-1 enzyme activity. Gastrin-stimulated AGS-GR cell migration in both scratch wound and Boyden chamber assays was inhibited by MMP-1 immunoneutralization. We conclude that MMP-1 expression is a target of gastrin implicated in mucosal remodeling. PMID:25977510

  10. PP2C phosphatase activity is coupled to cAMP-mediated pathway in rat parotid acinar cells.

    PubMed

    Yokoyama, N; Kobayashi, T; Tamura, S; Sugiya, H

    1995-07-01

    A 26 kDa particulate protein is phosphorylated during stimulation of amylase secretion by a beta-adrenergic agonist in the rat parotid gland. Previous study has shown that PP2C phosphatase is involved in dephosphorylation of this 26 kDa protein [Yokoyama, N. et al. (1994) Biochem. Biophys. Res. Commun. 200, 497-503]. In this study, immunotransblot analysis using anti-PP2C phosphatase antibody showed that PP2C phosphatase was found prominently in the cystolic fractions and less in secretory granule membranes. When cells were stimulated by isoproterenol, cytosolic PP2C phosphatase activity increased to 145% at 5 min and returned to basal level at 30 min. Forskolin increased PP2C phosphatase activity. H89 inhibited increase of PP2C phosphatase activity following beta-adrenergic stimulation. These results suggest that PP2C phosphatase activity is regulated by cAMP-mediated signaling following beta-adrenergic stimulation and participates in dephosphorylation of this 26 kDa protein.

  11. Probiotics promote endocytic allergen degradation in gut epithelial cells

    SciTech Connect

    Song, Chun-Hua; Liu, Zhi-Qiang; Huang, Shelly; Zheng, Peng-Yuan; Yang, Ping-Chang

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Knockdown of A20 compromised the epithelial barrier function. Black-Right-Pointing-Pointer The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Black-Right-Pointing-Pointer Antigens transported across A20-deficient HT-29 monolayers conserved antigenicity. Black-Right-Pointing-Pointer Probiotic proteins increased the expression of A20 in HT-29 cells. -- Abstract: Background and aims: Epithelial barrier dysfunction plays a critical role in the pathogenesis of allergic diseases; the mechanism is to be further understood. The ubiquitin E3 ligase A20 (A20) plays a role in the endocytic protein degradation in the cells. This study aims to elucidate the role of A20 in the maintenance of gut epithelial barrier function. Methods: Gut epithelial cell line, HT-29 cell, was cultured into monolayers to evaluate the barrier function in transwells. RNA interference was employed to knock down the A20 gene in HT-29 cells to test the role of A20 in the maintenance of epithelial barrier function. Probiotic derived proteins were extracted from the culture supernatants using to enhance the expression of A20 in HT-29 cells. Results: The results showed that the knockdown of A20 compromised the epithelial barrier function in HT-29 monolayers, mainly increased the intracellular permeability. The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Allergens collected from the transwell basal chambers of A20-deficient HT-29 monolayers still conserved functional antigenicity. Treating with probiotic derived proteins increased the expression of A20 in HT-29 cells and promote the barrier function. Conclusion: A20 plays an important role in the maintenance of epithelial barrier function as shown by HT-29 monolayer. Probiotic derived protein increases the expression of A20 and promote the HT-29 monolayer barrier function.

  12. Epithelial cell guidance by self-generated EGF gradients†

    PubMed Central

    Scherber, Cally; Aranyosi, Alexander J.; Kulemann, Birte; Thayer, Sarah P.; Toner, Mehmet; Iliopoulos, Othon

    2012-01-01

    Cancer epithelial cells often migrate away from the primary tumor to invade into the surrounding tissues. Their migration is commonly assumed to be directed by pre-existent spatial gradients of chemokines and growth factors in the target tissues. Unexpectedly however, we found that the guided migration of epithelial cells is possible in vitro in the absence of pre-existent chemical gradients. We observed that both normal and cancer epithelial cells can migrate persistently and reach the exit along the shortest path from microscopic mazes filled with uniform concentrations of media. Using microscale engineering techniques and biophysical models, we uncovered a self-guidance strategy during which epithelial cells generate their own guiding cues under conditions of biochemical confinement. The self-guidance strategy depends on the balance between three interdependent processes: epidermal growth factor (EGF) uptake by the cells (U), the restricted transport of EGF through the structured microenvironment (T), and cell chemotaxis toward the resultant EGF gradients (C). The UTC self-guidance strategy can be perturbed by inhibition of signalling through EGF-receptors and appears to be independent from chemokine signalling. Better understanding of the UTC self-guidance strategy could eventually help devise new ways for modulating epithelial cell migration and delaying cancer cell invasion or accelerating wound healing. PMID:22314635

  13. Lingual Epithelial Stem Cells and Organoid Culture of Them

    PubMed Central

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-01

    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine. PMID:26828484

  14. Starved epithelial cells uptake extracellular matrix for survival

    PubMed Central

    Muranen, Taru; Iwanicki, Marcin P.; Curry, Natasha L.; Hwang, Julie; DuBois, Cory D.; Coloff, Jonathan L.; Hitchcock, Daniel S.; Clish, Clary B.; Brugge, Joan S.; Kalaany, Nada Y.

    2017-01-01

    Extracellular matrix adhesion is required for normal epithelial cell survival, nutrient uptake and metabolism. This requirement can be overcome by oncogene activation. Interestingly, inhibition of PI3K/mTOR leads to apoptosis of matrix-detached, but not matrix-attached cancer cells, suggesting that matrix-attached cells use alternate mechanisms to maintain nutrient supplies. Here we demonstrate that under conditions of dietary restriction or growth factor starvation, where PI3K/mTOR signalling is decreased, matrix-attached human mammary epithelial cells upregulate and internalize β4-integrin along with its matrix substrate, laminin. Endocytosed laminin localizes to lysosomes, results in increased intracellular levels of essential amino acids and enhanced mTORC1 signalling, preventing cell death. Moreover, we show that starved human fibroblasts secrete matrix proteins that maintain the growth of starved mammary epithelial cells contingent upon epithelial cell β4-integrin expression. Our study identifies a crosstalk between stromal fibroblasts and epithelial cells under starvation that could be exploited therapeutically to target tumours resistant to PI3K/mTOR inhibition. PMID:28071763

  15. Development of human epithelial cell systems for radiation risk assessment

    NASA Astrophysics Data System (ADS)

    Yang, C. H.; Craise, L. M.

    1994-10-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-LET radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic transformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

  16. Development of human epithelial cell systems for radiation risk assessment

    NASA Technical Reports Server (NTRS)

    Yang, C. H.; Craise, L. M.

    1994-01-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-Linear Energy Transfer (LET) radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic tranformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

  17. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

    The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

  18. Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature-responsive closed culture device.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Kikuchi, Tetsutaro; Kitano, Yuriko; Watanabe, Hiroya; Mizutani, Manabu; Nozaki, Takayuki; Senda, Naoko; Saitoh, Kazuo; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-05-01

    Temperature-responsive culture surfaces make it possible to harvest transplantable carrier-free cell sheets. Here, we applied temperature-responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony-forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature-responsive closed culture devices. The results were similar to those obtained using temperature-responsive culture inserts. These results indicate that the novel temperature-responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets.

  19. Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling

    PubMed Central

    Kamimoto, Kenji; Kaneko, Kota; Kok, Cindy Yuet-Yin; Okada, Hajime; Miyajima, Atsushi; Itoh, Tohru

    2016-01-01

    Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity. DOI: http://dx.doi.org/10.7554/eLife.15034.001 PMID:27431614

  20. Cholera toxin stimulation of human mammary epithelial cells in culture

    SciTech Connect

    Stampfer, M.R.

    1982-06-01

    Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system.

  1. Vulnerability of Normal Human Mammary Epithelial Cells to Oncogenic Transformation

    DTIC Science & Technology

    2012-04-01

    diverse transformed HMEC lines with defined genetic alterations may aid the identification of potential therapeutic treatments , including...human model systems to test potential therapeutics, could facilitate individualized treatment and possibly prevention. The main variables thought to...epithelial cells. Middle, corresponding cell culture models used in this study. Red, treatment or genetic manipulation used. Cell models are described in

  2. Mechanobiology in Lung Epithelial Cells: Measurements, Perturbations, and Responses

    PubMed Central

    Waters, Christopher M.; Roan, Esra; Navajas, Daniel

    2015-01-01

    Epithelial cells of the lung are located at the interface between the environment and the organism and serve many important functions including barrier protection, fluid balance, clearance of particulate, initiation of immune responses, mucus and surfactant production, and repair following injury. Because of the complex structure of the lung and its cyclic deformation during the respiratory cycle, epithelial cells are exposed to continuously varying levels of mechanical stresses. While normal lung function is maintained under these conditions, changes in mechanical stresses can have profound effects on the function of epithelial cells and therefore the function of the organ. In this review, we will describe the types of stresses and strains in the lungs, how these are transmitted, and how these may vary in human disease or animal models. Many approaches have been developed to better understand how cells sense and respond to mechanical stresses, and we will discuss these approaches and how they have been used to study lung epithelial cells in culture. Understanding how cells sense and respond to changes in mechanical stresses will contribute to our understanding of the role of lung epithelial cells during normal function and development and how their function may change in diseases such as acute lung injury, asthma, emphysema, and fibrosis. PMID:23728969

  3. Ozone exposed epithelial cells modify cocultured natural killer cells

    PubMed Central

    Müller, Loretta; Brighton, Luisa E.

    2013-01-01

    Ozone (O3) causes significant adverse health effects worldwide. Nasal epithelial cells (NECs) are among the first sites within the respiratory system to be exposed to inhaled air pollutants. They recruit, activate, and interact with immune cells via soluble mediators and direct cell-cell contacts. Based on our recent observation demonstrating the presence of natural killer (NK) cells in nasal lavages, the goal of this study was to establish a coculture model of NECs and NK cells and examine how exposure to O3 modifies this interaction. Flow cytometry analysis was used to assess immunophenotypes of NK cells cocultured with either air- or O3-exposed NECs. Our data show that coculturing NK cells with O3-exposed NECs decreased intracellular interferon-γ (IFN-γ), enhanced, albeit not statistically significant, IL-4, and increased CD16 expression on NK cells compared with air controls. Additionally, the cytotoxicity potential of NK cells was reduced after coculturing with O3-exposed NECs. To determine whether soluble mediators released by O3-exposed NECs caused this shift, apical and basolateral supernatants of air- and O3-exposed NECs were used to stimulate NK cells. While the conditioned media of O3-exposed NECs alone did not reduce intracellular IFN-γ, O3 enhanced the expression of NK cell ligands ULBP3 and MICA/B on NECs. Blocking ULBP3 and MICA/B reversed the effects of O3-exposed NECs on IFN-γ production in NK cells. Taken together, these data showed that interactions between NECs and NK cells in the context of O3 exposure changes NK cell activity via direct cell-cell interactions and is dependent on ULBP3/MICA/B expressed on NECs. PMID:23241529

  4. Cells of Origin of Epithelial Ovarian Cancers

    DTIC Science & Technology

    2015-09-01

    lethal malignancy of the female reproductive system, largely due to the fact that most EOCs are diagnosed only after the cancer has metastasized into the...Epithelial ovarian cancer (EOC) is the most lethal malignancy of the female reproductive system, largely due to the fact that most EOCs are diagnosed only

  5. Epithelial cell division in the Xenopus laevis embryo during gastrulation.

    PubMed

    Hatte, Guillaume; Tramier, Marc; Prigent, Claude; Tassan, Jean-Pierre

    2014-01-01

    How vertebrate epithelial cells divide in vivo and how the cellular environment influences cell division is currently poorly understood. A sine qua non condition to study cell division in situ is the ease of observation of cell division. This is fulfilled in the Xenopus embryo at the gastrula stage where polarized epithelial cells divide with a high frequency at the surface of the organism. Recently, using this model system, we have shown that epithelial cells divide by asymmetric furrowing and that the mode of cell division is regulated during development. Here, we further characterize epithelial cell division in situ. To this end, we used confocal microscopy to study epithelial cell division in the ectoderm of the Xenopus laevis gastrula. Cell division was followed either by indirect immunofluorescence in fixed embryos or by live imaging of embryos transiently expressing diverse fluorescent proteins. Here, we show that during cytokinesis, the plasma membranes of the two daughter cells are usually separated by a gap. For most divisions, daughter cells make contacts basally at a distance from the furrow tip which creates an inverted teardrop-like shaped volume tightly associated with the furrow. At the end of cytokinesis, the inverted teardrop is resorbed; thus it is a transient structure. Several proteins involved in cytokinesis are localized at the tip of the inverted teardrop suggesting that the formation of the gap could be an active process. We also show that intercalation of neighboring cells between daughter cells occasionally occurs during cytokinesis. Our results reveal an additional level of complexity in the relationship between dividing cells and also with their neighboring cells during cytokinesis in the Xenopus embryo epithelium.

  6. Epithelial-mesenchymal transitions of bile duct epithelial cells in primary hepatolithiasis.

    PubMed

    Zhao, Lijin; Yang, Rigao; Cheng, Long; Wang, Maijian; Jiang, Yan; Wang, Shuguang

    2010-07-01

    The purpose of this study was to explore the role of epithelial-mesenchymal transition in the pathogenesis of hepatolithiasis. Thirty-one patients with primary hepatolithiasis were enrolled in this study. Expressions of E-cadherin, alpha-catenin, alpha-SMA, vimentin, S100A4, TGF-beta1 and P-smad2/3 in hepatolithiasis bile duct epithelial cells were examined by immunohistochemistry staining. The results showed that the expressions of the epithelial markers E-cadherin and alpha-catenin were frequently lost in hepatolithiasis (32.3% and 25.9% of cases, respectively), while the mesenchymal markers vimentin, alpha-SMA and S100A4 were found to be present in hepatolithiasis (35.5%, 29.0%, and 32.3% of cases, respectively). The increased mesenchymal marker expression was correlated with decreased epithelial marker expression. The expressions of TGF-beta1 and P-smad2/3 in hepatolithiasis were correlated with the expression of S100A4. These data indicate that TGF-beta1-mediated epithelial-mesenchymal transition might be involved in the formation of hepatolithiasis.

  7. Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

    SciTech Connect

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2008-06-26

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a 'cuboidal' epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-{beta}-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.

  8. Secretion of IL-13 by airway epithelial cells enhances epithelial repair via HB-EGF.

    PubMed

    Allahverdian, Sima; Harada, Norihiro; Singhera, Gurpreet K; Knight, Darryl A; Dorscheid, Delbert R

    2008-02-01

    Inappropriate repair after injury to the epithelium generates persistent activation, which may contribute to airway remodeling. In the present study we hypothesized that IL-13 is a normal mediator of airway epithelial repair. Mechanical injury of confluent airway epithelial cell (AEC) monolayers induced expression and release of IL-13 in a time-dependent manner coordinate with repair. Neutralizing of IL-13 secreted from injured epithelial cells by shIL-13Ralpha2.FC significantly reduced epithelial repair. Moreover, exogenous IL-13 enhanced epithelial repair and induced epidermal growth factor receptor (EGFR) phosphorylation. We examined secretion of two EGFR ligands, epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), after mechanical injury. Our data showed a sequential release of the EGF and HB-EGF by AEC after injury. Interestingly, we found that IL-13 induces HB-EGF, but not EGF, synthesis and release from AEC. IL-13-induced EGFR phosphorylation and the IL-13-reparative effect on AEC are mediated via HB-EGF. Finally, we demonstrated that inhibition of EGFR tyrosine kinase activity by tyrphostin AG1478 increases IL-13 release after injury, suggesting negative feedback between EGFR and IL-13 during repair. Our data, for the first time, showed that IL-13 plays an important role in epithelial repair, and that its effect is mediated through the autocrine release of HB-EGF and activation of EGFR. Dysregulation of EGFR phosphorylation may contribute to a persistent repair phenotype and chronically increased IL-13 release, and in turn result in airway remodeling.

  9. Biomaterial surface proteomic signature determines interaction with epithelial cells.

    PubMed

    Abdallah, Mohamed-Nur; Tran, Simon D; Abughanam, Ghada; Laurenti, Marco; Zuanazzi, David; Mezour, Mohamed A; Xiao, Yizhi; Cerruti, Marta; Siqueira, Walter L; Tamimi, Faleh

    2017-03-01

    Cells interact with biomaterials indirectly through extracellular matrix (ECM) proteins adsorbed onto their surface. Accordingly, it could be hypothesized that the surface proteomic signature of a biomaterial might determine its interaction with cells. Here, we present a surface proteomic approach to test this hypothesis in the specific case of biomaterial-epithelial cell interactions. In particular, we determined the surface proteomic signature of different biomaterials exposed to the ECM of epithelial cells (basal lamina). We revealed that the biomaterial surface chemistry determines the surface proteomic profile, and subsequently the interaction with epithelial cells. In addition, we found that biomaterials with surface chemistries closer to that of percutaneous tissues, such as aminated PMMA and aminated PDLLA, promoted higher selective adsorption of key basal lamina proteins (laminins, nidogen-1) and subsequently improved their interactions with epithelial cells. These findings suggest that mimicking the surface chemistry of natural percutaneous tissues can improve biomaterial-epithelial integration, and thus provide a rationale for the design of improved biomaterial surfaces for skin regeneration and percutaneous medical devices.

  10. Fibrin glue inhibits migration of ocular surface epithelial cells.

    PubMed

    Yeung, A M; Faraj, L A; McIntosh, O D; Dhillon, V K; Dua, H S

    2016-10-01

    PurposeFibrin glue has been used successfully in numerous ophthalmic surgical procedures. Recently, fibrin glue has been used in limbal stem cell transplantation to reduce both operative time and to negate the need for sutures. The aim of this study was to determine the effects of fibrin glue on epithelial cell migration in vitro.MethodsCorneoscleral rims were split to retain the epithelial layer, Bowman's layer, and anterior stroma. Rims were cut into eight equal-sized pieces and were placed directly on culture plates or affixed with fibrin glue. Rims were maintained in culture for 25 days and epithelial cell growth was monitored. Cells were photographed to measure area or growth and immunofluorescence staining of explants for fibrin was performed.ResultsExplants that were glued demonstrated significantly delayed epithelial cell growth and migration as compared with explants without glue. By day 16, all fibrin glue had dissolved and coincided with onset of cell growth from glued explants. Cell growth commenced between days 3 and 4 for control explants without glue and around days 14-16 for explants with fibrin glue.ConclusionsFibrin glue delays epithelial cell migration by acting as a physical barrier and can potentially interfere with explant-derived limbal epithelial cell migration on to the corneal surface. We propose that glue should be used to attach the conjunctival frill of the limbal explant but care should be taken to ensure that the glue does not wrap around the explant if used to secure the explant as well. Strategic use of glue, to attach the recessed conjunctiva, can be advantageous in delaying conjunctival cell migration and reducing the need for sequential sector conjunctival epitheliectomy.

  11. Salivary epithelial cells: an unassuming target site for gene therapeutics

    PubMed Central

    Perez, Paola; Rowzee, Anne M.; Zheng, Changyu; Adriaansen, Janik; Baum, Bruce J.

    2010-01-01

    Salivary glands are classical exocrine glands whose external secretions result in the production of saliva. However, in addition to the secretion of exocrine proteins, salivary epithelial cells are also capable of secreting proteins internally, into the bloodstream. This brief review examines the potential for using salivary epithelial cells as a target site for in situ gene transfer, with an ultimate goal of producing therapeutic proteins for treating both systemic and upper gastrointestinal tract disorders. The review discusses the protein secretory pathways reported to be present in salivary epithelial cells, the viral gene transfer vectors shown useful for transducing these cells, model transgenic secretory proteins examined, and some clinical conditions that might benefit from such salivary gland gene transfer. PMID:20219693

  12. Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils.

    PubMed

    Hosoki, Koa; Kainuma, Keigo; Toda, Masaaki; Harada, Etsuko; Chelakkot-Govindalayathila, Ayshwarya-Lakshmi; Roeen, Ziaurahman; Nagao, Mizuho; D'Alessandro-Gabazza, Corina N; Fujisawa, Takao; Gabazza, Esteban C

    2014-07-04

    Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.

  13. Epithelial cell apoptosis facilitates Entamoeba histolytica infection in the gut.

    PubMed

    Becker, Stephen M; Cho, Kyou-Nam; Guo, Xiaoti; Fendig, Kirsten; Oosman, Mohammed N; Whitehead, Robert; Cohn, Steven M; Houpt, Eric R

    2010-03-01

    Entamoeba histolytica is the protozoan parasite that causes amebic colitis. The parasite triggers apoptosis on contact with host cells; however, the biological significance of this event during intestinal infection is unclear. We examined the role of apoptosis in a mouse model of intestinal amebiasis. Histopathology revealed that abundant epithelial cell apoptosis occurred in the vicinity of amoeba in histological specimens. Epithelial cell apoptosis occurred rapidly on co-culture with amoeba in vitro as measured by annexin positivity, DNA degradation, and mitochondrial dysfunction. Administration of the pan caspase inhibitor ZVAD decreased the rate and severity of amebic infection in CBA mice by all measures (cecal culture positivity, parasite enzyme-linked immunosorbent assay, and histological scores). Similarly, caspase 3 knockout mice on the resistant C57BL/6 background exhibited even lower cecal parasite antigen burden and culture positive rates than wild type mice. The permissive effect of apoptosis on infection could be tracked to the epithelium, in that transgenic mice that overexpressed Bcl-2 in epithelial cells were more resistant to infection as measured by cecal parasite enzyme-linked immunosorbent assay and histological scores. We concluded that epithelial cell apoptosis in the intestine facilitates amebic infection in this mouse model. The parasite's strategy for inducing apoptosis may point to key virulence factors, and therapeutic maneuvers to diminish epithelial apoptosis may be useful in amebic colitis.

  14. Immunolocalization of epithelial and mesenchymal matrix constituents in association with inner enamel epithelial cells.

    PubMed

    Bosshardt, D D; Nanci, A

    1998-02-01

    After crown formation, the enamel organ reorganizes into Hertwig's epithelial root sheath (HERS). Although it is generally accepted that HERS plays an inductive role during root formation, it also has been suggested that it may contribute enamel-related proteins to cementum matrix. By analogy to the enamel-free area (EFA) in rat molars, in which epithelial cells express not only enamel proteins but also "typical" mesenchymal matrix constituents, it has been proposed that HERS cells may also have the potential to produce cementum proteins. To test this hypothesis, we examined the nature of the first matrix layer deposited along the cervical portion of root dentin and the characteristics of the associated cells. Rat molars were processed for postembedding colloidal gold immunolabeling with antibodies to amelogenin (AMEL), ameloblastin (AMBN), bone sialoprotein (BSP), and osteopontin (OPN). To minimize the possibility of false-negative results, several antibodies to AMEL were used. The labelings were compared with those obtained at the EFA. Initial cementum matrix was consistently observed at a time when epithelial cells from HERS covered most of the forming root surface. Cells with mesenchymal characteristics were rarely seen in proximity to the matrix. Both the EFA matrix and initial cementum exhibited collagen fibrils and were intensely immunoreactive for BSP and OPN. AMEL and AMBN were immunodetected at the EFA but not over the initial cementum proper. These two proteins were, however, present at the cervical-most portion of the root where enamel matrix extends for a short distance between dentin and cementum. These data suggest that epithelial cells along the root surface are likely responsible for the deposition of the initial cementum matrix and therefore, like the cells at the EFA, may be capable of producing mesenchymal proteins.

  15. Damage to pancreatic acinar cells and preservation of islets of Langerhans in a rat model of acute pancreatitis induced by Karwinskia humboldtiana (buckthorn).

    PubMed

    Carcano-Diaz, Katya; Garcia-Garcia, Aracely; Segoviano-Ramirez, Juan Carlos; Rodriguez-Rocha, Humberto; Loera-Arias, Maria de Jesus; Garcia-Juarez, Jaime

    2016-09-01

    Karwinskia humboldtiana (Kh) is a poisonous plant that grows in some regions of the American continent. Consuming large amounts of Kh fruit results in acute intoxication leading to respiratory failure, culminating in death within days. There is evidence of histological damage to the lungs, liver, and kidneys following accidental and experimental Kh intoxication. To date, the microscopic effect of Kh consumption on the pancreas has not been described. We examined the early effects of Kh fruit on pancreatic tissue at different stages of acute intoxication in the Wistar rat. We found progressive damage confined to the exocrine pancreas, starting with a reduction in the number of zymogen granules, loss of acinar architecture, the presence of autophagy-like vesicles, apoptosis and inflammatory infiltrate. The pancreatic pathology culminated in damaged acini characterized by necrosis and edema, with a complete loss of lobular architecture. Interestingly, the morphology of the islets of Langerhans was conserved throughout our evaluations. Taken together, our results indicate the damage induced by a high dose of Kh fruit in the Wistar rat is consistent with an early acute necrotizing pancreatitis that exclusively affects the exocrine pancreas. Therefore, this system might be useful as an animal model to study the treatment of pancreatic diseases. More importantly, as the islets of Langerhans were preserved, the active compounds of Kh fruit could be utilized for the treatment of acinar pancreatic cancer. Further studies might provide insight into the severity of acute Kh intoxication in humans and influence the design of treatments for pancreatic diseases and acinar pancreatic cancer.

  16. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

    This report presents a model developed to study growth and differentiation of primary cultures of ureteric epithelial cells from embryonic C57BL/6N mouse urinary tracts. Single cells were resuspended in medium and plated onto transwells coated with collagen IV and laminin. Basa...

  17. Cooperative Interactions During Human Mammary Epithelial Cell Immortalization

    DTIC Science & Technology

    2005-07-01

    Immortal Transformation of Cultured Human Mammary Epithelial Cells. Cellular Oncology, 26:248-251, 2004. Rodier , F., Kim, S-H., Nijjar, T., Yaswen, P...Promoter, Mol. Cell Biol.: 25:3923-3933, 2005. Goldstein, J, Rodier , F, Garbe, J, Stampfer, M, Campisi, J, Caspase-independent cytochrome c release is a

  18. Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers

    PubMed Central

    St Johnston, Daniel

    2016-01-01

    Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium1,2. Here we test this assumption in three types of Drosophila epithelia; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside of the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells appears to be driven by lateral adhesion, which pulls cells born outside the epithelia layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions. PMID:26414404

  19. Transcriptional PROFILING OF MUCOCILIARY DIFFERENTIATION IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of...

  20. Identification of Phosphorylation Sites on Extracellular Corneal Epithelial Cell Maspin

    PubMed Central

    Narayan, Malathi; Mirza, Shama P.; Twining, Sally S.

    2011-01-01

    Maspin, a 42-kDa non classical serine protease inhibitor (serpin) is expressed by epithelial cells of various tissues including the cornea. The protein localizes to the nucleus and cytosol, and is present in the extracellular space. While extracellular maspin regulates corneal stromal fibroblast adhesion and inhibits angiogenesis during wound healing in the cornea, the molecular mechanism of its extracellular functions is unclear. We hypothesized that identifying post-translational modifications of maspin, such as phosphorylation, may help decipher its mode of action. The focus of this study was on the identification of phosphorylation sites on extracellular maspin, since the extracellular form of the molecule is implicated in several functions. Multi-stage fragmentation mass spectrometry was used to identify sites of phosphorylation on extracellular corneal epithelial cell maspin. A total of eight serine and threonine phosphorylation sites (Thr50, Ser97, Thr118, Thr157, Ser240, Ser298, Thr310, Ser316) were identified on the extracellular forms of the molecule. Phosphorylation of tyrosine residues on extracellular maspin was not detected on extracellular maspin from corneal epithelial cell, in contrast to breast epithelial cells. This study provides the basis for further investigation into the functional role of phosphorylation of corneal epithelial maspin. PMID:21365746

  1. Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

    PubMed Central

    West, John D; Dorà, Natalie J; Collinson, J Martin

    2015-01-01

    In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell (LESC) hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient (or transit) amplifying cells (TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell (CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed

  2. Porphyromonas gingivalis genes isolated by screening for epithelial cell attachment.

    PubMed Central

    Duncan, M J; Emory, S A; Almira, E C

    1996-01-01

    Porphyromonas gingivalis is associated with chronic and severe periodontitis in adults. P. gingivalis and the other periodontal pathogens colonize and interact with gingival epithelial cells, but the genes and molecular mechanisms involved are unknown. To dissect the first steps in these interactions, a P. gingivalis expression library was screened for clones which bound human oral epithelial cells. Insert DNA from the recombinant clones did not contain homology to the P. gingivalis fimA gene, encoding fimbrillin, the subunit protein of fimbriae, but showed various degrees of homology to certain cysteine protease-hemagglutinin genes. The DNA sequence of one insert revealed three putative open reading frames which appeared to be in an operon. The relationship between P. gingivalis attachment to epithelial cells and the activities identified by the screen is discussed. PMID:8751909

  3. Coronavirus entry and release in polarized epithelial cells: a review.

    PubMed

    Cong, Yingying; Ren, Xiaofeng

    2014-09-01

    Most coronaviruses cause respiratory or intestinal infections in their animal or human host. Hence, their interaction with polarized epithelial cells plays a critical role in the onset and outcome of infection. In this paper, we review the knowledge regarding the entry and release of coronaviruses, with particular emphasis on the severe acute respiratory syndrome and Middle East respiratory syndrome coronaviruses. As these viruses approach the epithelial surfaces from the apical side, it is not surprising that coronavirus cell receptors are exposed primarily on the apical domain of polarized epithelial cells. With respect to release, all possibilities appear to occur. Thus, most coronaviruses exit through the apical surface, several through the basolateral one, although the Middle East respiratory syndrome coronavirus appears to use both sides. These observations help us understand the local or systematic spread of the infection within its host as well as the spread of the virus within the host population.

  4. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells.

  5. Oxidized alginate hydrogels as niche environments for corneal epithelial cells.

    PubMed

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-10-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2-0.8 µm) than unmodified gels (pore diameter: 0.05-0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy.

  6. Intestinal epithelial cells and their role in innate mucosal immunity.

    PubMed

    Maldonado-Contreras, A L; McCormick, Beth A

    2011-01-01

    The mucosal surfaces of the respiratory, gastrointestinal and urogenital tracts are covered by a layer of epithelial cells that are responsible for sensing and promoting a host immune response in order to establish the limits not only for commensal microorganisms but also for foreign organisms or particles. This is a remarkable task as the human body represents a composite of about 10 trillion human-self cells plus non-self cells from autochthonous or indigenous microbes that outnumber human cells 10:1. Hence, the homeostasis of epithelial cells that line mucosal surfaces relies on a fine-tuned immune system that patrols the boundaries between human and microbial cells. In the case of the intestine, the epithelial layer is composed of at least six epithelial cell lineages that act as a physiological barrier in addition to aiding digestion and the absorption of nutrients, water and electrolytes. In this review, we highlight the immense role of the intestinal epithelium in coordinating the mucosal innate immune response.

  7. Oxidized alginate hydrogels as niche environments for corneal epithelial cells

    PubMed Central

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-01-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2–0.8 µm) than unmodified gels (pore diameter: 0.05–0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy. © 2013 The Authors. Journal of Biomedical Materials Research Part A Published byWiley Periodicals, Inc. Part A: 102A: 3393–3400, 2014. PMID:24142706

  8. EBV BMRF-2 facilitates cell-to-cell spread of virus within polarized oral epithelial cells

    PubMed Central

    Xiao, Jianqiao; Palefsky, Joel M.; Herrera, Rossana; Berline, Jennifer; Tugizov, Sharof M.

    2009-01-01

    We previously reported that the Epstein-Barr virus (EBV) BMRF-2 protein plays an important role in EBV infection of polarized oral epithelial cells by interacting with β1 and αv family integrins. Here we show that infection of polarized oral epithelial cells with B27-BMRF-2low recombinant virus, expressing a low level of BMRF-2, resulted in significantly smaller plaques compared with infection by parental B95-8 virus. BMRF-2 localized in the trans-Golgi network (TGN) and basolateral sorting vesicles and was transported to the basolateral membranes of polarized epithelial cells. Mutation of the tyrosine- and dileucine-containing basolateral sorting signal, YLLV, in the cytoplasmic domain of BMRF-2 led to the failure of its accumulation in the TGN and its basolateral transport. These data show that BMRF-2 may play an important role in promoting the spread of EBV progeny virions through lateral membranes of oral epithelial cells. PMID:19394065

  9. TRPV channels as thermosensory receptors in epithelial cells.

    PubMed

    Lee, Hyosang; Caterina, Michael J

    2005-10-01

    Temperature-sensitive transient receptor potential vanilloid (TRPV) ion channels are critical contributors to normal pain and temperature sensation and therefore represent attractive targets for pain therapy. When these channels were first discovered, most attention was focused on their potential contributions to direct thermal activation of peripheral sensory neurons. However, recent anatomical, physiological, and behavioral studies have provided evidence that TRPV channels expressed in skin epithelial cells may also contribute to thermosensation in vitro and in vivo. Here, we review these studies and speculate on possible communication mechanisms from cutaneous epithelial cells to sensory neurons.

  10. 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.

  11. Epithelial neoplasia in Drosophila entails switch to primitive cell states.

    PubMed

    Khan, Sumbul J; Bajpai, Anjali; Alam, Mohammad Atif; Gupta, Ram P; Harsh, Sneh; Pandey, Ravi K; Goel-Bhattacharya, Surbhi; Nigam, Aditi; Mishra, Arati; Sinha, Pradip

    2013-06-11

    Only select cell types in an organ display neoplasia when targeted oncogenically. How developmental lineage hierarchies of these cells prefigure their neoplastic propensities is not yet well-understood. Here we show that neoplastic Drosophila epithelial cells reverse their developmental commitments and switch to primitive cell states. In a context of alleviated tissue surveillance, for example, loss of Lethal giant larvae (Lgl) tumor suppressor in the wing primordium induced epithelial neoplasia in its Homothorax (Hth)-expressing proximal domain. Transcriptional profile of proximally transformed mosaic wing epithelium and functional tests revealed tumor cooperation by multiple signaling pathways. In contrast, lgl(-) clones in the Vestigial (Vg)-expressing distal wing epithelium were eliminated by cell death. Distal lgl(-) clones, however, could transform when both tissue surveillance and cell death were compromised genetically and, alternatively, when the transcription cofactor of Hippo signaling pathway, Yorkie (Yki), was activated, or when Ras/EGFR signaling was up-regulated. Furthermore, transforming distal lgl(-) clones displayed loss of Vg, suggesting reversal of their terminal cell fate commitment. In contrast, reinforcing a distal (wing) cell fate commitment in lgl(-) clones by gaining Vg arrested their neoplasia and induced cell death. We also show that neoplasia in both distal and proximal lgl(-) clones could progress in the absence of Hth, revealing Hth-independent wing epithelial neoplasia. Likewise, neoplasia in the eye primordium resulted in loss of Elav, a retinal cell marker; these, however, switched to an Hth-dependent primitive cell state. These results suggest a general characteristic of "cells-of-origin" in epithelial cancers, namely their propensity for switch to primitive cell states.

  12. Extracellular cleavage of E-cadherin promotes epithelial cell extrusion.

    PubMed

    Grieve, Adam G; Rabouille, Catherine

    2014-08-01

    Epithelial cell extrusion and subsequent apoptosis is a key mechanism to prevent the accumulation of excess cells. By contrast, when driven by oncogene expression, apical cell extrusion is followed by proliferation and represents an initial step of tumorigenesis. E-cadherin (E-cad), the main component of adherens junctions, has been shown to be essential for epithelial cell extrusion, but its mechanistic contribution remains unclear. Here, we provide clear evidence that cell extrusion can be driven by the cleavage of E-cad, both in a wild-type and an oncogenic environment. We first show that CDC42 activation in a single epithelial cell results in its efficient matrix metalloproteinase (MMP)-sensitive extrusion through MEK signalling activation and this is supported by E-cad cleavage. Second, using an engineered cleavable form of E-cad, we demonstrate that, by itself, truncation of extracellular E-cad at the plasma membrane promotes apical extrusion. We propose that extracellular cleavage of E-cad generates a rapid change in cell-cell adhesion that is sufficient to drive apical cell extrusion. Whereas in normal epithelia, extrusion is followed by apoptosis, when combined with active oncogenic signalling, it is coupled to cell proliferation.

  13. Airway epithelial cell response to human metapneumovirus infection

    SciTech Connect

    Bao, X.; Liu, T.; Spetch, L.; Kolli, D.; Garofalo, R.P.; Casola, A.

    2007-11-10

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and type I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-{kappa}B, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators.

  14. [Methotrexate as inducer of proinflammatory cytokines by epithelial cells].

    PubMed

    Morón-Medina, Alejandra; Viera, Ninoska; de Morales, Thaís Rojas; Alcocer, Sirley; Bohorquez, Dinorath

    2014-03-01

    Methotrexate (MTX), a drug commonly used in childhood cancer, has also been indicated as a cytotoxic agent of the oral mucosa, which can trigger the inflammatory process and increase the vascularity of epithelial tissues during the early stages of oral mucositis. The aim of this study was to determine the production of proinflammatory cytokines IL-1beta, IL-6 y TNF-alpha in epithelial cell cultures treated with MTX. Epithelial cells of human larynx, obtained from the cell line Hep-2, were cultured with different doses of MTX during different incubation times. The drug cytotoxicity was analyzed by means of the colorimetric test, which is based on the metabolic reduction of the bromide of 3-(4, 5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol (MTT); and the proinflammatory cytokines production by the test enzyme-linked immunosorbent assay (ELISA). Cultures of HEp-2 cells showed increased production of proinflammatory cytokines at 72 hours with 0.32 microM of MTX. These results suggest that depending on the dose and exposure time, MTX alters the physiology of human epithelial cells, which may play an important role during the phases of initiation and development of oral mucositis.

  15. Hyperoxia-induced signal transduction pathways in pulmonary epithelial cells

    PubMed Central

    Zaher, Tahereh E.; Miller, Edmund J.; Morrow, Dympna M. P.; Javdan, Mohammad; Mantell, Lin L.

    2007-01-01

    Mechanical ventilation with hyperoxia is necessary to treat critically ill patients. However, prolonged exposure to hyperoxia leads to the generation of excessive reactive oxygen species (ROS), which can cause acute inflammatory lung injury. One of the major effects of hyperoxia is the injury and death of pulmonary epithelium, which is accompanied by increased levels of pulmonary proinflammatory cytokines and excessive leukocyte infiltration. A thorough understanding of the signaling pathways leading to pulmonary epithelial cell injury/death may provide some insights into the pathogenesis of hyperoxia-induced acute inflammatory lung injury. This review focuses on epithelial responses to hyperoxia and some of the major factors regulating pathways to epithelial cell injury/death, and proinflammatory responses upon exposure to hyperoxia. We discuss in detail some of the most interesting players, such as, NF-κB, that can modulate both proinflammatory responses and cell injury/death of lung epithelial cells. A better appreciation for the functions of these factors will no doubt help us to delineate the pathways to hyperoxic cell death and proinflammatory responses. PMID:17349918

  16. TMIGD1 is a novel adhesion molecule that protects epithelial cells from oxidative cell injury.

    PubMed

    Arafa, Emad; Bondzie, Philip A; Rezazadeh, Kobra; Meyer, Rosana D; Hartsough, Edward; Henderson, Joel M; Schwartz, John H; Chitalia, Vipul; Rahimi, Nader

    2015-10-01

    Oxidative damage to renal tubular epithelial cells is a fundamental pathogenic mechanism implicated in both acute kidney injury and chronic kidney diseases. Because epithelial cell survival influences the outcome of acute kidney injury and chronic kidney diseases, identifying its molecular regulators could provide new insight into pathobiology and possible new therapeutic strategies for these diseases. We have identified transmembrane and immunoglobulin domain-containing 1 (TMIGD1) as a novel adhesion molecule, which is highly conserved in humans and other species. TMIGD1 is expressed in renal tubular epithelial cells and promotes cell survival. The extracellular domain of TMIGD1 contains two putative immunoglobulin domains and mediates self-dimerization. Our data suggest that TMIGD1 regulates transepithelial electric resistance and permeability of renal epithelial cells. TMIGD1 controls cell migration, cell morphology, and protects renal epithelial cells from oxidative- and nutrient-deprivation-induced cell injury. Hydrogen peroxide-induced oxidative cell injury downregulates TMIGD1 expression and targets it for ubiquitination. Moreover, TMIGD1 expression is significantly affected in both acute kidney injury and in deoxy-corticosterone acetate and sodium chloride (deoxy-corticosterone acetate salt)-induced chronic hypertensive kidney disease mouse models. Taken together, we have identified TMIGD1 as a novel cell adhesion molecule expressed in kidney epithelial cells that protects kidney epithelial cells from oxidative cell injury to promote cell survival.

  17. AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells.

    PubMed

    Yoshinaga, Tomoyo; Uwabe, Kenichiro; Naito, Shoichi; Higashino, Kenichi; Nakano, Toru; Numata, Yoshito; Kihara, Akio

    2016-01-01

    Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-β1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-β signaling pathway. Our findings regarding the effects of AM251 on the TGF-β signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis.

  18. AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells

    PubMed Central

    Yoshinaga, Tomoyo; Uwabe, Kenichiro; Naito, Shoichi; Higashino, Kenichi; Nakano, Toru; Numata, Yoshito

    2016-01-01

    Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-β1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-β signaling pathway. Our findings regarding the effects of AM251 on the TGF-β signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis. PMID:27936102

  19. Rhinovirus Disrupts the Barrier Function of Polarized Airway Epithelial Cells

    PubMed Central

    Sajjan, Umadevi; Wang, Qiong; Zhao, Ying; Gruenert, Dieter C.; Hershenson, Marc B.

    2008-01-01

    Rationale: Secondary bacterial infection following rhinovirus (RV) infection has been recognized in chronic obstructive pulmonary disease. Objectives: We sought to understand mechanisms by which RV infection facilitates secondary bacterial infection. Methods: Primary human airway epithelial cells grown at air–liquid interface and human bronchial epithelial (16HBE14o-) cells grown as polarized monolayers were infected apically with RV. Transmigration of bacteria (nontypeable Haemophilus influenzae and others) was assessed by colony counting and transmission electron microscopy. Transepithelial resistance (RT) was measured by using a voltmeter. The distribution of zona occludins (ZO)-1 was determined by immunohistochemistry and immunoblotting. Measurements and Main Results: Epithelial cells infected with RV showed 2-log more bound bacteria than sham-infected cultures, and bacteria were recovered from the basolateral media of RV- but not sham-infected cells. Infection of polarized airway epithelial cell cultures with RV for 24 hours caused a significant decrease in RT without causing cell death or apoptosis. Ultraviolet-treated RV did not decrease RT, suggesting a requirement for viral replication. Reduced RT was associated with increased paracellular permeability, as determined by flux of fluorescein isothiocyanate (FITC)-inulin. Neutralizing antibodies to tumor necrosis factor (TNF)-α, IFN-γ and IL-1β reversed corresponding cytokine-induced reductions in RT but not that induced by RV, indicating that the RV effect is independent of these proinflammatory cytokines. Confocal microscopy and immunoblotting revealed the loss of ZO-1 from tight junction complexes in RV-infected cells. Intranasal inoculation of mice with RV1B also caused the loss of ZO-1 from the bronchial epithelium tight junctions in vivo. Conclusions: RV facilitates binding, translocation, and persistence of bacteria by disrupting airway epithelial barrier function. PMID:18787220

  20. Epithelial cell adhesion and gastrointestinal colonization of Lactobacillus in poultry.

    PubMed

    Spivey, Megan A; Dunn-Horrocks, Sadie L; Duong, Tri

    2014-11-01

    Administration of probiotic Lactobacillus cultures is an important alternative to the use of antibiotic growth promoters and has been demonstrated to improve animal health, growth performance, and preharvest food safety in poultry production. Whereas gastrointestinal colonization is thought to be critical to their probiotic functionality, factors important to Lactobacillus colonization in chickens are not well understood. In this study we investigate epithelial cell adhesion in vitro and colonization of Lactobacillusin vivo in broiler chickens. Adhesion of Lactobacillus cultures to epithelial cells was evaluated using the chicken LMH cell line. Lactobacillus cultures were able adhere effectively to LMH cells relative to Bacillus subtilis and Salmonella Typhimurium. Epithelial cell adhesion was similar for Lactobacillus crispatus TDCC 75, L. cristpatus TDCC 76, and Lactobacillus gallinarum TDCC 77, and all 3 were more adherent than L. gallinarum TDCC 78. However, when colonization was evaluated in the ileum and cecum of broiler chicks, L. crispatus TDCC 75 and L. gallinarum TDCC 77 were more persistent than L. crispatus TDCC 76 and L. gallinarum TDCC 78. The reduction of growth in medium supplemented with oxgal was greater for L. gallinarum TDCC 78 than L. gallinarum TDCC 77, suggesting that whereas adhesion was similar for the 2 strains, the difference in colonization between L. gallinarum strains may be due in part to their bile sensitivity. This study demonstrates that whereas adhesion to epithelial cells may be important in predicting gastrointestinal colonization, other factors including bile tolerance may also contribute to the colonization of Lactobacillus in poultry. Additionally, the chicken LMH cell line is expected to provide a platform for investigating mechanisms of Lactobacillus adhesion to epithelial tissue and evaluating the probiotic potential Lactobacillus in poultry.

  1. Live pancreatic acinar imaging of exocytosis using syncollin-pHluorin.

    PubMed

    Fernandez, Nestor A; Liang, Tao; Gaisano, Herbert Y

    2011-06-01

    In this report, a novel live acinar exocytosis imaging technique is described. An adenovirus was engineered, encoding for an endogenous zymogen granule (ZG) protein (syncollin) fused to pHluorin, a pH-dependent green fluorescent protein (GFP). Short-term culture of mouse acini infected with this virus permits exogenous adenoviral protein expression while retaining acinar secretory competence and cell polarity. The syncollin-pHluorin fusion protein was shown to be correctly localized to ZGs, and the pH-dependent fluorescence of pHluorin was retained. Coupled with the use of a spinning disk confocal microscope, the syncollin-pHluorin fusion protein exploits the ZG luminal pH changes that occur during exocytosis to visualize exocytic events of live acinar cells in real-time with high spatial resolution in three dimensions. Apical and basolateral exocytic events were observed on stimulation of acinar cells with maximal and supramaximal cholecystokinin concentrations, respectively. Sequential exocytic events were also observed. Coupled with the use of transgenic mice and/or adenovirus-mediated protein expression, this syncollin-pHluorin imaging method offers a superior approach to studying pancreatic acinar exocytosis. This assay can also be applied to acinar disease models to elucidate the mechanisms implicated in pancreatitis.

  2. Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture

    PubMed Central

    Gidfar, Sanaz; Milani, Farnoud Y.; Milani, Behrad Y.; Shen, Xiang; Eslani, Medi; Putra, Ilham; Huvard, Michael J.; Sagha, Hossein; Djalilian, Ali R.

    2017-01-01

    Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated β-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay. The results demonstrated that primary HCEC treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-β-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of HCEC prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells. PMID:28054657

  3. Cell associated urokinase activity and colonic epithelial cells in health and disease.

    PubMed Central

    Gibson, P R; van de Pol, E; Doe, W F

    1991-01-01

    It is not known if urokinase-type plasminogen activator (uPA) is associated with normal colonic epithelial cells. The aims of this study were to determine if normal colonic epithelial cells have uPA activity and whether this is concentrated at the cell membrane. In addition, the contribution of colonic epithelial cell associated uPA activity to disease related pertubations of mucosal uPA activity were examined. A highly enriched population of colonic epithelial cells was isolated from resected colon or biopsy specimens by an enzymatic technique. uPA activity was measured in cell homogenates by a specific and sensitive colorimetric method and expressed relative to cellular DNA. In two experiments subcellular fractionation of colonic epithelial cells was performed by nitrogen cavitation followed by ultracentrifugation over a linear sucrose gradient. The fractions collected were analysed for uPA and organelle-specific enzyme activities. Normal colonic epithelial cells have cell associated uPA activity (mean (SEM) 5.6 (1.1) IU/mg, n = 18). This colocalised with fractions enriched for leucine-beta-naphthylamidase and 5'-nucleotidase, markers of plasma membrane. uPA activities in epithelial cells from cancerous colons (9.8 (3.1) n = 7) or from mucosa affected by inflammatory bowel disease (3.8 (0.7) n = 15) were not significantly different from normal (paired t test), while that in epithelial cells from greatly inflamed mucosa was similar to that from autologous normal or mildly inflamed areas (4.4 (1.2) v 5.9 (3.6), n = 9). Thus normal colonic epithelial cells have cell associated uPA activity which is concentrated on the plasma membranes, suggesting the presence of uPA receptors. Increased mucosal levels of uPA previously reported in patients with inflammatory bowel disease are not due to increased colonic epithelial cell associated uPA. PMID:1650741

  4. Modulation of Candida albicans attachment to human epithelial cells by bacteria and carbohydrates.

    PubMed Central

    Centeno, A; Davis, C P; Cohen, M S; Warren, M M

    1983-01-01

    The effects of carbohydrates (mannose and dextrose). Escherichia coli 07KL. and Klebsiella pneumoniae on Candida albicans attachment to epithelial cells was studied. Dextrose had no effect on yeast attachment to epithelial cells. Conversely, mannose significantly decreased both yeast and piliated bacterial attachment (E. coli 07KL, heavily piliated K. pneumoniae) whereas having no effect on nonpiliated K. pneumoniae attachment to epithelial cells. The number of yeasts attaching to epithelial cells was enhanced by preincubation of epithelial cells with piliated strains of bacteria, whereas preincubation with nonpiliated strains of bacteria had no effect on yeast attachment. Scanning electron microscopy showed that piliated bacteria and yeasts were juxtaposed on the epithelial cell surface. These data suggest that certain piliated strains of bacteria can enhance C. albicans attachment to epithelial cells and that type 1 pili of bacteria can be a factor in the enhanced attachment of C. albicans to epithelial cells. Images PMID:6132878

  5. NITROTYROSINE ATTENUATES RSV-INDUCED INFLAMMATION IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Nitrotyrosine attenuates RSV-induced inflammation in airway epithelial cells. Joleen Soukup, Zuowei Li, Susanne Becker and Yuh-Chin Huang. NHEERL, ORD, USEPA, RTP, North Carolina, CEMALB, University of North Carolina, Chapel Hill, North Carolina

    Nitrotyrosine (NO2Tyr) is a...

  6. A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-11-01

    Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

  7. Requirements for invasion of epithelial cells by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Sreenivasan, P K; Meyer, D H; Fives-Taylor, P M

    1993-01-01

    Actinobacillus actinomycetemcomitans, an oral bacterium implicated in human periodontal disease, was recently demonstrated to invade cultured epithelial cells (D. H. Meyer, P. K. Sreenivasan, and P. M. Fives-Taylor, Infect. Immun. 59:2719-2726, 1991). This report characterizes the requirements for invasion of KB cells by A. actinomycetemcomitans. The roles of bacterial and host factors were investigated by using selective agents that influence specific bacterial or host cell functions. Inhibition of bacterial protein synthesis decreased invasion, suggesting the absence of a preformed pool of proteins involved in A. actinomycetemcomitans invasion. Inhibition of bacterial and eukaryotic energy synthesis also decreased invasion, confirming that A. actinomycetemcomitans invasion is an active process. Bacterial adherence to KB cells was indicated by scanning electron microscopy of infected KB cells. Further, the addition of A. actinomycetemcomitans-specific serum to the bacterial inoculum reduced invasion substantially, suggesting a role for bacterial attachment in invasion. Many of the adherent bacteria invaded the epithelial cells under optimal conditions. Inhibitors of receptor-mediated endocytosis inhibited invasion by A. actinomycetemcomitans. Like that of many facultatively intracellular bacteria, A. actinomycetemcomitans invasion was not affected by eukaryotic endosomal acidification. These are the first published observations describing the requirements for epithelial cell invasion by a periodontopathogen. They demonstrate that A. actinomycetemcomitans utilizes a mechanism similar to those used by many but not all invasive bacteria to gain entry into eukaryotic cells. Images PMID:8454326

  8. Transport Mechanism of Nicotine in Primary Cultured Alveolar Epithelial Cells.

    PubMed

    Takano, Mikihisa; Nagahiro, Machi; Yumoto, Ryoko

    2016-02-01

    Nicotine is absorbed from the lungs into the systemic circulation during cigarette smoking. However, there is little information concerning the transport mechanism of nicotine in alveolar epithelial cells. In this study, we characterized the uptake of nicotine in rat primary cultured type II (TII) and transdifferentiated type I-like (TIL) epithelial cells. In both TIL and TII cells, [(3)H]nicotine uptake was time and temperature-dependent, and showed saturation kinetics. [(3)H]Nicotine uptake in these cells was not affected by Na(+), but was sensitive to extracellular and intracellular pH, suggesting the involvement of a nicotine/proton antiport system. The uptake of [(3)H]nicotine in these cells was potently inhibited by organic cations such as clonidine, diphenhydramine, and pyrilamine, but was not affected by substrates and/or inhibitors of known organic cation transporters such as carnitine, 1-methyl-4-phenylpyridinium, and tetraethylammonium. In addition, the uptake of [(3)H]nicotine in TIL cells was stimulated by preloading the cells with unlabeled nicotine, pyrilamine, and diphenhydramine, but not with tetraethylammonium. These results suggest that a novel proton-coupled antiporter is involved in the uptake of nicotine in alveolar epithelial cells and its absorption from the lungs into the systemic circulation.

  9. Differentiation of cultured epithelial cells: Response to toxic agents

    SciTech Connect

    Rice, R.H.; LaMontagne, A.D.; Petito, C.T.; Rong, Xianhui )

    1989-03-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAmP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

  10. Keratin cytoskeletons in epithelial cells of internal organs

    PubMed Central

    Sun, Tung-Tien; Shih, Chiaho; Green, Howard

    1979-01-01

    An antiserum against human epidermal keratins was used to detect keratins in frozen sections of various rabbit and human tissues by indirect immunofluorescence. Strong staining was observed in all stratified squamous epithelia (epidermis, cornea, conjunctiva, tongue, esophagus, vagina, and anus), in epidermal appendages (hair follicle, sebaceous gland, ductal and myoepithelial cells of sweat glands), as well as in Hassall's corpuscles of the thymus, indicating that all contain abundant keratins. No staining by the antiserum was observed in fibroblasts, muscle of any type, cartilage, blood vessel, nerve tissue, iris or lens epithelium, or the glomerular or tubular cells of the kidney. In contrast, the antiserum stained the cells of most epithelia of the intestinal tract, urinary tract (urethra, bladder, ureter, collecting ducts of kidney), female genital tract (cervix, cervical glands, uterus, and oviduct), and respiratory tract (trachea and bronchi). Epithelial cells of the fine ductal system in the pancreas and submaxillary gland also stained well. When primary cultures of epithelial cells derived from bladder, intestine, kidney, and trachea were grown on glass coverslips and stained with anti-keratin, fiber networks similar to those of cultured keratinocytes were observed. These results show that keratins constitute a cytoskeleton in epithelial cells of diverse morphology and embryological origin. The stability of keratin filaments probably confers the structural strength necessary for cells covering a free surface. Keratin staining can be used to obtain information about the origin of cell lines. Images PMID:111242

  11. Modeling Alveolar Epithelial Cell Behavior In Spatially Designed Hydrogel Microenvironments

    NASA Astrophysics Data System (ADS)

    Lewis, Katherine Jean Reeder

    The alveolar epithelium consists of two cell phenotypes, elongated alveolar type I cells (AT1) and rounded alveolar type II cells (ATII), and exists in a complex three-dimensional environment as a polarized cell layer attached to a thin basement membrane and enclosing a roughly spherical lumen. Closely surrounding the alveolar cysts are capillary endothelial cells as well as interstitial pulmonary fibroblasts. Many factors are thought to influence alveolar epithelial cell differentiation during lung development and wound repair, including physical and biochemical signals from the extracellular matrix (ECM), and paracrine signals from the surrounding mesenchyme. In particular, disrupted signaling between the alveolar epithelium and local fibroblasts has been implicated in the progression of several pulmonary diseases. However, given the complexity of alveolar tissue architecture and the multitude of signaling pathways involved, designing appropriate experimental platforms for this biological system has been difficult. In order to isolate key factors regulating cellular behavior, the researcher ideally should have control over biophysical properties of the ECM, as well as the ability to organize multiple cell types within the scaffold. This thesis aimed to develop a 3D synthetic hydrogel platform to control alveolar epithelial cyst formation, which could then be used to explore how extracellular cues influence cell behavior in a tissue-relevant cellular arrangement. To accomplish this, a poly(ethylene glycol) (PEG) hydrogel network containing enzymatically-degradable crosslinks and bioadhesive pendant peptides was employed as a base material for encapsulating primary alveolar epithelial cells. First, an array of microwells of various cross-sectional shapes was photopatterned into a PEG gel containing photo-labile crosslinks, and primary ATII cells were seeded into the wells to examine the role of geometric confinement on differentiation and multicellular arrangement

  12. Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

    PubMed

    Rene, Hernandez-Delgadillo; Badireddy, Appala Raju; José, Martínez-Sanmiguel Juan; Francisco, Contreras-Cordero Juan; Israel, Martinez-Gonzalez Gustavo; Isela, Sánchez-Nájera Rosa; Chellam, Shankararaman; Claudio, Cabral-Romero

    2016-01-01

    Bismuth nanoparticles have many interesting properties to be applied in biomedical and medicinal sectors, however their safety in humans have not been comprehensively investigated. The objective of this research was to determine the cytotoxic effect of bismuth dimercaptopropanol nanoparticles (BisBAL NPs) on epithelial cells. The nanoparticles are composed of 18.7 nm crystallites on average and have a rhombohedral structure, agglomerating into chains-like or clusters of small nanoparticles. Based on MTT viability assay and fluorescence microscopy, cytotoxicity was not observed on monkey kidney cells after growing with 5 µM of BisBAL NPs for 24 h. Employing same techniques, identical results were obtained with human epithelial cells (HeLa), showing a not strain-dependent phenomenon. The absence of toxic effects on epithelial cells growing with BisBAL NPs was corroborated with long-time experiments (24-72 hrs.), showing no difference in comparison with growing control (cells without nanoparticles). Further, genotoxicity assays, comet assay and fluorescent microscopy and electrophoresis in bromide-stained agarose gel revealed no damage to genomic DNA of MA104 cells after 24 h. of exposition to BisBAL NPs. Finally, the effect of bismuth nanoparticles on protein synthesis was studied in cells growing with BisBAL NPs for 24 h. SDS-PAGE assays showed no difference between treated and untreated cells, suggesting that BisBAL NPs did not interfere with protein synthesis. Hence BisBAL NPs do not appear to exert cytotoxic effects suggesting their biological compatibility with epithelial cells.

  13. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    SciTech Connect

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang Zhang, Yi

    2013-11-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

  14. Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration.

    PubMed

    Lindemans, Caroline A; Calafiore, Marco; Mertelsmann, Anna M; O'Connor, Margaret H; Dudakov, Jarrod A; Jenq, Robert R; Velardi, Enrico; Young, Lauren F; Smith, Odette M; Lawrence, Gillian; Ivanov, Juliet A; Fu, Ya-Yuan; Takashima, Shuichiro; Hua, Guoqiang; Martin, Maria L; O'Rourke, Kevin P; Lo, Yuan-Hung; Mokry, Michal; Romera-Hernandez, Monica; Cupedo, Tom; Dow, Lukas E; Nieuwenhuis, Edward E; Shroyer, Noah F; Liu, Chen; Kolesnick, Richard; van den Brink, Marcel R M; Hanash, Alan M

    2015-12-24

    Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5(+) crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.

  15. Directed differentiation of airway epithelial cells of human bone marrow mesenchymal stem cells.

    PubMed

    Li, Jian-Dong

    2016-11-01

    The ability to generate lung and airway epithelial cells from human bone marrow mesenchymal stem cells (hBMSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening, and studies of human lung development. In this research, hBMSCs were cultured in specialized airway epithelial cell growth media for differentiation of airway epithelial cells, including keratinocyte growth factor transferrin, bovine pituitary extract, epinephrine, triiodothyronine and retinoic acid. The surfactant protein C, a specific marker of type II pneumocytes, and its corresponding protein were demonstrated by immunofluorescence and western blotting after differentiation of airway epithelial cells, respectively. These cells were then transferred into an induced acute lung injury model. The results showed that the hBMSCs could induce differentiation in airway epithelial cells under the special conditions of the medium, the result for surfactant protein C was positive in differentiated airway epithelial cells using immunofluorescence and western blotting, and these cells were successfully colonized in the injured lung airway. In conclusion, our research shows that a population of airway epithelial cells can be specifically generated from hBMSCs and that induced cells may be allowed to participate in tissue repair.

  16. COMPARISON OF PM-INDUCED GENE EXPRESSION PROFILES BETWEEN BRONCHIAL EPITHELIAL CELLS AND NASAL EPITHELIAL CELLS IN HUMAN

    EPA Science Inventory

    Epidemiologic studies have linked exposures to particulate matter (PM) and increased pulmonary mortality and morbidity. Bronchial epithelial cells (BEC) are the primary target of PM. PM exposure induces a wide array of biological responses in BEC. Primary human BEC, however, need...

  17. Concise review: limbal epithelial stem cell therapy: controversies and challenges.

    PubMed

    O'Callaghan, Anna R; Daniels, Julie T

    2011-12-01

    Limbal epithelial stem cells (LESCs) are a population of stem cells responsible for maintenance and repair of the corneal surface. Injury and disease can result in a deficiency of these stem cells, the vision affecting condition called limbal stem cell deficiency (LSCD) in which the cornea becomes opaque, vascularized, and inflamed. Cultured LESC therapy was first described in 1997;29:19231932-19231932.and LESCs cultured from either patients or donors have been used to successfully treat LSCD. In this review, some of the challenges and controversies associated with cultured LESC therapy will be discussed including alternative stem cell sources.

  18. Targeting Epithelial Cell Migration to Accelerate Wound Healing

    DTIC Science & Technology

    2012-02-01

    consisting of the proteins Rsu1, Integrin Linked Kinase (ILK), PINCH, and Parvin. The correct association of these proteins in a functional complex...impacting integrin function and actin polymerization. 15. SUBJECT TERMS Wound healing, cell migration, protein kinase C, protein kinase A 16. SECURITY...epithelial cell migration in wound healing. In addition, the correct association of these proteins in a functional complex depends on their phosphorylation

  19. Ultraviolet Irradiation-Induced Volume Alteration of Corneal Epithelial Cells

    PubMed Central

    Wang, Ling; Lu, Luo

    2016-01-01

    Purpose The purpose of the study is to understand how extracellular stresses, such as ultraviolet (UV) irradiation, affect corneal epithelial cells. Cell volume changes, damage to corneal epithelial integrity, and cellular responses were assessed after exposure to UVC stresses. Methods Primary human and rabbit corneal epithelial cells were exposed to UVC light in culture conditions. Ultraviolet C irradiation–induced changes in cell size and volume were measured by real-time microscopy and self-quenching of the fluorescent dye calcein, respectively. The effects of UVC irradiation on Src and focal adhesion kinase (FAK) phosphorylation and FAK-dependent integrin signaling were detected by ELISA, immunoblotting, and immunostaining. Results Ultraviolet C irradiation induced both size and volume shifts in human and rabbit corneal epithelial cells. Ultraviolet C irradiation-induced decrease of cell volume elicited activation of Src and FAK, characterized by increased phosphorylations of SrcY416, FAKY397, and FAKY925. In addition, immunostaining studies showed UVC irradiation–induced increases in phosphorylation of FAK and formation of integrin β5 clustering. Application of Kv channel blockers, including 4-aminopyridine (4-AP), α-DTX, and depressing substance-1 (BDS-1), effectively suppressed UVC irradiation–induced cell volume changes, and subsequently inhibited UVC irradiation–induced phosphorylation of Src/FAK, and formation of integrin β5 clustering, suggesting UVC irradiation–induced volume changes and Src/FAK activation. Hyperosmotic pressure–induced volume decreases were measured in comparison with effects of UVC irradiation on volume and Src/FAK activation. However, Kv channel blocker, 4-AP, had no effect on hyperosmotic pressure–induced responses. Conclusions The present study demonstrates that UVC irradiation–induced decreases in cell volume lead to Src/FAK activation due to a rapid loss of K ions through membrane Kv channels. PMID:27978555

  20. Limbal Stromal Tissue Specific Stem Cells and Their Differentiation Potential to Corneal Epithelial Cells.

    PubMed

    Katikireddy, Kishore Reddy; Jurkunas, Ula V

    2016-01-01

    From the derivation of the first human embryonic stem (hES) cell line to the development of induced pluripotent stem (iPS) cells; it has become evident that tissue specific stem cells are able to differentiate into a specific somatic cell types. The understanding of key processes such as the signaling pathways and the role of the microenvironment in epidermal/epithelial development has provided important clues for the derivation of specific epithelial cell types.Various differentiation protocols/methods were used to attain specific epithelial cell types. Here, we describe in detail the procedure to follow for isolation of tissue specific stem cells, mimicking their microenvironment to attain stem cell characteristics, and their potential differentiation to corneal epithelial cells.

  1. Review: Corneal epithelial stem cells, their niche and wound healing

    PubMed Central

    2013-01-01

    Stem cells emerged as a concept during the second half of 19th century, first as a theoretical entity, but then became one of the most promising research fields in cell biology. This work describes the most important characteristics of adult stem cells, including the experimental criteria used to identify them, and discusses current knowledge that led to the proposal that stem cells existed in different parts of the eye, such as the retina, lens, conjunctiva, corneal stroma, Descemet’s membrane, and the subject of this review: the corneal epithelium. Evidence includes results that support the presence of corneal epithelial stem cells at the limbus, as well as the major obstacles to isolating them as pure cell populations. Part of this review describes the variation in the basement membrane composition between the limbus and the central cornea, to show the importance of the corneal stem cell niche, its structure, and the participation of extracellular matrix (ECM) components in regulating corneal stem cell compartment. Results obtained by various laboratories suggest that the extracellular matrix plays a central role in regulating stem cell commitment, corneal differentiation, and participation in corneal wound healing, in addition to other environmental signals such as cytokines and growth factors. The niche could define cell division patterns in corneal stem cell populations, establishing whether stem cells divide asymmetrically or symmetrically. Characterization and understanding of the factors that regulate corneal epithelial stem cells should open up new paths for developing new therapies and strategies for accelerating and improving corneal wound healing. PMID:23901244

  2. Established thymic epithelial progenitor/stem cell-like cell lines differentiate into mature thymic epithelial cells and support T cell development.

    PubMed

    Chen, Pengfei; Zhang, Jun; Zhan, Yu; Su, Juanjuan; Du, Yarui; Xu, Guoliang; Shi, Yufang; Siebenlist, Ulrich; Zhang, Xiaoren

    2013-01-01

    Common thymic epithelial progenitor/stem cells (TEPCs) differentiate into cortical and medullary thymic epithelial cells (TECs), which are required for the development and selection of thymocytes. Mature TEC lines have been widely established. However, the establishment of TEPC lines is rarely reported. Here we describe the establishment of thymic epithelial stomal cell lines, named TSCs, from fetal thymus. TSCs express some of the markers present on tissue progenitor/stem cells such as Sca-1. Gene expression profiling verifies the thymic identity of TSCs. RANK stimulation of these cells induces expression of autoimmune regulator (Aire) and Aire-dependent tissue-restricted antigens (TRAs) in TSCs in vitro. TSCs could be differentiated into medullary thymic epithelial cell-like cells with exogenously expressed NF-κB subunits RelB and p52. Importantly, upon transplantation under the kidney capsules of nude mice, TSCs are able to differentiate into mature TEC-like cells that can support some limited development of T cells in vivo. These findings suggest that the TSC lines we established bear some characteristics of TEPC cells and are able to differentiate into functional TEC-like cells in vitro and in vivo. The cloned TEPC-like cell lines may provide useful tools to study the differentiation of mature TEC cells from precursors.

  3. In vitro methods to culture primary human breast epithelial cells.

    PubMed

    Raouf, Afshin; Sun, Yu Jia

    2013-01-01

    Current evidence suggests that much like leukemia, breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore, knowledge about their nature will provide a platform to develop therapies to eliminate these breast cancer stem cells. This concept highlights the need to understand the mechanisms that regulate the normal functions of the breast stem cells and their immediate progeny as alterations to these same mechanisms can cause these primitive cells to act as cancer stem cells. The study of the primitive cell functions relies on the ability to isolate them from primary sources of breast tissue. This chapter describes processing of discarded tissue from reduction mammoplasty samples as sources of normal primary human breast epithelial cells and describes cell culture systems to grow single-cell suspensions prepared from these reduction samples in vitro.

  4. Microtubule organization is determined by the shape of epithelial cells

    PubMed Central

    Gomez, Juan Manuel; Chumakova, Lyubov; Bulgakova, Natalia A.; Brown, Nicholas H.

    2016-01-01

    Interphase microtubule organization is critical for cell function and tissue architecture. In general, physical mechanisms are sufficient to drive microtubule organization in single cells, whereas cells within tissues are thought to utilize signalling mechanisms. By improving the imaging and quantitation of microtubule alignment within developing Drosophila embryos, here we demonstrate that microtubule alignment underneath the apical surface of epithelial cells follows cell shape. During development, epidermal cell elongation and microtubule alignment occur simultaneously, but by perturbing cell shape, we discover that microtubule organization responds to cell shape, rather than the converse. A simple set of microtubule behaviour rules is sufficient for a computer model to mimic the observed responses to changes in cell surface geometry. Moreover, we show that microtubules colliding with cell boundaries zip-up or depolymerize in an angle-dependent manner, as predicted by the model. Finally, we show microtubule alignment responds to cell shape in diverse epithelia. PMID:27779189

  5. Type II alveolar epithelial cell in vitro culture in aerobiosis.

    PubMed

    Aerts, C; Voisin, C; Wallaert, B

    1988-08-01

    A method of Type II alveolar epithelial cell culture in aerobiosis has been developed. Isolation of Type II cells was performed by digesting guinea-pig lung tissue with crude trypsin and elastase and using discontinuous Percoll density gradients. The Type II cells, as identified by light and electron microscopy, were cultured in aerobiosis for up to six days, in direct contact with the atmosphere in conditions mimicking those present in the lower respiratory tract. Significant activities of cellular superoxide dismutase (SOD), manganese dependent superoxide dismutase (Mn-SOD), catalase and glutathione peroxidase (GSH-Px) were found at the time of isolation. In contrast, cell glutathione content varied widely from one experiment to another. Changes of antioxidant enzymes were evaluated during cell culture in aerobiosis. SOD, Mn-SOD and catalase were significantly decreased after three days but were not significantly different between a three day and six day culture. Antioxidant changes did not influence the cell culture. In marked contrast, decrease in cell glutathione was associated with rapid cell death, whereas good cell survival was obtained at high levels of cell glutathione. Cell culture in aerobiosis will permit a precise evaluation of the effects of gases, particularly oxidant gases, on a primary culture of Type II alveolar epithelial cells.

  6. Molecular mechanisms of membrane polarity in renal epithelial cells.

    PubMed

    Campo, C; Mason, A; Maouyo, D; Olsen, O; Yoo, D; Welling, P A

    2005-01-01

    Exciting discoveries in the last decade have cast light onto the fundamental mechanisms that underlie polarized trafficking in epithelial cells. It is now clear that epithelial cell membrane asymmetry is achieved by a combination of intracellular sorting operations, vectorial delivery mechanisms and plasmalemma-specific fusion and retention processes. Several well-defined signals that specify polarized segregation, sorting, or retention processes have, now, been described in a number of proteins. The intracellular machineries that decode and act on these signals are beginning to be described. In addition, the nature of the molecules that associate with intracellular trafficking vesicles to coordinate polarized delivery, tethering, docking, and fusion are also becoming understood. Combined with direct visualization of polarized sorting processes with new technologies in live-cell fluorescent microscopy, new and surprising insights into these once-elusive trafficking processes are emerging. Here we provide a review of these recent advances within an historically relevant context.

  7. Expression of inducible nitric oxide in human lung epithelial cells.

    PubMed

    Robbins, R A; Barnes, P J; Springall, D R; Warren, J B; Kwon, O J; Buttery, L D; Wilson, A J; Geller, D A; Polak, J M

    1994-08-30

    Nitric oxide (NO) is increased in the exhaled air of subjects with several airway disorders. To determine if cytokines could stimulate epithelial cells accounting for the increased NO, the capacity of the proinflammatory cytokines (cytomix: tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma) to increase inducible nitric oxide synthase (iNOS) was investigated in A549 and primary cultures of human bronchial epithelial cells. Cytomix induced a time-dependent increase in nitrite levels in culture supernatant fluids (p < 0.05). Increased numbers of cells stained for iNOS and increased iNOS mRNA was detected in the cytokine-stimulated cells compared to control (p < 0.05). Dexamethasone diminished the cytokine-induced increase in nitrite, iNOS by immunocytochemistry, and iNOS mRNA. These data demonstrate that cytokines, such as those released by mononuclear cells, can induce lung epithelial iNOS expression and NO release, and that this is attenuated by dexamethasone.

  8. Effects of weaning on intestinal crypt epithelial cells in piglets

    PubMed Central

    Yang, Huansheng; Xiong, Xia; Wang, Xiaocheng; Li, Tiejun; Yin, Yulong

    2016-01-01

    Intestinal epithelial cells in the crypt proliferate in piglets in response to weaning. However, the underlying mechanism has been unclear. We examined 40 piglets from eight litters (five piglets per litter) that were weaned at the age of 14 d, and one piglet from each litter was randomly selected for closer investigation. Based on the distended intestinal sac method, we isolated crypt epithelial cells from the mid-jejunum on Days 0, 1, 3, 5, and 7 post-weaning. Protein expression was analyzed using either isobaric tags for relative and absolute quantification or western blotting. Proteins related to the cell cycle, organization of the cellular macromolecular complex subunit, localization of cellular macromolecules, Golgi vesicle transport, fatty acid metabolism, oxidative phosphorylation, and translational initiation were mainly down-regulated, while those involved in glycolysis, cell cycle arrest, protein catabolism, and cellular amino acid metabolism were up-regulated. The amount of proteins active in the mTOR signaling pathway was generally decreased over time. These results indicate that weaning influences energy metabolism, cellular macromolecule organization and localization, and protein metabolism, thereby affecting the proliferation of intestinal epithelial cells in weaned piglets. Moreover, those cellular processes are possibly controlled by that signaling pathway. PMID:27830738

  9. Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells*

    PubMed Central

    Cai, Zhenyu; Shi, Zhongcheng; Sanchez, Amir; Zhang, Tingting; Liu, Mingyao; Yang, Jianghua; Wang, Fen; Zhang, Dekai

    2009-01-01

    As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions. PMID:19801549

  10. Spraying Respiratory Epithelial Cells to Coat Tissue-Engineered Constructs

    PubMed Central

    Thiebes, Anja Lena; Albers, Stefanie; Klopsch, Christian; Jockenhoevel, Stefan; Cornelissen, Christian G.

    2015-01-01

    Abstract Applying cells in a spray can overcome current hurdles in coating tissue engineered constructs with a thin layer of endo- or epithelial cells. We report here a structured study on the influences of spray application with a medical spray device on vascular smooth muscle cells (vSMCs) and respiratory epithelial cells (RECs) with and without fibrin gel. Next to viability and cytotoxicity assays, the in vitro differentiation capacity after spray processing was analyzed. For vSMC, no influence of air pressures till 0.8 bar could be shown, whereas the viability decreased for higher pressures. The viability of RECs was reduced to 88.5% with 0.4 bar air pressure. Lactate dehydrogenase-levels in the culture medium increased the first day after spraying but normalized afterward. In the short term, no differences by means of morphology and expression-specific markers for vSMCs and RECs were seen between the control and study group. In addition, in a long-term study for 28 days with the air–liquid interface, RECs differentiated and built up an organized epithelial layer with ciliary development that was comparable to the control for cells sprayed without fibrin gel. When spraying within fibrin gel, ciliary development was lower at 28 days. Thus, spraying of vSMCs and RECs was proved to be a suitable method for tissue engineering. Especially for RECs, this application is of special significance when coating luminal structures or other unfavorable topographies. PMID:26309803

  11. Asbestos exposure increases human bronchial epithelial cell fibrinolytic activity.

    PubMed

    Gross, T J; Cobb, S M; Gruenert, D C; Peterson, M W

    1993-03-01

    Chronic exposure to asbestos fibers results in fibrotic lung disease. The distal pulmonary epithelium is an early target of asbestos-mediated injury. Local plasmin activity may be important in modulating endoluminal inflammatory responses in the lung. We studied the effects of asbestos exposure on cell-mediated plasma clot lysis as a marker of pericellular plasminogen activation. Exposing human bronchial epithelial (HBE) cells to 100 micrograms/ml of asbestos fibers for 24 h resulted in increased plasma clot lysis. Fibrinolytic activity was augmented in a dose-dependent fashion, was not due to secreted protease, and occurred only when there was direct contact between the plasma clot and the epithelial monolayer. Further analysis showed that asbestos exposure increased HBE cell-associated urokinase-type plasminogen activator (uPA) activity in a time-dependent manner. The increased cell-associated PA activity could be removed by acid washing. The increase in PA activity following asbestos exposure required new protein synthesis because it was abrogated by treatment with either cycloheximide or actinomycin D. Therefore, asbestos exposure increases epithelial-mediated fibrinolysis by augmenting expression of uPA activity at the cell surface by mechanisms that require new RNA and protein synthesis. These observations suggest a novel mechanism whereby exposure of the distal epithelium to inhaled particulates may result in a chronic inflammatory response that culminates in the development of fibrotic lung disease.

  12. Radiogenic transformation of human mammary epithelial cells in vitro

    NASA Technical Reports Server (NTRS)

    Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

    1996-01-01

    Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

  13. Generation of Spheres from Dental Epithelial Stem Cells

    PubMed Central

    Natsiou, Despoina; Granchi, Zoraide; Mitsiadis, Thimios A.; Jimenez-Rojo, Lucia

    2017-01-01

    The in vitro three-dimensional sphere model has already been established as an important tool in fundamental sciences. This model facilitates the study of a variety of biological processes including stem cell/niche functions and tissue responses to injury and drugs. Here we describe the complete protocol for the in vitro formation of spheres originated from the epithelium of rodent incisors. In addition, we show that in these spheres cell proliferation is maintained, as well as the expression of several key molecules characterizing stem cells such as Sox2 and p63. These epithelial dentospheres could be used as an in vitro model system for stem cell research purposes. PMID:28154538

  14. Hepatic stellate cells promote upregulation of epithelial cell adhesion molecule and epithelial-mesenchymal transition in hepatic cancer cells.

    PubMed

    Nagahara, Teruya; Shiraha, Hidenori; Sawahara, Hiroaki; Uchida, Daisuke; Takeuchi, Yasuto; Iwamuro, Masaya; Kataoka, Junro; Horiguchi, Shigeru; Kuwaki, Takeshi; Onishi, Hideki; Nakamura, Shinichiro; Takaki, Akinobu; Nouso, Kazuhiro; Yamamoto, Kazuhide

    2015-09-01

    Microenvironment plays an important role in epithelial-mesenchymal transition (EMT) and stemness of cells in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) is known as a tumor stemness marker of HCC. To investigate the relationship between microenvironment and stemness, we performed an in vitro co-culture assay. Four HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) were co-cultured with the TWNT-1 immortalized hepatic stellate cells (HSCs), which create a microenvironment with HCC. Cell proliferation ability was analyzed by flow cytometry (FCM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while migration ability was assessed by a wound healing assay. Expression of EpCAM was analyzed by immunoblotting and FCM. HCC cell lines were co-cultured with TWNT-1 treated with small interfering RNA (siRNA) for TGF-β and HB-EGF; we then analyzed proliferation, migration ability and protein expression using the methods described above. Proliferation ability was unchanged in HCC cell lines co-cultured with TWNT-1. Migration ability was increased in HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) directly (216.2±67.0, 61.0±22.0, 124.0±66.2 and 51.5±40.3%) and indirectly (102.5±22.0, 84.6±30.9, 86.1±25.7 and 73.9±29.7%) co-cultured with TWNT-1 compared with the HCC uni-culture. Immunoblot analysis revealed increased EpCAM expression in the HCC cell lines co-cultured with TWNT-1. Flow cytometry revealed that the population of E-cadherin-/N-cadherin+ and EpCAM-positive cells increased and accordingly, EMT and stemness in the HCC cell line were activated. These results were similar in the directly and indirectly co-cultured samples, indicating that humoral factors were at play. Conversely, HCC cell lines co-cultured with siRNA‑treated TWNT-1 showed decreased migration ability, a decreased population of EpCAM-positive and E-cadherin-/N-cadherin+ cells. Taken together, humoral factors secreted from TWNT-1

  15. Progenitor Cells in Proximal Airway Epithelial Development and Regeneration

    PubMed Central

    Lynch, Thomas J.; Engelhardt, John F.

    2015-01-01

    Multiple distinct epithelial domains are found throughout the airway that are distinguishable by location, structure, function, and cell-type composition. Several progenitor cell populations in the proximal airway have been identified to reside in confined microenvironmental niches including the submucosal glands (SMGs), which are embedded in the tracheal connective tissue between the surface epithelium and cartilage, and basal cells that reside within the surface airway epithelium (SAE). Current research suggests that regulatory pathways that coordinate development of the proximal airway and establishment of progenitor cell niches may overlap with pathways that control progenitor cell responses during airway regeneration following injury. SMGs have been shown to harbor epithelial progenitor cells, and this niche is dysregulated in diseases such as cystic fibrosis. However, mechanisms that regulate progenitor cell proliferation and maintenance within this glandular niche are not completely understood. Here we discuss glandular progenitor cells during development and regeneration of the proximal airway and compare properties of glandular progenitors to those of basal cell progenitors in the SAE. Further investigation into glandular progenitor cell control will provide a direction for interrogating therapeutic interventions to correct aberrant conditions affecting the SMGs in diseases such as cystic fibrosis, chronic bronchitis, and asthma. PMID:24818588

  16. Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

    PubMed Central

    Mizuno, Takako; Sridharan, Anusha; Du, Yina; Guo, Minzhe; Wikenheiser-Brokamp, Kathryn A.; Perl, Anne-Karina T.; Funari, Vincent A.; Gokey, Jason J.; Stripp, Barry R.; Whitsett, Jeffrey A.

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease. PMID:27942595

  17. Building Epithelial Tissues from Skin Stem Cells

    PubMed Central

    Fuchs, E.; Nowak, J.A.

    2009-01-01

    The skin epidermis and its appendages provide a protective barrier that guards against loss of fluids, physical trauma, and invasion by harmful microbes. To perform these functions while confronting the harsh environs of the outside world, our body surface undergoes constant rejuvenation through homeostasis. In addition, it must be primed to repair wounds in response to injury. The adult skin maintains epidermal homeostasis, hair regeneration, and wound repair through the use of its stem cells. What are the properties of skin stem cells, when do they become established during embryogenesis, and how are they able to build tissues with such remarkably distinct architectures? How do stem cells maintain tissue homeostasis and repair wounds and how do they regulate the delicate balance between proliferation and differentiation? What is the relationship between skin cancer and mutations that perturbs the regulation of stem cells? In the past 5 years, the field of skin stem cells has bloomed as we and others have been able to purify and dissect the molecular properties of these tiny reservoirs of goliath potential. We report here progress on these fronts, with emphasis on our laboratory’s contributions to the fascinating world of skin stem cells. PMID:19022769

  18. Epigenetics in Intestinal Epithelial Cell Renewal

    PubMed Central

    Roostaee, Alireza; Benoit, Yannick D.; Boudjadi, Salah

    2016-01-01

    A controlled balance between cell proliferation and differentiation is essential to maintain normal intestinal tissue renewal and physiology. Such regulation is powered by several intracellular pathways that are translated into the establishment of specific transcription programs, which influence intestinal cell fate along the crypt‐villus axis. One important check‐point in this process occurs in the transit amplifying zone of the intestinal crypts where different signaling pathways and transcription factors cooperate to manage cellular proliferation and differentiation, before secretory or absorptive cell lineage terminal differentiation. However, the importance of epigenetic modifications such as histone methylation and acetylation in the regulation of these processes is still incompletely understood. There have been recent advances in identifying the impact of histone modifications and chromatin remodelers on the proliferation and differentiation of normal intestinal crypt cells. In this review we discuss recent discoveries on the role of the cellular epigenome in intestinal cell fate, development, and tissue renewal. J. Cell. Physiol. 231: 2361–2367, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:27061836

  19. Elevated tropomyosin expression is associated with epithelial-mesenchymal transition of lens epithelial cells.

    PubMed

    Kubo, Eri; Hasanova, Nailia; Fatma, Nigar; Sasaki, Hiroshi; Singh, Dhirendra P

    2013-01-01

    Injury to lens epithelial cells (LECs) leads to epithelial-mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1α/2β suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of α-smooth muscle actin (α-SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1α/2β following EMT/PCO formation. Expression of Tpm1α/2β was up-regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non-cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2β mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1α/2β, as evidenced by increased expression of α-SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm-based inhibitors for postponing PCO and cataractogenesis.

  20. Azithromycin kills invasive Aggregatibacter actinomycetemcomitans in gingival epithelial cells.

    PubMed

    Lai, Pin-Chuang; Walters, John D

    2013-03-01

    Aggregatibacter actinomycetemcomitans invades periodontal pocket epithelium and is therefore difficult to eliminate by periodontal scaling and root planing. It is susceptible to azithromycin, which is taken up by many types of mammalian cells. This led us to hypothesize that azithromycin accumulation by gingival epithelium could enhance the killing of intraepithelial A. actinomycetemcomitans. [(3)H]azithromycin transport by Smulow-Glickman gingival epithelial cells and SCC-25 oral epithelial cells was characterized. To test our hypothesis, we infected cultured Smulow-Glickman cell monolayers with A. actinomycetemcomitans (Y4 or SUNY 465 strain) for 2 h, treated them with gentamicin to eliminate extracellular bacteria, and then incubated them with azithromycin for 1 to 4 h. Viable intracellular bacteria were released, plated, and enumerated. Azithromycin transport by both cell lines exhibited Michaelis-Menten kinetics and was competitively inhibited by l-carnitine and several other organic cations. Cell incubation in medium containing 5 μg/ml azithromycin yielded steady-state intracellular concentrations of 144 μg/ml in SCC-25 cells and 118 μg/ml in Smulow-Glickman cells. Azithromycin induced dose- and time-dependent intraepithelial killing of both A. actinomycetemcomitans strains. Treatment of infected Smulow-Glickman cells with 0.125 μg/ml azithromycin killed approximately 29% of the intraepithelial CFU of both strains within 4 h, while treatment with 8 μg/ml azithromycin killed ≥82% of the CFU of both strains (P < 0.05). Addition of carnitine inhibited the killing of intracellular bacteria by azithromycin (P < 0.05). Thus, human gingival epithelial cells actively accumulate azithromycin through a transport system that facilitates the killing of intraepithelial A. actinomycetemcomitans and is shared with organic cations.

  1. Differentiation of cultured epithelial cells: response to toxic agents.

    PubMed Central

    Rice, R H; LaMontagne, A D; Petito, C T; Rong, X H

    1989-01-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Great differences were evident even among those cells derived from stratified squamous epithelia (epidermal, esophageal, vaginal, forestomach) despite their expression of aryl hydrocarbon hydroxylase activities to similar degrees. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAMP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Although expressing keratinocyte character (transglutaminase activity and envelope forming ability), the cells thus retain some hormonal character that may be modulated by cAMP-dependent kinase activity. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents. Images FIGURE 1. FIGURE 4. PMID:2466642

  2. 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

  3. CCL20, (gamma)(delta) T cells, and IL-22 in corneal epithelial healing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6 (+) IL-17(+) gamma delta T cells migrate into the epithelium. Gamma delta T-cell-deficient (TCRd(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mR...

  4. Left–right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    PubMed Central

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-01-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left–right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left–right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left–right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction. PMID:26656655

  5. Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

    PubMed Central

    Joshi, Sagar D.; Davidson, Lance A.

    2010-01-01

    Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis. PMID:20498627

  6. XB130 promotes bronchioalveolar stem cell and Club cell proliferation in airway epithelial repair and regeneration

    PubMed Central

    Toba, Hiroaki; Wang, Yingchun; Bai, Xiaohui; Zamel, Ricardo; Cho, Hae-Ra; Liu, Hongmei; Lira, Alonso; Keshavjee, Shaf; Liu, Mingyao

    2015-01-01

    Proliferation of bronchioalveolar stem cells (BASCs) is essential for epithelial repair. XB130 is a novel adaptor protein involved in the regulation of epithelial cell survival, proliferation and migration through the PI3K/Akt pathway. To determine the role of XB130 in airway epithelial injury repair and regeneration, a naphthalene-induced airway epithelial injury model was used with XB130 knockout (KO) mice and their wild type (WT) littermates. In XB130 KO mice, at days 7 and 14, small airway epithelium repair was significantly delayed with fewer number of Club cells (previously called Clara cells). CCSP (Club cell secreted protein) mRNA expression was also significantly lower in KO mice at day 7. At day 5, there were significantly fewer proliferative epithelial cells in the KO group, and the number of BASCs significantly increased in WT mice but not in KO mice. At day 7, phosphorylation of Akt, GSK-3β, and the p85α subunit of PI3K was observed in airway epithelial cells in WT mice, but to a much lesser extent in KO mice. Microarray data also suggest that PI3K/Akt-related signals were regulated differently in KO and WT mice. An inhibitory mechanism for cell proliferation and cell cycle progression was suggested in KO mice. XB130 is involved in bronchioalveolar stem cell and Club cell proliferation, likely through the PI3K/Akt/GSK-3β pathway. PMID:26360608

  7. Oral Epithelial Cell Responses to Multispecies Microbial Biofilms

    PubMed Central

    Peyyala, R.; Kirakodu, S.S.; Novak, K.F.; Ebersole, J.L

    2013-01-01

    This report describes the use of a novel model of multispecies biofilms to stimulate profiles of cytokines/chemokines from oral epithelial cells that contribute to local inflammation in the periodontium. Streptococcus gordonii (Sg)/S. oralis (So)/S. sanguinis (Ss) and Sg/Fusobacterium nucleatum (Fn)/Porphyromonas gingivalis (Pg) biofilms elicited significantly elevated levels of IL-1α and showed synergistic stimulatory activity compared with an additive effect of the 3 individual bacteria. Only the Sg/Actinomyces naeslundii (An)/Fn multispecies biofilms elicited IL-6 levels above those of control. IL-8 was a primary response to the Sg/An/Fn biofilms, albeit the level was not enhanced compared with a predicted composite level from the monospecies challenges. These results represent some of the first data documenting alterations in profiles of oral epithelial cell responses to multispecies biofilms. PMID:23300185

  8. Total pancreatectomy for metachronous mixed acinar-ductal carcinoma in a remnant pancreas.

    PubMed

    Shonaka, Tatsuya; Inagaki, Mitsuhiro; Akabane, Hiromitsu; Yanagida, Naoyuki; Shomura, Hiroki; Yanagawa, Nobuyuki; Oikawa, Kensuke; Nakano, Shiro

    2014-09-07

    In October 2009, a 71-year-old female was diagnosed with a cystic tumor in the tail of the pancreas with an irregular dilatation of the main pancreatic duct in the body and tail of the pancreas. A distal pancreatectomy with splenectomy, and partial resection of the duodenum, jejunum and transverse colon was performed. In March 2011, a follow-up computed tomography scan showed a low density mass at the head of the remnant pancreas. We diagnosed it as a recurrence of the tumor and performed a total pancreatectomy for the remnant pancreas. In the histological evaluation of the resected specimen of the distal pancreas, the neoplastic cells formed an acinar and papillary structure that extended into the main pancreatic duct. Mucin5AC, α1-antitrypsin (α-AT) and carcinoembryonic antigen (CEA) were detected in the tumor cells by immunohistochemistry. In the resected head of the pancreas, the tumor was composed of both acinar and ductal elements with a mottled pattern. The proportions of each element were approximately 40% and 60%, respectively. Strongly positive α-AT cells were detected in the acinar element. Some tumor cells were also CEA positive. However, the staining for synaptophysin and chromogranin A was negative in the tumor cells. Ultimately, we diagnosed the tumor as a recurrence of mixed acinar-ductal carcinoma in the remnant pancreas. In conclusion, we report here a rare case of repeated pancreatic resection for multicentric lesions of mixed acinar-ductal carcinoma of the pancreas.

  9. Maintenance of Epithelial Stem Cells by Cbl Proteins

    DTIC Science & Technology

    2013-09-01

    our research findings during the entire grant period (Sept. 2010 – Aug. 2013). 1. Analysis of Cbl functions in progenitor-type mammary epithelial...catenin pathway, but further investigation is required to establish this. 2. Analysis of Cbl functions in vivo using gene mutant mouse models We...Nandwani N, Gu H, Band V, Band H. Rapidly fatal myeloproliferative disorders in mice with deletion of Casitas B-cell lymphoma (Cbl) and Cbl-b in

  10. Norepinephrine potentiates proinflammatory responses of human vaginal epithelial cells.

    PubMed

    Brosnahan, Amanda J; Vulchanova, Lucy; Witta, Samantha R; Dai, Yuying; Jones, Bryan J; Brown, David R

    2013-06-15

    The vaginal epithelium provides a barrier to pathogens and recruits immune defenses through the secretion of cytokines and chemokines. Several studies have shown that mucosal sites are innervated by norepinephrine-containing nerve fibers. Here we report that norepinephrine potentiates the proinflammatory response of human vaginal epithelial cells to products produced by Staphylococcus aureus, a pathogen that causes menstrual toxic shock syndrome. The cells exhibit immunoreactivity for catecholamine synthesis enzymes and the norepinephrine transporter. Moreover, the cells secrete norepinephrine and dopamine at low concentrations. These results indicate that norepinephrine may serve as an autocrine modulator of proinflammatory responses in the vaginal epithelium.

  11. Interaction between submicron COD crystals and renal epithelial cells

    PubMed Central

    Peng, Hua; Ouyang, Jian-Ming; Yao, Xiu-Qiong; Yang, Ru-E

    2012-01-01

    Objectives This study aims to investigate the adhesion characteristics between submicron calcium oxalate dihydrate (COD) with a size of 150 ± 50 nm and African green monkey kidney epithelial cells (Vero cells) before and after damage, and to discuss the mechanism of kidney stone formation. Methods Vero cells were oxidatively injured by hydrogen peroxide to establish a model of injured cells. Scanning electron microscopy was used to observe Vero–COD adhesion. Inductively coupled plasma emission spectrometry was used to quantitatively measure the amount of adhered COD microcrystals. Nanoparticle size analyzer and laser scanning confocal microscopy were performed to measure the change in the zeta potential on the Vero cell surface and the change in osteopontin expression during the adhesion process, respectively. The level of cell injury was evaluated by measuring the changes in malonaldehyde content, and cell viability during the adhesion process. Results The adhesion capacity of Vero cells in the injury group to COD microcrystals was obviously stronger than that of Vero cells in the control group. After adhesion to COD, cell viability dropped, both malonaldehyde content and cell surface zeta potential increased, and the fluorescence intensity of osteopontin decreased because the osteopontin molecules were successfully covered by COD. Submicron COD further damaged the cells during the adhesion process, especially for Vero cells in the control group, leading to an elevated amount of attached microcrystals. Conclusion Submicron COD can further damage injured Vero cells during the adhesion process. The amount of attached microcrystals is proportional to the degree of cell damage. The increased amount of microcrystals that adhered to the injured epithelial cells plays an important role in the formation of early-stage kidney stones. PMID:22973095

  12. Erythropoietin Induces an Epithelial to Mesenchymal Transition-like Process in Mammary Epithelial Cells MCF10A.

    PubMed

    Ordoñez-Moreno, Alejandra; Rodriguez-Monterrosas, Cecilia; Cortes-Reynosa, Pedro; Perez-Carreon, Julio Isael; Perez Salazar, Eduardo

    2017-03-01

    Anemia is associated with chemotherapy treatment in cancer patients. Erythropoietin (EPO) has been used to treat anemia of cancer patients, because it stimulates erythropoiesis. However, treatment of breast cancer patients with EPO has been associated with poor prognosis and decrease of survival. Epithelial to mesenchymal transition (EMT) is a process by which epithelial cells are transdifferentiated to a mesenchymal state. It has been implicated in tumor progression, because epithelial cells acquire the capacity to execute the multiple steps of invasion/metastasis process. However, the role of EPO on EMT process in human mammary epithelial cells has not been studied. In the present study, we demonstrate that EPO promotes a decrease of E-cadherin expression, an increase of N-cadherin, vimentin and Snail2 expression, activation of FAK and Src kinases and an increase of MMP-2 and MMP-9 secretions. Moreover, EPO induces an increase of NFκB DNA binding activity, an increase of binding of p50 and p65 NFκB subunits to Snail1 promoter, migration and invasion in mammary non-tumorigenic epithelial cells MCF10A. In summary, these findings demonstrate, for the first time, that EPO induces an EMT-like process in mammary non-tumorigenic epithelial cells. This article is protected by copyright. All rights reserved.

  13. Epithelial stem cells are formed by small-particles released from particle-producing cells

    PubMed Central

    Kong, Wuyi; Zhu, Xiao Ping; Han, Xiu Juan; Nuo, Mu; Wang, Hong

    2017-01-01

    Recent spatiotemporal report demonstrated that epidermal stem cells have equal potential to divide or differentiate, with no asymmetric cell division observed. Therefore, how epithelial stem cells maintain lifelong stem-cell support still needs to be elucidated. In mouse blood and bone marrow, we found a group of large cells stained strongly for eosin and containing coiled-tubing-like structures. Many were tightly attached to each other to form large cellular clumps. After sectioning, these large cell-clumps were composed of not cells but numerous small particles, however with few small “naked” nuclei. The small particles were about 2 to 3 μm in diameter and stained dense red for eosin, so they may be rich in proteins. Besides the clumps composed of small particles, we identified clumps formed by fusion of the small particles and clumps of newly formed nucleated cells. These observations suggest that these small particles further fused and underwent cellularization. E-cadherin was expressed in particle-fusion areas, some “naked” nuclei and the newly formed nucleated cells, which suggests that these particles can form epithelial cells via fusion and nuclear remodeling. In addition, we observed similar-particle fusion before epithelial cellularization in mouse kidney ducts after kidney ischemia, which suggests that these particles can be released in the blood and carried to the target tissues for epithelial-cell regeneration. Oct4 and E-cadherin expressed in the cytoplasmic areas in cells that were rich in protein and mainly located in the center of the cellular clumps, suggesting that these newly formed cells have become tissue-specific epithelial stem cells. Our data provide evidence that these large particle-producing cells are the origin of epithelial stem cells. The epithelial stem cells are newly formed by particle fusion. PMID:28253358

  14. A dynamic podosome-like structure of epithelial cells.

    PubMed

    Spinardi, Laura; Rietdorf, Jens; Nitsch, Lucio; Bono, Maria; Tacchetti, Carlo; Way, Michael; Marchisio, Pier Carlo

    2004-05-01

    Focal contacts and hemidesmosomes are cell-matrix adhesion structures of cultured epithelial cells. While focal contacts link the extracellular matrix to microfilaments, hemidesmosomes make connections with intermediate filaments. We have analyzed hemidesmosome assembly in 804G carcinoma cells. Our data show that hemidesmosomes are organized around a core of actin filaments that appears early during cell adhesion. These actin structures look similar to podosomes described in cells of mesenchymal origin. These podosome-like structures are distinct from focal contacts and specifically contain Arp3 (Arp2/3 complex), cortactin, dynamin, gelsolin, N-WASP, VASP, Grb2 and src-like kinase(s). The integrin alpha3beta1 is localized circularly around F-actin cores and co-distributes with paxillin, vinculin, and zyxin. We also show that the maintenance of the actin core and hemidesmosomes is dependent on actin polymerization, src-family kinases, and Grb2, but not on microtubules. Video microscopy analysis reveals that assembly of hemidesmosomes is preceded by recruitment of beta4 integrin subunit to the actin core before its positioning at hemidesmosomes. When 804G cells are induced to migrate, actin cores as well as hemidesmosomes disappear and beta4 integrin subunit becomes co-localized with dynamic actin at leading edges. We show that podosome-like structures are not unique to cells of mesenchymal origin, but also appear in epithelial cells, where they seem to be related to basement membrane adhesion.

  15. Airway Epithelial Cell Cilia and Obstructive Lung Disease

    PubMed Central

    Yaghi, Asma; Dolovich, Myrna B.

    2016-01-01

    Airway epithelium is the first line of defense against exposure of the airway and lung to various inflammatory stimuli. Ciliary beating of airway epithelial cells constitutes an important part of the mucociliary transport apparatus. To be effective in transporting secretions out of the lung, the mucociliary transport apparatus must exhibit a cohesive beating of all ciliated epithelial cells that line the upper and lower respiratory tract. Cilia function can be modulated by exposures to endogenous and exogenous factors and by the viscosity of the mucus lining the epithelium. Cilia function is impaired in lung diseases such as COPD and asthma, and pharmacologic agents can modulate cilia function and mucus viscosity. Cilia beating is reduced in COPD, however, more research is needed to determine the structural-functional regulation of ciliary beating via all signaling pathways and how this might relate to the initiation or progression of obstructive lung diseases. Additionally, genotypes and how these can influence phenotypes and epithelial cell cilia function and structure should be taken into consideration in future investigations. PMID:27845721

  16. Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells.

    PubMed Central

    Corbeil, L B; Hodgson, J L; Jones, D W; Corbeil, R R; Widders, P R; Stephens, L R

    1989-01-01

    Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells (VECs) in vitro was investigated with fresh washed bovine VECs and log-phase cultures of T. foetus. Observation under phase-contrast microscopy showed that T. foetus usually adhered first by the posterior flagellum and later by the body. Significantly more keratinized squamous epithelial cells were detected with attached parasites than nonkeratinized round epithelial cells. The optimal pH range for attachment was 6.0 to 7.5, with peak attachment at pH 6.5 for squamous VECs. Surface-reactive bovine antiserum to T. foetus prevented adherence to bovine squamous VECs. Inhibition of adherence occurred at nonagglutinating, nonimmobilizing serum dilutions. Antiserum fractions enriched for immunoglobulin G1 inhibited adherence, but fractions enriched for immunoglobulin G2 did not. The inhibitory antiserum was specific for several medium- to high-molecular-weight membrane antigens as detected in Western blots (immunoblots). The ability of surface-reactive antibodies to prevent adherence and to agglutinate and immobilize T. foetus indicates that they may be protective. Images PMID:2471692

  17. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2007-10-01

    robust and reproducible methodology to detect, quantify and isolate stem cells in normal human mammary tissue, using a xenotransplantation system...covers the first year of the grant, during which substantial progress has been made in the development and validation of the xenotransplantation assay...Subrenal xenotransplantation surgery. The hair on the back of anesthetized mice was shaved, and the skin swabbed with 70% alcohol. An anterior to

  18. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2008-10-01

    9 Appendix……………………………………………………………………………… 10 Eirew,P., Stingl,J., Raouf,A., Turashvili,G., Aparicio ,S., Emerman,J.T., and Eaves,C.J. A...Peter Eirew, John Stingl, Afshin Raouf, Gulisa Turashvili, Samuel Aparicio , Joanne Emerman and Connie Eaves. A method for quantifying normal human...Eirew, Afshin Raouf, John Stingl, Gulisa Turashvili, Allen Delaney, Joanne Emerman, Marco Marra and Samuel Aparicio . “Stem Cells in the Mammary Gland

  19. Synthesis of sulfated oligosaccharides by cystic fibrosis trachea epithelial cells.

    PubMed

    Mendicino, J; Sangadala, S

    1999-11-01

    The mucin glycoproteins in tracheal mucus of patients with cystic fibrosis is more highly sulfated than the corresponding secretions from healthy individuals [16]. In order to further characterize these differences in sulfation and possibly also glycosylation patterns, we compared the structures of sulfated mucin oligosaccharides synthesized by continuously cultured human tracheal cells transformed by simian virus 40. The synthesis of highly sulfated oligosaccharide chains in mucins secreted by normal human epithelial and submucosal cell lines were compared with mucins formed by cystic fibrosis tracheal epithelial and submucosal cell lines. The epithelial cell lines from cystic fibrosis trachea showed a higher rate of sulfate uptake and a significantly higher rate of synthesis and sulfation of high molecular weight chains. Mucins synthesized by each cell line in the presence of 35SO4 were isolated and oligosaccharide chains were released by beta-elimination and separated by ion exchange chromatography and gel filtration. The sulfated high molecular weight chains synthesized by the cystic fibrosis cell lines were characterized by methylation analysis and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GlcNAc in a ratio of 1:2:2.2 and only one galactosaminitol residue for about every 150-200 sugar residues present. The average molecular size of oligosaccharide chains in these fractions was between 30,000-40,000 daltons. These studies show that increased sulfation of oligosaccharides in mucins synthesized by cells from cystic fibrosis trachea is accompanied by a significant increase in the extension of a basic branched structure present in many of the lower molecular weight oligosaccharides.

  20. Slugging their way to immortality: driving mammary epithelial cells into a stem cell-like state.

    PubMed

    Soady, Kelly; Smalley, Matthew J

    2012-09-10

    Delineating the molecular factors that define and maintain the mammary stem cell state is vital for understanding normal development and tumourigenesis. A recent study by Guo and colleagues identifies two master transcriptional regulators of mammary stem cells, Slug and Sox9, ectopic expression of which confers stem cell attributes on differentiated mammary epithelial cells. Slug and Sox9 expression was also shown to determine in vivo metastatic potential of human breast cancer cell lines. Understanding these factors in the context of normal lineage differentiation is an important step toward elucidating the mammary epithelial cell hierarchy and the origins of cancer stem cells.

  1. Human induced pluripotent stem cell differentiation and direct transdifferentiation into corneal epithelial-like cells

    PubMed Central

    Cieślar-Pobuda, Artur; Rafat, Mehrdad; Knoflach, Viktoria; Skonieczna, Magdalena; Hudecki, Andrzej; Małecki, Andrzej; Urasińska, Elżbieta; Ghavami, Seaid; Łos, Marek J.

    2016-01-01

    The corneal epithelium is maintained by a small pool of tissue stem cells located at the limbus. Through certain injuries or diseases this pool of stem cells may get depleted. This leads to visual impairment. Standard treatment options include autologous or allogeneic limbal stem cell (LSC) transplantation, however graft rejection and chronic inflammation lowers the success rate over long time. Induced pluripotent stem (iPS) cells have opened new possibilities for treating various diseases with patient specific cells, eliminating the risk of immune rejection. In recent years, several protocols have been developed, aimed at the differentiation of iPS cells into the corneal epithelial lineage by mimicking the environmental niche of limbal stem cells. However, the risk of teratoma formation associated with the use of iPS cells hinders most applications from lab into clinics. Here we show that the differentiation of iPS cells into corneal epithelial cells results in the expression of corneal epithelial markers showing a successful differentiation, but the process is long and the level of gene expression for the pluripotency markers does not vanish completely. Therefore we set out to determine a direct transdifferentiation approach to circumvent the intermediate state of pluripotency (iPS-stage). The resulting cells, obtained by direct transdifferentiation of fibroblasts into limbal cells, exhibited corneal epithelial cell morphology and expressed corneal epithelial markers. Hence we shows for the first time a direct transdifferentiation of human dermal fibroblasts into the corneal epithelial lineage that may serve as source for corneal epithelial cells for transplantation approaches. PMID:27275539

  2. Influences of nanomaterials on the barrier function of epithelial cells.

    PubMed

    Ali, Shariq; Rytting, Erik

    2014-01-01

    Recent advances in nanotechnology have led to exciting opportunities in medicine, energy, manufacturing, and other fields. Nevertheless, it is important to adequately assess the potential impacts of nanomaterial exposure. This chapter focuses on the interactions of nanomaterials with epithelial barriers in the lungs, intestine, kidneys, skin, and placenta. Methods for determining transepithelial electrical resistance and paracellular permeability are described. Effects on cell viability and barrier integrity depend on the chemical nature of the nanomaterial, nanoparticle size, surface coatings, and concentration. Disruption of tight junctions can affect permeability and interfere with normal regulatory processes of the epithelial barrier. Future research is needed to better understand the possibilities and the limits of novel approaches in nanotechnology.

  3. Efficient cultivation conditions for human limbal epithelial cells.

    PubMed

    Kim, Mee Kum; Lee, Jae Lim; Oh, Joo Youn; Shin, Mi Sun; Shin, Kyeong Seon; Wee, Won Ryang; Lee, Jin Hak; Park, Ki Sook; Son, Young Sook

    2008-10-01

    To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding cassette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

  4. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-03-15

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  5. 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

  6. Inflammation and the Intestinal Barrier: Leukocyte–Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair

    PubMed Central

    Luissint, Anny-Claude; Parkos, Charles A.; Nusrat, Asma

    2017-01-01

    The intestinal tract is lined by a single layer of columnar epithelial cells that forms a dynamic, permeable barrier allowing for selective absorption of nutrients, while restricting access to pathogens and food-borne antigens. Precise regulation of epithelial barrier function is therefore required for maintaining mucosal homeostasis and depends, in part, on barrier-forming elements within the epithelium and a balance between pro- and anti-inflammatory factors in the mucosa. Pathologic states, such as inflammatory bowel disease, are associated with a leaky epithelial barrier, resulting in excessive exposure to microbial antigens, recruitment of leukocytes, release of soluble mediators, and ultimately mucosal damage. An inflammatory microenvironment affects epithelial barrier properties and mucosal homeostasis by altering the structure and function of epithelial intercellular junctions through direct and indirect mechanisms. We review our current understanding of complex interactions between the intestinal epithelium and immune cells, with a focus on pathologic mucosal inflammation and mechanisms of epithelial repair. We discuss leukocyte–epithelial interactions, as well as inflammatory mediators that affect the epithelial barrier and mucosal repair. Increased knowledge of communication networks between the epithelium and immune system will lead to tissue-specific strategies for treating pathologic intestinal inflammation. PMID:27436072

  7. The acinar differentiation determinant PTF1A inhibits initiation of pancreatic ductal adenocarcinoma

    PubMed Central

    Krah, Nathan M; De La O, Jean-Paul; Swift, Galvin H; Hoang, Chinh Q; Willet, Spencer G; Chen Pan, Fong; Cash, Gabriela M; Bronner, Mary P; Wright, Christopher VE; MacDonald, Raymond J; Murtaugh, L Charles

    2015-01-01

    Understanding the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) may provide therapeutic strategies for this deadly disease. Recently, we and others made the surprising finding that PDAC and its preinvasive precursors, pancreatic intraepithelial neoplasia (PanIN), arise via reprogramming of mature acinar cells. We therefore hypothesized that the master regulator of acinar differentiation, PTF1A, could play a central role in suppressing PDAC initiation. In this study, we demonstrate that PTF1A expression is lost in both mouse and human PanINs, and that this downregulation is functionally imperative in mice for acinar reprogramming by oncogenic KRAS. Loss of Ptf1a alone is sufficient to induce acinar-to-ductal metaplasia, potentiate inflammation, and induce a KRAS-permissive, PDAC-like gene expression profile. As a result, Ptf1a-deficient acinar cells are dramatically sensitized to KRAS transformation, and reduced Ptf1a greatly accelerates development of invasive PDAC. Together, these data indicate that cell differentiation regulators constitute a new tumor suppressive mechanism in the pancreas. DOI: http://dx.doi.org/10.7554/eLife.07125.001 PMID:26151762

  8. Epithelial cells from smokers modify dendritic cell responses in the context of influenza infection

    EPA Science Inventory

    Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza...

  9. ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

  10. Radial transport along the human acinar tree.

    PubMed

    Henry, F S; Tsuda, A

    2010-10-01

    A numerical model of an expanding asymmetric alveolated duct was developed and used to investigate lateral transport between the central acinar channel and the surrounding alveoli along the acinar tree. Our results indicate that some degree of recirculation occurs in all but the terminal generations. We found that the rate of diffusional transport of axial momentum from the duct to the alveolus was by far the largest contributor to the resulting momentum in the alveolar flow but that the magnitude of the axial momentum is critical in determining the nature of the flow in the alveolus. Further, we found that alveolar flow rotation, and by implication chaotic mixing, is strongest in the entrance generations. We also found that the expanding alveolus provides a pathway by which particles with little intrinsic motion can enter the alveoli. Thus, our results offer a possible explanation for why submicron particles deposit preferentially in the acinar-entrance region.

  11. RADIAL TRANSPORT ALONG THE HUMAN ACINAR TREE

    PubMed Central

    Henry, F.S.; Tsuda, A.

    2013-01-01

    A numerical model of an expanding asymmetric alveolated duct was developed and used to investigate lateral transport between the central acinar channel and the surrounding alveoli along the acinar tree. Our results indicate that some degree of recirculation occurs in all but the terminal generations. We found that the rate of diffusional transport of axial momentum, from the duct to the alveolus, was by far the largest contributor to the resulting momentum in the alveolar flow but that the magnitude of the axial momentum is critical in determining the nature of the flow in the alveolus. Further, we found that alveolar flow rotation, and by implication chaotic mixing, are strongest in the entrance generations. We also found that the expanding alveolus provides a pathway by which particles with little intrinsic motion can enter the alveoli. Thus, our results offer a possible explanation for why submicron particles deposit preferentially in acinar entrance region. PMID:20887011

  12. Stereological Quantification of Cell-Cycle Kinetics and Mobilization of Epithelial Stem Cells during Wound Healing.

    PubMed

    Martínez-Martínez, Eduardo; Uribe-Querol, Eileen; Galván-Hernández, Claudio I; Gutiérrez-Ospina, Gabriel

    2016-01-01

    We describe a stereology method to obtain reliable estimates of the total number of proliferative and migratory epithelial cells after wounding. Using pulse and chase experiments with halogenated thymidine analogs such as iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU), it is possible to track epithelial populations with heterogeneous proliferative characteristics through skin compartments. The stereological and tissue processing methods described here apply widely to address important questions of skin stem-cell biology.

  13. Protocadherin-1 Localization and Cell-Adhesion Function in Airway Epithelial Cells in Asthma

    PubMed Central

    Faura Tellez, Grissel; Willemse, Brigitte W. M.; Brouwer, Uilke; Nijboer-Brinksma, Susan; Vandepoele, Karl; Noordhoek, Jacobien A.; Heijink, Irene; de Vries, Maaike; Smithers, Natalie P.; Postma, Dirkje S.; Timens, Wim; Wiffen, Laura; van Roy, Frans; Holloway, John W.; Lackie, Peter M.; Nawijn, Martijn C.; Koppelman, Gerard H.

    2016-01-01

    Background The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma. Methods We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance. Results PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding. Conclusions In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair. PMID:27701444

  14. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    SciTech Connect

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  15. Effects of amniotic epithelial cell transplantation in endothelial injury

    PubMed Central

    Vácz, Gabriella; Cselenyák, Attila; Cserép, Zsuzsanna; Benkő, Rita; Kovács, Endre; Pankotai, Eszter; Lindenmair, Andrea; Wolbank, Susanne; Schwarz, Charlotte M.; Horváthy, Dénes B.; Kiss, Levente; Hornyák, István; Lacza, Zsombor

    2016-01-01

    Purpose Human amniotic epithelial cells (hAECs) are promising tools for endothelial repair in vascular regenerative medicine. We hypothesized that these epithelial cells are capable of repairing the damaged endothelial layer following balloon injury of the carotid artery in adult male rats. Results Two days after injury, the transplanted hAECs were observed at the luminal side of the arterial wall. Then, 4 weeks after the injury, significant intimal thickening was observed in both untreated and cell implanted vessels. Constriction was decreased in both implanted and control animals. Immunohistochemical analysis showed a few surviving cells in the intact arterial wall, but no cells were observed at the site of injury. Interestingly, acetylcholine-induced dilation was preserved in the intact side and the sham-transplanted injured arteries, but it was a trend toward decreased vasodilation in the hAECs’ transplanted vessels. Conclusion We conclude that hAECs were able to incorporate into the arterial wall without immunosuppression, but failed to improve vascular function, highlighting that morphological implantation does not necessarily result in functional benefits and underscoring the need to understand other mechanisms of endothelial regeneration. PMID:28180006

  16. In vitro adhesion of Escherichia coli to porcine small intestinal epithelial cells: pili as adhesive factors.

    PubMed Central

    Isaacson, R E; Fusco, P C; Brinton, C C; Moon, H W

    1978-01-01

    Escherichia coli strains with pili (K99 or 987P) known to facilitate intestinal colonization adhered in vitro to porcine intestinal epithelial cells. These strains adhered equally to both ileal and jejunal epithelial cells. A laboratory E. coli strain that has type 1 pili also adhered to porcine intestinal epithelial cells. When nonpiliated cells derived from 987P+, K99+, or type 1 pilus+ strains were used for in vitro adhesion assays, they failed to adhere. The attachment of piliated bacteria to epithelial cells was a saturable process that plateaued at 30 to 40 bacterial cells attached per epithelial cell. Competitive inhibition of bacterial cell attachment to epithelial cells with purified pili showed that only purified 987P competed against the 987P+ strain and only purified type 1 pili competed against the type 1 pilus+ strain. Competition between a K99+ strain and K99 was not consistently achieved. K99+, 987P+, and type 1 pilus+ bacteria could be prevented from adhering to epithelial cells by Fab fragments specific for K99, 987P, or type 1 pili, respectively. Fab fragments specific for non-K99 bacterial surface antigens did not inhibit adhesion of the K99+ strain. It is concluded that adhesion of E. coli to porcine intestinal epithelial cells in vitro is mediated by pili and that the epithelial cells used apparently had different receptors for different pili. PMID:357285

  17. In vitro adhesion of Escherichia coli to porcine small intestinal epithelial cells: pili as adhesive factors.

    PubMed

    Isaacson, R E; Fusco, P C; Brinton, C C; Moon, H W

    1978-08-01

    Escherichia coli strains with pili (K99 or 987P) known to facilitate intestinal colonization adhered in vitro to porcine intestinal epithelial cells. These strains adhered equally to both ileal and jejunal epithelial cells. A laboratory E. coli strain that has type 1 pili also adhered to porcine intestinal epithelial cells. When nonpiliated cells derived from 987P+, K99+, or type 1 pilus+ strains were used for in vitro adhesion assays, they failed to adhere. The attachment of piliated bacteria to epithelial cells was a saturable process that plateaued at 30 to 40 bacterial cells attached per epithelial cell. Competitive inhibition of bacterial cell attachment to epithelial cells with purified pili showed that only purified 987P competed against the 987P+ strain and only purified type 1 pili competed against the type 1 pilus+ strain. Competition between a K99+ strain and K99 was not consistently achieved. K99+, 987P+, and type 1 pilus+ bacteria could be prevented from adhering to epithelial cells by Fab fragments specific for K99, 987P, or type 1 pili, respectively. Fab fragments specific for non-K99 bacterial surface antigens did not inhibit adhesion of the K99+ strain. It is concluded that adhesion of E. coli to porcine intestinal epithelial cells in vitro is mediated by pili and that the epithelial cells used apparently had different receptors for different pili.

  18. LOXL2 induces aberrant acinar morphogenesis via ErbB2 signaling

    PubMed Central

    2013-01-01

    Introduction Lysyl oxidase-like 2 (LOXL2) is a matrix-remodeling enzyme that has been shown to play a key role in invasion and metastasis of breast carcinoma cells. However, very little is known about its role in normal tissue homeostasis. Here, we investigated the effects of LOXL2 expression in normal mammary epithelial cells to gain insight into how LOXL2 mediates cancer progression. Methods LOXL2 was expressed in MCF10A normal human mammary epithelial cells. The 3D acinar morphogenesis of these cells was assessed, as well as the ability of the cells to form branching structures on extracellular matrix (ECM)-coated surfaces. Transwell-invasion assays were used to assess the invasive properties of the cells. Clinically relevant inhibitors of ErbB2, lapatinib and Herceptin (traztuzumab), were used to investigate the role of ErbB2 signaling in this model. A retrospective study on a previously published breast cancer patient dataset was carried out by using Disease Specific Genomic Analysis (DSGA) to investigate the correlation of LOXL2 mRNA expression level with metastasis and survival of ErbB2-positive breast cancer patients. Results Fluorescence staining of the acini revealed increased proliferation, decreased apoptosis, and disrupted polarity, leading to abnormal lumen formation in response to LOXL2 expression in MCF10A cells. When plated onto ECM, the LOXL2-expressing cells formed branching structures and displayed increased invasion. We noted that LOXL2 induced ErbB2 activation through reactive oxygen species (ROS) production, and ErbB2 inhibition by using Herceptin or lapatinib abrogated the effects of LOXL2 on MCF10A cells. Finally, we found LOXL2 expression to be correlated with decreased overall survival and metastasis-free survival in breast cancer patients with ErbB2-positive tumors. Conclusions These findings suggest that LOXL2 expression in normal epithelial cells can induce abnormal changes that resemble oncogenic transformation and cancer progression

  19. Bimodal Analysis of Mammary Epithelial Cell Migration in Two Dimensions

    PubMed Central

    Potdar, Alka A.; Lu, Jenny; Jeon, Junhwan; Weaver, Alissa M.; Cummings, Peter T.

    2013-01-01

    Cell migration paths of mammary epithelial cells (expressing different versions of the promigratory tyrosine kinase receptor Her2/Neu) were analyzed within a bimodal framework that is a generalization of the run-and-tumble description applicable to bacterial migration. The mammalian cell trajectories were segregated into two types of alternating modes, namely, the “directional-mode” (mode I, the more persistent mode, analogous to the bacterial run phase) and the “re-orientation-mode” (mode II, the less persistent mode, analogous to the bacterial tumble phase). Higher resolution (more pixel information, relative to cell size) and smaller sampling intervals (time between images) were found to give a better estimate of the deduced single cell dynamics (such as directional-mode time and turn angle distribution) of the various cell types from the bimodal analysis. The bimodal analysis tool permits the deduction of short-time dynamics of cell motion such as the turn angle distributions and turn frequencies during the course of cell migration compared to standard methods of cell migration analysis. We find that the two-hour mammalian cell tracking data do not fall into the diffusive regime implying that the often-used random motility expressions for mammalian cell motion (based on assuming diffusive motion) are invalid over the time steps (fraction of minute) typically used in modeling mammalian cell migration. PMID:18982450

  20. Xenobiotic induction of quinone oxidoreductase activity in lens epithelial cells.

    PubMed

    Tumminia, S J; Rao, P V; Zigler, J S; Russell, P

    1993-12-08

    Xenobiotic regulatory elements have been identified for enzymes which ameliorate oxidative damage in cells. Zeta (zeta)-crystallin, a taxon-specific enzyme/crystallin shown to be a novel NADPH-dependent quinone reductase, is found in a number of tissues and cell types. This study shows that zeta-crystallin is present in mouse lens epithelium, as well as in the alpha TN4 mouse lens epithelial cell line. To determine whether zeta-crystallin is an inducible quinone reductase, cell cultures were exposed to the xenobiotics, 1,2-naphthoquinone and beta-naphthoflavone. Assays of cellular homogenates showed that quinone reductase activity was stimulated greater than 70% and 90%, respectively, over the control cells. This observed activity was sensitive to dicumarol, a potent inhibitor of quinone reductase activity. 1,2-Naphthoquinone- and beta-naphthoflavone-exposed cells were found to exhibit 1.47- and 1.68-fold increases, respectively, in zeta-crystallin protein concentration. A comparable increase in zeta-crystallin mRNA was indicative of an induction in zeta-crystallin expression in response to naphthalene challenge. Lens epithelial cells were also checked for DT-diaphorase, a well-known cellular protective enzyme which can catalyze the two-electron reduction of quinones. Slot blot analyses indicated that alpha TN4 cells exposed to 1,2-naphthoquinone and beta-naphthoflavone exhibited 2.71- and 6.81-fold increases in DT-diaphorase concentration when compared to the control cells. The data suggest that while DT-diaphorase is most likely responsible for the majority of the observed increase in quinone reductase activity, the zeta-crystallin gene also undergoes activation which is apparently mediated by a xenobiotic-responsive element.

  1. Cholinergic regulation of epithelial sodium channels in rat alveolar type 2 epithelial cells.

    PubMed

    Takemura, Yoshizumi; Helms, My N; Eaton, Amity F; Self, Julie; Ramosevac, Semra; Jain, Lucky; Bao, Hui-Fang; Eaton, Douglas C

    2013-03-15

    We and others have shown that epithelial Na(+) channels (ENaC) in alveolar type 2 (AT2) cells are activated by β2 agonists, steroid hormones, elevated oxygen tension, and by dopamine. Although acetylcholine receptors (AChRs) have been previously described in the lung, there are few reports of whether cholinergic agonists alter sodium transport in the alveolar epithelium. Therefore, we investigated how cholinergic receptors regulate ENaC activity in primary cultures of rat AT2 cells using cell-attached patch-clamp recordings to assess ENaC activity. We found that the muscarinic agonists, carbachol (CCh) and oxotremorine, activated ENaC in a dose-dependent manner but that nicotine did not. CCh-induced activation of ENaC was blocked by atropine. Western blotting and immunohistochemistry suggested that muscarinic M2 and M3 receptors (mAChRs) but not nicotinic receptors were present in AT2 cells. Endogenous RhoA and GTP-RhoA increased in response to CCh and the increase was reduced by pretreatment with atropine. We showed that Y-27632, an inhibitor of Rho-associated protein kinase (ROCK), abolished endogenous ENaC activity and inhibited the activation of ENaC by CCh. We also showed that ROCK signaling was necessary for ENaC stability in 2F3 cells, a model for AT2 cells. Our results showed that muscarinic agonists activated ENaC in rat AT2 cells through M2 and/or M3 mAChRs probably via a RhoA/ROCK signaling pathway.

  2. Nerve growth factor modulate proliferation of cultured rabbit corneal endothelial cells and epithelial cells.

    PubMed

    Li, Xinyu; Li, Zhongguo; Qiu, Liangxiu; Zhao, Changsong; Hu, Zhulin

    2005-01-01

    In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

  3. Force dependence of phagosome trafficking in retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Daniel, Rebekah; Koll, Andrew T.; Altman, David

    2014-09-01

    Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of forces through the use of in vivo optical trapping. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking.

  4. 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

  5. Quantification of regenerative potential in primary human mammary epithelial cells.

    PubMed

    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

    2015-09-15

    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 CD49f(hi)/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.

  6. Limbal stem cells: Central concepts of corneal epithelial homeostasis.

    PubMed

    Yoon, Jinny J; Ismail, Salim; Sherwin, Trevor

    2014-09-26

    A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent studies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface.

  7. Density dependent polarized secretion of a prostatic epithelial cell line.

    PubMed

    Djakiew, D; Pflug, B; Delsite, R; Lynch, J H; Onoda, M

    1992-01-01

    The polarized secretions (apical/basal) of newly synthesized total protein and proteases from prostatic epithelial sheets of PA-III cells grown in dual compartment chambers were investigated at various cell densities and culture conditions. PA-III cells grown in a serum free defined medium (SFDM) form morphologically polarized monolayers of epithelial cells. These cells secreted their 35S-methionine labeled total protein in a predominantly apical direction (apical/basal ratio, 4-8 fold), with a lesser proportion of protein secreted apically at lower cell densities of the PA-III cell monolayer. PA-III cells grown in 5% fetal calf serum (FCS) are morphologically squamous, comparable to the anaplastic phenotype, and exhibited an inversion of polarized total protein secretion (apical/basal ratio, 0.4-0.9 fold), with an increased proportion of total protein secreted in a basal direction at lower cell densities. Since the culture of PA-III cells in FCS may approximate the anaplastic phenotype we investigated the polarized secretion of proteases from these cells at various cell densities, and compared them with the secretory pattern of protease secretion from polarized PA-III cells cultured in SFDM. At lower cell densities of the PA-III cells grown in FCS the polarity of protease secretion was inverted such that metalloproteinases, tissue type plasminogen activator, and a 72 kD gelatinase were secreted in a predominantly basal direction, as well as urokinase and a gelatinase of 26 kD that were secreted more or less equally into the apical and basal compartments of the chambers. On the other hand, for cultures of PA-III cells grown in SFDM the aforementioned proteases exhibited predominantly an apically directed polarity of secretion. These results suggest that the anaplastic phenotype characterized by a loss of polarized structure may also be characterized by a functional loss or inversion of polarized secretion. The consequences of such a loss or inversion of polarized

  8. Gradient isolation of glial cells: evidence that flat epithelial cells are astroglial cell precursors.

    PubMed

    Meller, K

    1987-07-01

    Discontinuous gradients of metrizamide were used to separate the cell components of monolayers of primary cultures of embryonic rat brains. These primary cell cultures were of two types: long-term cultures (more than a year) of embryonic rat brain, which contained several glial cell types, and monolayers of cell cultures (several weeks old), which contained a complex population of cells, including neuronal elements. The gradient separation produces fractions of pure flat epithelial cells that are able to survive and proliferate. After a few days, all flat epithelial cells become confluent and show a positive reaction to glial fibrillary acidic protein (GFAP); this indicates that these cells astroglial precursor cells. Following their maintenance in vitro for several months, all cultures give rise to a pure population of astrocytes identified not only by their characteristic morphology, but also by their content of GFAP. It is proposed that the differentiation controls are dependent on cell interactions that are influenced by the composition of the cell population and/or the molecular growth and differentiation factors released by these cells into the medium.

  9. A Common Stem Cell for Murine Cortical and Medullary Thymic Epithelial Cells?

    PubMed Central

    Van Soest, Peter; Platenburg, Peter Paul; Van Ewijk, Willem

    1995-01-01

    We have addressed the question whether the epithelial stroma in the thymus is derived from a common stem cell or whether cortical and medullary epithelial cells are derived from different embryonic stem cells emerging, for example, from endoderm and ectoderm. By the use of rapidly expanding cultures of thymic epithelial cells (TEC) from 14 to 16 day-old murine fetuses and by specific antibodies against cortical and medullary epithelium, respectively, we were able to demonstrate a small subpopulation of double-labeled TEC in the cultures. These cells were not present in TEC cultures initiated from thymuses of neonatal mice. Double-labeled TEC were also found in tissue sections from fetal thymuses. These findings may indicate that TEC populations of the cortex and the medulla are derived from a common stem cell, with potential for differentiation toward both cortical and medullary TEC. PMID:9700364

  10. Barrier Epithelial Cells and the Control of Type 2 Immunity.

    PubMed

    Hammad, Hamida; Lambrecht, Bart N

    2015-07-21

    Type-2-cell-mediated immunity, rich in eosinophils, basophils, mast cells, CD4(+) T helper 2 (Th2) cells, and type 2 innate lymphoid cells (ILC2s), protects the host from helminth infection but also drives chronic allergic diseases like asthma and atopic dermatitis. Barrier epithelial cells (ECs) represent the very first line of defense and express pattern recognition receptors to recognize type-2-cell-mediated immune insults like proteolytic allergens or helminths. These ECs mount a prototypical response made up of chemokines, innate cytokines such as interleukin-1 (IL-1), IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), as well as the alarmins uric acid, ATP, HMGB1, and S100 proteins. These signals program dendritic cells (DCs) to mount Th2-cell-mediated immunity and in so doing boost ILC2, basophil, and mast cell function. Here we review the general mechanisms of how different stimuli trigger type-2-cell-mediated immunity at mucosal barriers and how this leads to protection or disease.

  11. Proteomic Analysis of Nasal Epithelial Cells from Cystic Fibrosis Patients

    PubMed Central

    Papon, Jean-François; Chhuon, Cerina; Zadigue, Patricia; Prulière-Escabasse, Virginie; Amselem, Serge; Escudier, Estelle; Coste, André; Edelman, Aleksander

    2014-01-01

    The pathophysiology of cystic fibrosis (CF) lung disease remains incompletely understood. New explanations for the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in cultured human nasal epithelial cells (HNEC). To that aim, we compared the level of protein expressions in primary cultures of HNEC from nasal polyps secondary to CF (CFNP, n = 4), primary nasal polyps (NP, n = 8) and control mucosa (CTRL, n = 4) using isobaric tag for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography (LC)-MS-MS. The analysis of the data revealed 42 deregulated protein expressions in CFNP compared to NP and CTRL, suggesting that these alterations are related to CF. Overall, AmiGo analysis highlighted six major pathways important for cell functions that seem to be impaired: metabolism, G protein process, inflammation and oxidative stress response, protein folding, proteolysis and structural proteins. Among them, glucose and fatty acid metabolic pathways could be impaired in CF with nine deregulated proteins. Our proteomic study provides a reproducible set of differentially expressed proteins in airway epithelial cells from CF patients and reveals many novel deregulated proteins that could lead to further studies aiming to clarify the involvement of such proteins in CF pathophysiology. PMID:25268127

  12. Comparative proteomics reveals human pluripotent stem cell-derived limbal epithelial stem cells are similar to native ocular surface epithelial cells

    PubMed Central

    Mikhailova, Alexandra; Jylhä, Antti; Rieck, Jochen; Nättinen, Janika; Ilmarinen, Tanja; Veréb, Zoltán; Aapola, Ulla; Beuerman, Roger; Petrovski, Goran; Uusitalo, Hannu; Skottman, Heli

    2015-01-01

    Limbal epithelial stem cells (LESCs) are tissue-specific stem cells responsible for renewing the corneal epithelium. Acute trauma or chronic disease affecting LESCs may disrupt corneal epithelial renewal, causing vision threatening and painful ocular surface disorders, collectively referred to as LESC deficiency (LESCD). These disorders cannot be treated with traditional corneal transplantation and therefore alternative cell sources for successful cell-based therapy are needed. LESCs derived from human pluripotent stem cells (hPSCs) are a prospective source for ocular surface reconstruction, yet critical evaluation of these cells is crucial before considering clinical applications. In order to quantitatively evaluate hPSC-derived LESCs, we compared protein expression in native human corneal cells to that in hPSC-derived LESCs using isobaric tag for relative and absolute quantitation (iTRAQ) technology. We identified 860 unique proteins present in all samples, including proteins involved in cell cycling, proliferation, differentiation and apoptosis, various LESC niche components, and limbal and corneal epithelial markers. Protein expression profiles were nearly identical in LESCs derived from two different hPSC lines, indicating that the differentiation protocol is reproducible, yielding homogeneous cell populations. Their protein expression profile suggests that hPSC-derived LESCs are similar to the human ocular surface epithelial cells, and possess LESC-like characteristics. PMID:26423138

  13. Roles of limbal microvascular net and limbal stroma in regulating maintenance of limbal epithelial stem cells.

    PubMed

    Huang, Minghai; Wang, Bowen; Wan, Pengxia; Liang, Xuanwei; Wang, Xiaoran; Liu, Ying; Zhou, Qiang; Wang, Zhichong

    2015-02-01

    Knowledge of the microenvironment (niche) of stem cells is helpful for stem-cell-based regenerative medicine. In the eye, limbal epithelial stem cells (corneal epithelial stem cells) provide the self-renewal capacity of the corneal epithelium and are essential for maintaining corneal transparency and vision. Limbal epithelial stem cell deficiency results in significant visual deterioration. Successful treatment of this type of blinding disease requires studies of the limbal epithelial stem cells and their microenvironment. We investigate the function of the limbal microvascular net and the limbal stroma in the maintenace of the limbal epithelial stem cell niche in vivo and examine the regulation of limbal epithelial stem cell survival, proliferation and differentiation in vivo. We assess the temporal and spatial changes in the expression patterns of the following markers during a six-month follow-up of various rabbit limbal autograft transplantation models: vascular endothelial cell marker CD31, corneal epithelium differentiation marker K3, limbal epithelial stem-cell-associated markers P63 and ABCG2 and proliferating cell nuclear marker Ki67. Our results suggest that limbal epithelial stem cells cannot maintain their stemness or proliferation without the support of the limbal microvascular net microenvironment. Thus, both the limbal microvascular net and the limbal stroma play important roles as components of the limbal epithelial stem cell niche maintaining limbal epithelial stem cell survival and proliferation and the avoidance of differentiation. The limbal stroma constitutes the structural basis of the limbal epithelial stem cell niche and the limbal microvascular net is a requirement for this niche. These new insights should aid the eventual construction of tissue-engineered cornea for corneal blind patients in the future.

  14. Nonhematopoietic cells are the primary source of bone marrow-derived lung epithelial cells.

    PubMed

    Kassmer, Susannah H; Bruscia, Emanuela M; Zhang, Ping-Xia; Krause, Diane S

    2012-03-01

    Previous studies have demonstrated that bone marrow (BM)-derived cells differentiate into nonhematopoietic cells of multiple tissues. To date, it remains unknown which population(s) of BM cells are primarily responsible for this engraftment. To test the hypothesis that nonhematopoietic stem cells in the BM are the primary source of marrow-derived lung epithelial cells, either wild-type hematopoietic or nonhematopoietic BM cells were transplanted into irradiated surfactant-protein-C (SPC)-null mice. Donor-derived, SPC-positive type 2 pneumocytes were predominantly detected in the lungs of mice receiving purified nonhematopoietic cells and were absent from mice receiving purified hematopoietic stem and progenitor cells. We conclude that cells contained in the nonhematopoietic fraction of the BM are the primary source of marrow-derived lung epithelial cells. These nonhematopoietic cells may represent a primitive stem cell population residing in adult BM.

  15. Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

    PubMed

    Sun, Chi-Chin; Chiu, Hsiao-Ting; Lin, Yi-Fang; Lee, Kuo-Ying; Pang, Jong-Hwei Su

    2015-01-01

    Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

  16. Small molecule and RNAi induced phenotype transition of expanded and primary colonic epithelial cells

    PubMed Central

    Sharbati, Jutta; Hanisch, Carlos; Pieper, Robert; Einspanier, Ralf; Sharbati, Soroush

    2015-01-01

    Recent progress in mammalian intestinal epithelial cell culture led to novel concepts of tissue modeling. Especially the development of phenotypically stable cell lines from individual animals enables an investigation of distinct intestinal loci and disease states. We here report primary and prolonged culture of normal porcine epithelial cells from colon for cell line development. In addition, a novel primary three-dimensional intestinal culture system is presented, which generated organoids composed of a highly polarized epithelial layer lining a core of subepithelial tissue. Cellular characterization of monolayer cell lines revealed epithelial identity and pointed to a proliferative crypt cell phenotype. We evaluated both RNAi and chemical approaches to induce epithelial differentiation in generated cell lines by targeting promoters of epithelial to mesenchymal transition (EMT). By in silico prediction and ectopic expression, miR-147b was proven to be a potent trigger of intestinal epithelial cell differentiation. Our results outline an approach to generate phenotypically stable cell lines expanded from primary colonic epithelial cultures and demonstrate the relevance of miR-147b and chemical inhibitors for promoting epithelial differentiation features. PMID:26223582

  17. Potential role of corneal epithelial cell-derived exosomes in corneal wound healing and neovascularization

    PubMed Central

    Han, Kyu-Yeon; Tran, Jennifer A.; Chang, Jin-Hong; Azar, Dimitri T.; Zieske, James D.

    2017-01-01

    Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions. PMID:28165027

  18. Purification and characterization of protein phosphatase 2C in rat parotid acinar cells: two forms of Mg(2+)-activated histone phosphatase and phosphorylation by cAMP-dependent protein kinase.

    PubMed

    Yokoyama, N; Kobayashi, T; Tamura, S; Sugiya, H

    1996-07-01

    Two forms of Mg(2+)-activated histone phosphatase activities were partially purified from rat parotid acinar cells using Mono Q and gel filtration chromatography. Both enzymes activities were dependent on the presence of Mg2+, showing little activity in the presence of EDTA. The activities fractionated on the Mono Q column into two peaks: the first was a minor peak of histone phosphatase activity; the second was a major peak. These two peaks eluted at distinct positions on the gel filtration column. The molecular masses of the two peak fractions corresponded to 46 and 55 kDa, respectively on SDS-gels. The first 46-kDa peak immunoreacted with anti-PP2Calpha phosphatase antibody and like PP2Calpha phosphatase could be phosphorylated by cAMP-dependent protein kinase. The second 55-kDa peak showed neither reactivity with anti-PP2Calpha phosphatase antibody nor phosphorylability by cAMP-dependent protein kinase, but retained a Mg2+ or Mn2+ dependence for its histone phosphatase activity. Ca2+ showed a strong inhibition on this activity. On the basis of these observations, we have identified the first peak enzyme as PP2Calpha phosphatase and the second peak as a novel PP2C-like phosphatase.

  19. Enterotoxigenic Escherichia coli infection induces intestinal epithelial cell autophagy.

    PubMed

    Tang, Yulong; Li, Fengna; Tan, Bie; Liu, Gang; Kong, Xiangfeng; Hardwidge, Philip R; Yin, Yulong

    2014-06-25

    The morbidity and mortality in piglets caused by enterotoxigenic Escherichia coli (ETEC) results in large economic losses to the swine industry, but the precise pathogenesis of ETEC-associated diseases remains unknown. Intestinal epithelial cell autophagy serves as a host defense against pathogens. We found that ETEC induced autophagy, as measured by both the increased punctae distribution of GFP-LC3 and the enhanced conversion of LC3-I to LC3-II. Inhibiting autophagy resulted in decreased survival of IPEC-1 cells infected with ETEC. ETEC triggered autophagy in IPEC-1 cells through a pathway involving the mammalian target of rapamycin (mTOR), the extracellular signal-regulated kinases 1/2 (ERK1/2), and the AMP-activated protein kinase (AMPK).

  20. PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells.

    PubMed

    Zhang, Hanying; Okamoto, Miyako; Panzhinskiy, Evgeniy; Zawada, W Michael; Das, Mita

    2014-04-01

    Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases.

  1. PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells

    PubMed Central

    Zhang, Hanying; Okamoto, Miyako; Panzhinskiy, Evgeniy; Zawada, W. Michael

    2014-01-01

    Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases. PMID:24500281

  2. Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting

    PubMed Central

    Lowell, Clifford A.

    2017-01-01

    In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 × 106 ATII cells per mouse lung. The cell preps are highly pure and viable and can be used for genomic or proteomic analyses or cultured ex vivo to understand their roles in various biological processes. PMID:28180137

  3. Heterogeneity of thymic epithelial cells in promoting T-lymphocyte differentiation in vivo.

    PubMed Central

    Gutierrez, J C; Palacios, R

    1991-01-01

    To study in vivo the contribution of different thymic epithelial cells to T-lymphocyte differentiation, we have established several nontransformed thymic epithelial cell lines and developed an in vivo assay, not involving exposure to drugs or radiation, that permitted us to study the capacity of these epithelial lines to support T-cell differentiation. We found that cell lines EA2 and ET, which express markers of cortical epithelial cells, produce interleukin 7 mRNA and after being injected into the spleens of young athymic nude mice support in vivo generation of CD4+CD8- T-cell receptor alpha beta+ T lymphocytes (ET line) or both CD4+CD8- and CD4-CD8+ T-cell receptor alpha beta+ T cells (EA2 line). Both cell lines also supported generation of T-cell receptor gamma delta+ T cells but appear not to support development of double-positive (CD4+CD8+) cells. One cell line, EB3, which expresses markers of medullary epithelial cells, produces interleukin 1 alpha RNA transcripts but does not support T-lymphocyte differentiation. The results provide direct evidence for functional heterogeneity of thymic epithelial cells in vivo and show the involvement of different cortical epithelial cells in the differentiation of T-cell progenitors into distinct thymocyte subsets. Images PMID:1988959

  4. Cell-autonomous requirements for Dlg-1 for lens epithelial cell structure and fiber cell morphogenesis.

    PubMed

    Rivera, Charlene; Yamben, Idella F; Shatadal, Shalini; Waldof, Malinda; Robinson, Michael L; Griep, Anne E

    2009-09-01

    Cell polarity and adhesion are thought to be key determinants in organismal development. In Drosophila, discs large (dlg) has emerged as an important regulator of epithelial cell proliferation, adhesion, and polarity. Herein, we investigated the role of the mouse homolog of dlg (Dlg-1) in the development of the mouse ocular lens. Tissue-specific ablation of Dlg-1 throughout the lens early in lens development led to an expansion and disorganization of the epithelium that correlated with changes in the distribution of adhesion and polarity factors. In the fiber cells, differentiation defects were observed. These included alterations in cell structure and the disposition of cell adhesion/cytoskeletal factors, delay in denucleation, and reduced levels of alpha-catenin, pERK1/2, and MIP26. These fiber cell defects were recapitulated when Dlg-1 was disrupted only in fiber cells. These results suggest that Dlg-1 acts in a cell autonomous manner to regulate epithelial cell structure and fiber cell differentiation.

  5. Opioid receptors on guinea-pig intestinal crypt epithelial cells.

    PubMed Central

    Lang, M E; Davison, J S; Bates, S L; Meddings, J B

    1996-01-01

    1. Opioid peptides promote net intestinal absorption via two mechanisms: stimulation of Na+ and Cl- absorption and inhibition of Cl- secretion. Although these transport changes are predominantly mediated by submucosal neurones, it is currently unclear whether opioid peptides can regulate enterocyte function directly. We therefore tested the hypothesis that enterocytes have specific opioid receptors. 2. Villus and crypt jejunal epithelial cells were isolated by the distended sac method from anaesthetized guinea-pigs. Flow cytometry was used to resolve enterocytes from other cell types and to determine whether binding of a fluorescently labelled opioid antagonist, naltrexone-FITC, could be prevented by unlabelled mu- and delta-opioid receptor agonists. A population of crypt enterocytes (approximately 21%) exhibited high-affinity naltrexone-FITC binding to both mu- and delta-type binding sites that was stereoselective and sodium dependent. Villus enterocytes did not exhibit any of these characteristics. 3. Basal cAMP production was elevated in both villus and crypt cells treated with IBMX (3-isobutyl-1-methylxanthine). Villus cells did not respond to 100 nM vasoactive intestinal peptide (VIP), nor were they affected by opioid peptides. In contrast, 100 nM VIP significantly increased cAMP production in crypt epithelial cells, which was significantly reduced by both morphiceptin and D-Ser2-Leu-Enk-Thr. This opioid-mediated effect was stereoselective and blocked by the opioid receptor antagonist naltrexone. 4. These experiments suggest that enterocytes isolated from the crypt epithelium of guineapigs have both mu- and delta-types of opioid receptors. It is possible that these cells participate in opioid-mediated regulation of intestinal secretion. Images Figure 12 PMID:8951719

  6. Adhesion and invasion of Streptococcus pneumoniae to primary and secondary respiratory epithelial cells

    PubMed Central

    Novick, Sara; Shagan, Marilous; Blau, Karin; Lifshitz, Sarit; Givon-Lavi, Noga; Grossman, Nili; Bodner, Lipa; Dagan, Ron; Nebenzahl, Yaffa Mizrachi

    2016-01-01

    The interaction between Streptococcus pneumoniae (S. pneumoniae) and the mucosal epithelial cells of its host is a prerequisite for pneumococcal disease development, yet the specificity of this interaction between different respiratory cells is not fully understood. In the present study, three areas were examined: i) The capability of the encapsulated S. pneumoniae serotype 3 strain (WU2) to adhere to and invade primary nasal-derived epithelial cells in comparison to primary oral-derived epithelial cells, A549 adenocarcinoma cells and BEAS-2B viral transformed bronchial cells; ii) the capability of the unencapsulated 3.8DW strain (a WU2 derivative) to adhere to and invade the same cells over time; and iii) the ability of various genetically-unrelated encapsulated and unencapsulated S. pneumoniae strains to adhere to and invade A549 lung epithelial cells. The results of the present study demonstrated that the encapsulated WU2 strain adhesion to and invasion of primary nasal epithelial cells was greatest, followed by BEAS-2B, A549 and primary oral epithelial cells. By contrast, the unencapsulated 3.8-DW strain invaded oral epithelial cells significantly more efficiently when compared to the nasal epithelial cells. In addition, unencapsulated S. pneumoniae strains adhered to and invaded the A459 cells significantly more efficiently than the encapsulated strains; this is consistent with previously published data. In conclusion, the findings presented in the current study indicated that the adhesion and invasion of the WU2 strain to primary nasal epithelial cells was more efficient compared with the other cultured respiratory epithelial cells tested, which corresponds to the natural course of S. pneumoniae infection and disease development. The target cell preference of unencapsulated strains was different from that of the encapsulated strains, which may be due to the exposure of cell wall proteins. PMID:27922699

  7. Three-Dimensional Cultures of Mouse Mammary Epithelial Cells

    PubMed Central

    Mroue, Rana; Bissell, Mina J.

    2013-01-01

    The mammary gland is an ideal “model organism” for studying tissue specificity and gene expression in mammals: it is one of the few organs that develop after birth and it undergoes multiple cycles of growth, differentiation and regression during the animal’s lifetime in preparation for the important function of lactation. The basic “functional differentiation” unit in the gland is the mammary acinus made up of a layer of polarized epithelial cells specialized for milk production surrounded by myoepithelial contractile cells, and the two-layered structure is surrounded by basement membrane. Much knowledge about the regulation of mammary gland development has been acquired from studying the physiology of the gland and of lactation in rodents. Culture studies, however, were hampered by the inability to maintain functional differentiation on conventional tissue culture plastic. We now know that the microenvironment, including the extracellular matrix and tissue architecture, plays a crucial role in directing functional differentiation of organs. Thus, in order for culture systems to be effective experimental models, they need to recapitulate the basic unit of differentiated function in the tissue or organ and to maintain its three-dimensional (3D) structure. Mouse mammary culture models evolved from basic monolayers of cells to an array of complex 3D systems that observe the importance of the microenvironment in dictating proper tissue function and structure. In this chapter, we focus on how 3D mouse mammary epithelial cultures have enabled investigators to gain a better understanding of the organization, development and function of the acinus, and to identify key molecular, structural, and mechanical cues important for maintaining mammary function and architecture. The accompanying chapter of Vidi et al. describes 3D models developed for human cells. Here, we describe how mouse primary epithelial cells and cell lines—essentially those we use in our

  8. NK cells are necessary for recovery of corneal CD11c+ dendritic cells after epithelial abrasion injury

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mechanisms controlling CD11c(+) MHCII(+) DCs during corneal epithelial wound healing were investigated in a murine model of corneal abrasion. Selective depletion of NKp46(+) CD3- NK cells that normally migrate into the cornea after epithelial abrasion resulted in >85% reduction of the epithelial CD1...

  9. Connexin expression in nonneoplastic human prostate epithelial cells.

    PubMed

    Saladino, Francesca; Carruba, Giuseppe; Quader, Salmaan T A; Amoroso, Maria; Di Cristina, Antoniette; Webber, Mukta M; Castagnetta, Luigi A M

    2002-06-01

    Expression of gap-junction proteins connexins (Cx), specifically Cx43, Cx32, and Cx26, in both nontumorigenic (RWPE-1) and tumorigenic (RWPE-2) human prostate epithelial cells as well as in two cell clones (WPEI-7 and WPEI-10) originating from the RWPE-1 cell line was investigated. The aim was to determine whether individual connexins are differentially expressed in cultured cells. Western blot analysis revealed striking differences in the expression of individual connexins in the cell lines studied. In particular, Cx43 is largely expressed in RWPE-1 and WPEI-10 cells, whereas Cx32 is expressed predominantly in RWPE-2 and WPEI-7 cells. In addition, both forskolin and estrone increase Cx43 expression levels in WPEI-10 cells, with no apparent effect on WPEI-7 cells. Conversely, forskolin and especially estrone induce a marked increase of Cx32 in WPEI-7 cells, whereas Cx32 expression is limitedly affected by both agents in WPEI-10 cells. Overall, expression levels of Cx43 and Cx32 appear to be inversely related, with RWPE-1 and WPEI-10 cells having a significantly higher Cx43 to Cx32 ratio than that observed in RWPE-2 and WPEI-7 cells. We recently reported that junctional communication could be rescued in RWPE-1 cells by either forskolin or estrone and that restoration of GJIC is associated with an increase of Cx43 or a decrease of Cx32, or both, eventually leading to a marked rise of the Cx43 to Cx32 ratio. Studies are currently ongoing in our laboratories to assess the potential effect of agents increasing the Cx43 to Cx32 ratio on GJIC activity in these systems.

  10. Genistein affects proliferation and migration of bovine oviductal epithelial cells.

    PubMed

    García, Daniela C; Valdecantos, Pablo A; Miceli, Dora C; Roldán-Olarte, Mariela

    2017-03-08

    Genistein is one of the most abundant isoflavones in soybean. This molecule induces cell cycle arrest and apoptosis in different normal and cancer cells. Genistein has been of considerable interest due to its adverse effects on bovine reproduction, altering estrous cycle, implantation and fetal development and producing subfertility or infertility. The objective of this work was to study the effects of genistein on the expression of selected genes involved in the regulation of cell cycle and apoptosis. Primary cultures of bovine oviductal epithelial cells (BOEC) were treated with different genistein concentrations (0.2, 2 and 10μM) to analyze CYCLIN B1, BCL-2 and BAX gene expression by Real-time RT-PCR. Results showed that genistein down-regulated CYCLIN B1 expression, affecting cell cycle progression, and caused a decrease in the BCL-2/BAX ratio starting at 2μM of genistein. In addition, in order to determine if genistein affects BOEC migration, in vitro wound healing assays were performed. A significant reduction in cell migration after 12h of culture was observed at both 0.2 and 10μM genistein concentrations. Also, in the presence of genistein the percentage of mitotic cells decreased, although apoptotic cells percentages were not affected. These findings indicate that genistein has an inhibitory effect on BOEC proliferation and migration, suggesting that it could influence the normal physiology of the oviductal epithelium.

  11. The PCP pathway regulates Baz planar distribution in epithelial cells

    PubMed Central

    Aigouy, Benoit; Le Bivic, André

    2016-01-01

    The localisation of apico-basal polarity proteins along the Z-axis of epithelial cells is well understood while their distribution in the plane of the epithelium is poorly characterised. Here we provide a systematic description of the planar localisation of apico-basal polarity proteins in the Drosophila ommatidial epithelium. We show that the adherens junction proteins Shotgun and Armadillo, as well as the baso-lateral complexes, are bilateral, i.e. present on both sides of cell interfaces. In contrast, we report that other key adherens junction proteins, Bazooka and the myosin regulatory light chain (Spaghetti squash) are unilateral, i.e. present on one side of cell interfaces. Furthermore, we demonstrate that planar cell polarity (PCP) and not the apical determinants Crumbs and Par-6 control Bazooka unilaterality in cone cells. Altogether, our work unravels an unexpected organisation and combination of apico-basal, cytoskeletal and planar polarity proteins that is different on either side of cell-cell interfaces and unique for the different contacts of the same cell. PMID:27624969

  12. PRRX2 as a novel TGF-β-induced factor enhances invasion and migration in mammary epithelial cell and correlates with poor prognosis in breast cancer.

    PubMed

    Juang, Yu-Lin; Jeng, Yung-Ming; Chen, Chi-Long; Lien, Huang-Chun

    2016-12-01

    TGF-β and cancer progression share a multifaceted relationship. Despite the knowledge of TGF-β biology in the development of cancer, several factors that mediate the cancer-promoting role of TGF-β continue to be identified. This study aimed to identify and characterise novel factors potentially related to TGF-β-mediated tumour aggression in breast cells. We treated the human mammary epithelial cell line MCF10A with TGF-β and identified TGF-β-dependent upregulation of PRRX2, the gene encoding paired-related homeobox 2 transcription factor. Overexpression of PRRX2 enhanced migration, invasion and anchorage-independent growth of MCF10A cells and induced partial epithelial mesenchymal transition (EMT), as determined by partial fibroblastoid morphology of cells, upregulation of EMT markers and partially disrupted acinar structure in a three-dimensional culture. We further identified PLAT, the gene encoding tissue-type plasminogen activator (tPA), as the highest differentially expressed gene in PRRX2-overexpressing MCF10A cells, and demonstrated direct binding and transactivation of the PLAT promoter by PRRX2. Furthermore, PLAT knockdown inhibited PRRX2-mediated enhanced migration and invasion, suggesting that tPA may mediate PRRX2-induced migration and invasion. Finally, the significant correlation of PRRX2 expression with poor survival in 118 primary breast tumour samples (P = 0.027) and the increased PRRX2 expression in metaplastic breast carcinoma samples, which is pathogenetically related to EMT, validated the biological importance of PRRX2-enhanced migration and invasion and PRRX2-induced EMT. Thus, our data suggest that upregulation of PRRX2 may be a mechanism contributing to TGF-β-induced invasion and EMT in breast cancer. © 2016 Wiley Periodicals, Inc.

  13. FOXO responses to Porphyromonas gingivalis in epithelial cells

    PubMed Central

    Wang, Qian; Sztukowska, Maryta; Ojo, Akintunde; Scott, David A.; Wang, Huizhi; Lamont, Richard J.

    2015-01-01

    Summary Porphyromonas gingivalis is a prominent periodontal, and emerging systemic, pathogen that redirects host cell signalling pathways and modulates innate immune responses. In this study, we show that P. gingivalis infection induces the dephosphorylation and activation of forkhead box-O (FOXO)1, 3 and 4 in gingival epithelial cells. In addition, immunofluorescence showed that FOXO1 accumulated in the nucleus of P. gingivalis-infected cells. Quantitative reverse transcription PCR demonstrated that transcription of genes involved in protection against oxidative stress (Cat, Sod2, Prdx3), inflammatory responses (IL1β) and anti-apoptosis (Bcl-6) was induced by P. gingivalis, while small-interfering RNA (siRNA)-mediated knockdown of FOXO1 suppressed the transcriptional activation of these genes. P. gingivalis-induced secretion of interleukin (IL)-1β and inhibition of apoptosis were also impeded by FOXO1 knockdown. Neutralization of reactive oxygen species (ROS) by N-acetyl-l-cysteine blocked the activation of FOXO1 by P. gingivalis and concomitantly suppressed the activation of oxidative stress responses, anti-apoptosis programmes and IL-β production. Inhibition of c-Jun-N-terminal kinase (JNK) either pharmacologically or by siRNA, reduced FOXO1 activation and downstream FOXO1-dependent gene regulation in response to P. gingivalis. The results indicate that P. gingivalis-induced ROS activate FOXO transcription factors through JNK signalling, and that FOXO1 controls oxidative stress responses, inflammatory cytokine production and cell survival. These data position FOXO as an important signalling node in the epithelial cell–P. gingivalis interaction, with particular relevance to cell fate and dysbiotic host responses. PMID:25958948

  14. Epithelial mechanobiology, skin wound healing, and the stem cell niche.

    PubMed

    Evans, Nicholas D; Oreffo, Richard O C; Healy, Eugene; Thurner, Philipp J; Man, Yu Hin

    2013-12-01

    Skin wound healing is a vital process that is important for re-establishing the epithelial barrier following disease or injury. Aberrant or delayed skin wound healing increases the risk of infection, causes patient morbidity, and may lead to the formation of scar tissue. One of the most important events in wound healing is coverage of the wound with a new epithelial layer. This occurs when keratinocytes at the wound periphery divide and migrate to re-populate the wound bed. Many approaches are under investigation to promote and expedite this process, including the topical application of growth factors and the addition of autologous and allogeneic tissue or cell grafts. The mechanical environment of the wound site is also of fundamental importance for the rate and quality of wound healing. It is known that mechanical stress can influence wound healing by affecting the behaviour of cells within the dermis, but it remains unclear how mechanical forces affect the healing epidermis. Tensile forces are known to affect the behaviour of cells within epithelia, however, and the material properties of extracellular matrices, such as substrate stiffness, have been shown to affect the morphology, proliferation, differentiation and migration of many different cell types. In this review we will introduce the structure of the skin and the process of wound healing. We will then discuss the evidence for the effect of tissue mechanics in re-epithelialisation and, in particular, on stem cell behaviour in the wound microenvironment and in intact skin. We will discuss how the elasticity, mechanical heterogeneity and topography of the wound extracellular matrix impact the rate and quality of wound healing, and how we may exploit this knowledge to expedite wound healing and mitigate scarring.

  15. Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells.

    PubMed

    Bhattacharya, Sujoy; Ray, Ramesh M; Johnson, Leonard R

    2014-03-01

    Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco-2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco-2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF-α/CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner.

  16. Cytokeratin 18 is necessary for initiation of TGF-β1-induced epithelial-mesenchymal transition in breast epithelial cells.

    PubMed

    Jung, Hyejung; Kim, Bomin; Moon, Byung In; Oh, Eok-Soo

    2016-12-01

    During epithelial-mesenchymal transition (EMT), epithelial cells lose key phenotypic markers (e.g., E-cadherin and cytokeratin 18) and acquire mesenchymal markers (e.g., N-cadherin and vimentin). Although the loss of cytokeratin 18 is a hallmark of EMT, the regulatory role of cytokeratin 18 in EMT is not yet fully understood. Here, we report that cytokeratin 18 is involved in the regulation of transforming growth factor-beta1 (TGF-β1)-induced EMT in breast epithelial cells. When MCF10A cells were treated with TGF-β1 for 24 h, considerable morphological changes, indicative of the early stages of EMT (e.g., loss of cell-cell contact), were observed and cytokeratin 18 was downregulated. However, E-cadherin levels were not altered until a later time point. This suggests that cytokeratin 18 may play an active role during the earlier stages of EMT. Consistent with this notion, siRNA-mediated knockdown of cytokeratin 18 delayed TGF-β1-mediated EMT, and the associated downregulation of E-cadherin reduced the phosphorylation/nuclear localization of smad 2/3 and decreased the expression levels of snail and slug (which inhibit E-cadherin expression in epithelial cells as an early response to TGF-β1). Taken together, these results suggest that cytokeratin 18 critically contributes to initiating TGF-β1-induced EMT via the smad 2/3-mediated regulation of snail and slug expression in breast epithelial cells.

  17. Elucidation of a Novel Cell Death Mechanism in Prostate Epithelial Cells

    DTIC Science & Technology

    2002-12-01

    surface. Galectin-1 binds to saccharide ligands on susceptible LNCaP cells to trigger cell death . Susceptibility to galectin-1 appears to depend on the...induced LNCaP cell death . Resistance to galectin-1 induced death correlates with markedly decreased expression of a specific glycosyltransferase, the...galectin-1 induced death, indicating that a common glycosylation pathway may control cell death in epithelial and lymphoid cells. Identification of a

  18. Elucidation of a Novel Cell Death Mechanism in Prostate Epithelial Cells

    DTIC Science & Technology

    2003-12-01

    surface. Galectin-1 binds to saccharide ligands on suscepibel LNCaP cells to trigger cell death . Susceptibility to galectin-1 appears to depend on the...induced LNcaP cell death . Resistance to galectin-1 induced death correlates with markedly decreased expression of a specific glycosyltransferase, the...galectin-1 induced death, indicating that a common glycosylation pathway may control cell death in epithelial and lymphoid cells. Identification of a

  19. Cigarette smoke impairs airway epithelial barrier function and cell-cell contact recovery.

    PubMed

    Heijink, I H; Brandenburg, S M; Postma, D S; van Oosterhout, A J M

    2012-02-01

    Cigarette smoking, the major cause of chronic obstructive pulmonary disease (COPD), induces aberrant airway epithelial structure and function. The underlying mechanisms are unresolved so far. We studied effects of cigarette smoke extract (CSE) on epithelial barrier function and wound regeneration in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) from COPD patients, nonsmokers and healthy smokers. We demonstrate that CSE rapidly and transiently impairs 16HBE barrier function, largely due to disruption of cell-cell contacts. CSE induced a similar, but stronger and more sustained, defect in PBECs. Application of the specific epidermal growth factor receptor (EGFR) inhibitor AG1478 showed that EGFR activation contributes to the CSE-induced defects in both 16HBE cells and PBECs. Furthermore, our data indicate that the endogenous protease calpain mediates these defects through tight junction protein degradation. CSE also delayed the reconstitution of 16HBE intercellular contacts during wound healing and attenuated PBEC barrier function upon wound regeneration. These findings were comparable between PBECs from smokers, healthy smokers and COPD patients. In conclusion, we demonstrate for the first time that CSE reduces epithelial integrity, probably by EGFR and calpain-dependent disruption of intercellular contacts. This may increase susceptibility to environmental insults, e.g. inhaled pathogens. Thus, EGFR may be a promising target for therapeutic strategies to improve mucosal barrier function in cigarette smoking-related disease.

  20. The yin and yang of intestinal epithelial cells in controlling dendritic cell function

    PubMed Central

    Iliev, Iliyan D.; Matteoli, Gianluca; Rescigno, Maria

    2007-01-01

    Recent work suggests that dendritic cells (DCs) in mucosal tissues are “educated” by intestinal epithelial cells (IECs) to suppress inflammation and promote immunological tolerance. After attack by pathogenic microorganisms, however, “non-educated” DCs are recruited from nearby areas, such as the dome of Peyer's patches (PPs) and the blood, to initiate inflammation and the ensuing immune response to the invader. Differential epithelial cell (EC) responses to commensals and pathogens may control these two tolorogenic and immunogenic functions of DCs. PMID:17893197

  1. Contact inhibition of phagocytosis in epithelial sheets: alterations of cell surface properties induced by cell-cell contacts.

    PubMed

    Vasiliev, J M; Gelfand, I M; Domnina, L V; Zacharova, O S; Ljubimov, A V

    1975-02-01

    Contact inhibition of phagocytosis was found to be characteristic for epithelial sheets formed in cultures by several cell types: normal and transformed mouse kidney cells, and differentiated mouse hepatoma cells. In these sheets most central cells surrounded by other cells had very low phagocytic activity. In contrast, marginal cells having a free edge were able to perform an active phagocytosis. Contact inhibition of phagocytosis was absent in dense cultures of mouse embryo fibroblasts and in cultures of anaplastic mouse hepatoma 22a. The upper surface of epithelial sheets was nonadhesive for prelabeled epithelial cells and fibroblasts. In contrast, the upper surface of dense cultures of mouse fibroblasts was adhesive for these cells. These and other data strengthen the suggestion that contact inhibition of phagocytosis is a result of different adhesiveness of the upper cell surface and of the surfaces near the free edge. Agents inhibiting cell surface movements at the free edges of marginal epithelial cells (cytochalasin, azide, sorbitol, low temperature) prevented adhesion of particles to these edges. Possibly, the surface of actively moving cytoplasmic processes is the only cell part that has adhesive properties necessary for the formation of attachments with other cellular and noncellular surfaces. In epithelial sheets, in contrast to fibroblast cultures, Colcemid did not activate movements of immobile contacting cell edges. These results indicate that mechanisms of contact immobilization of cell surface may be different in epithelium and fibroblasts. Firm contacts formed between epithelial cells are sufficient for stable immobilization of the surface; additional stabilization of the surface by microtubules is not essential. Fibroblasts do not form firm contacts and the Colcemid-sensitive stabilization process is essential for maintenance of the immobile state of their surfaces. Differences in the stability of cell surface immobilization produced by cell-cell

  2. Cytotoxicity of folpet fungicide on human bronchial epithelial cells.

    PubMed

    Canal-Raffin, Mireille; l'Azou, Béatrice; Jorly, Joana; Hurtier, Annabelle; Cambar, Jean; Brochard, Patrick

    2008-07-30

    Folpet, a widely used dicarboximide fungicide, has been detected in the ambient air of several vine-growing regions of France. It is present in particle form in the environment; however, no study exploring its potential health impact on airways and the respiratory system has been published. Here, the biological effect of these particles was investigated in vitro on human bronchial epithelial cells (16HBE14o-). To be close to the real-life conditions of exposure, Folpan 80WG, a commercial form of folpet, was tested. Folpan 80WG particles showed dose- and time-dependent cytotoxic effects on 16HBE14o- cells. This effect was compared to that produced by technical-grade folpet and both were found to induce a toxicity with similar IC(50) values after 24h of exposure. After 4h and at least until 48h of exposure, the IC(50) values of Folpan 80WG particles were between 2.4 and 2.8 microg/cm(2). Investigation of the cytotoxicity found that Folpan 80WG particles at 1.85 microg/cm(2) induced an increase in ROS production from the first hour of exposure. Evidence that oxidative processes occur in folpet-exposed cells was confirmed by the presence of membrane lipid peroxidation. Furthermore, early apoptosis and late apoptosis/necrosis were both present after the first hour of exposure. These findings indicate that exposure to Folpan 80WG particles result in a rapid cytotoxic effect on human bronchial epithelial cells in vitro that could be in part explained by oxidative stress, characterised by membrane lipid peroxidation and ROS production.

  3. Emodin suppresses TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells through Notch signaling pathway.

    PubMed

    Gao, Rundi; Chen, Ruilin; Cao, Yu; Wang, Yuan; Song, Kang; Zhang, Ya; Yang, Junchao

    2017-03-01

    Pulmonary fibrosis is characterized by the destruction of lung tissue architecture and the formation of fibrous foci, currently has no satisfactory treatment. Emodin is a component of Chinese herb that has been reported to be medicament on pancreatic fibrosis and liver fibrosis. However, its role in pulmonary fibrosis has not been established yet. In the present study, we investigated the hypothesis that Emodin plays an inhibitory role in TGF-β1 induced epithelial-mesenchymal transition (EMT) of alveolar epithelial cell, and Emodin exerts its effect through the Notch signaling pathway. Emodin inhibits the proliferation of Rat alveolar type II epithelial cells RLE-6TN in a concentration-dependent manner; reduces the expression of Collagen I, α-SMA and Vimentin, promotes the expression of E-cadherin. Moreover, Emodin could regulate the expression patterns of the Notch signaling pathway-related factors and reduce the Notch-1 nucleus translocation. Knockdown of Notch-1 enhances the inhibitory effect of Emodin on TGF-β1-induced EMT in RLE-6TN cells. In conclusion, the data of the present study suggests that Emodin suppresses TGF-β1-induced EMT in alveolar epithelial cells through Notch signaling pathway and shows the potential to be effective in the treatment of pulmonary fibrosis.

  4. Interactions between airway epithelial cells and dendritic cells during viral infections using an in vitro co-culture model

    EPA Science Inventory

    Rationale: Historically, single cell culture models have been limited in pathological and physiological relevance. A co-culture model of dendritic cells (DCs) and differentiated human airway epithelial cells was developed to examine potential interactions between these two cell t...

  5. ICAM-1 is necessary for epithelial recruitment of gammadelta T cells and efficient corneal wound healing.

    PubMed

    Byeseda, Sarah E; Burns, Alan R; Dieffenbaugher, Sean; Rumbaut, Rolando E; Smith, C Wayne; Li, Zhijie

    2009-08-01

    Wound healing and inflammation are both significantly reduced in mice that lack gammadelta T cells. Here, the role of epithelial intercellular adhesion molecule-1 (ICAM-1) in gammadelta T cell migration in corneal wound healing was assessed. Wild-type mice had an approximate fivefold increase in epithelial gammadelta T cells at 24 hours after epithelial abrasion. ICAM-1(-/-) mice had 50.9% (P < 0.01) fewer gammadelta T cells resident in unwounded corneal epithelium, which failed to increase in response to epithelial abrasion. Anti-ICAM-1 blocking antibody in wild-type mice reduced epithelial gammadelta T cells to a number comparable to that of ICAM-1(-/-) mice, and mice deficient in lymphocyte function-associated antigen-1 (CD11a/CD18), a principal leukocyte receptor for ICAM-1, exhibited a 48% reduction (P < 0.01) in peak epithelial gammadelta T cells. Re-epithelialization and epithelial cell division were both significantly reduced ( approximately 50% at 18 hours, P < 0.01) after abrasion in ICAM-1(-/-) mice versus wild-type, and at 96 hours, recovery of epithelial thickness was only 66% (P < 0.01) of wild-type. ICAM-1 expression by corneal epithelium in response to epithelial abrasion appears to be critical for accumulation of gammadelta T cells in the epithelium, and deficiency of ICAM-1 significantly delays wound healing. Since gammadelta T cells are necessary for efficient epithelial wound healing, ICAM-1 may contribute to wound healing by facilitating gammadelta T cell migration into the corneal epithelium.

  6. Long-term homeostasis and wound healing in an in vitro epithelial stem cell niche model

    PubMed Central

    Miyashita, Hideyuki; Niwano, Hiroko; Yoshida, Satoru; Hatou, Shin; Inagaki, Emi; Tsubota, Kazuo; Shimmura, Shigeto

    2017-01-01

    Cultures of epithelial cells are limited by the proliferative capacity of primary cells and cell senescence. Herein we show that primary human epithelial cell sheets cultured without dermal equivalents maintained homeostasis in vitro for at least 1 year. Transparency of these sheets enabled live observation of pigmented melanocytes and Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) labeled epithelial cells during wound healing. Cell turn over and KRT15 expression pattern stabilized within 3 months, when KRT15 bright clusters often associated with niche-like melanocytes became apparent. EdU labels were retained in a subset of epithelial cells and melanocytes after 6 months chasing, suggesting their slow cell cycling property. FUCCI-labeling demonstrated robust cell migration and proliferation following wounding. Transparency and long-term (1 year) homeostasis of this model will be a powerful tool for the study of wound healing and cell linage tracing. PMID:28233843

  7. Bivariate flow cytometric analysis of murine intestinal epithelial cells for cytokinetic studies

    SciTech Connect

    Pallavicini, M.G.; Ng, C.R.; Gray, J.W.

    1984-01-01

    The heterogeneous nature of the small intestine and the lack of methods to obtain pure crypt populations has, in the past, limited the application of standard flow cytometric analysis for cytokinetic studies of the proliferating crypts. The authors describe a flow cytometric technique to discriminate crypt and villus cells in an epithelial cell suspension on the basis of cell length, and to measure the DNA content of the discriminated subpopulations. These data indicate that bivariate analysis of a mixed epithelial cell suspension can be used to distinguish mature villus cells, G/sub 1/ crypt cells, and S-phase crypt cells. In addition, continuous labeling studies suggest that the position of a cell on the cell length axis reflects epithelial cell maturity. The authors applied this flow cytometric technique to determine the cytokinetic nature of epithelial cells obtained by sequential digestion of the small intestine. 22 references, 4 figures, 2 tables.

  8. TGF-beta suppresses EGF-induced MAPK signaling and proliferation in asthmatic epithelial cells.

    PubMed

    Semlali, Abdelhabib; Jacques, Eric; Plante, Sophie; Biardel, Sabrina; Milot, Julie; Laviolette, Michel; Boulet, Louis-Philippe; Chakir, Jamila

    2008-02-01

    Epithelial damage is an important pathophysiologic feature of asthma. Bronchial epithelium damage results in release of growth factors such as transforming growth factor (TGF)-beta(1) that may affect epithelial cell proliferation. The objective of our study is to evaluate the importance of TGF-beta(1) in regulating epithelial cell repair in asthma. We evaluated the effect of TGF-beta(1) on epidermal growth factor (EGF)-induced proliferation and downstream signaling in epithelial cells obtained from subjects with asthma compared with cells from healthy subjects. Cell proliferation was evaluated by bromodeoxyuridine incorporation. EGF receptor (EGFR), mitogen-activated protein kinase, TGF-beta receptors, Smads, Smad anchor for receptor activation (SARA), and cyclin-dependant kinase inhibitors were evaluated by Western blot. TGF-beta(1) and receptor expression were measured by RT-PCR and by enzyme-linked immunosorbent assay. Proliferation of epithelial cells at baseline and after EGF stimulation was significantly reduced in cells derived from subjects with asthma compared with cells obtained from healthy control subjects. EGF-induced ERK1/2 phosphorylation was reduced in epithelial cells from subjects with asthma compared with cells from healthy control subjects. This was paralleled with a reduced EGFR phosphorylation. Addition of TGF-beta(1) significantly decreased EGF-induced cell proliferation. TGF-beta(1) production was higher in asthmatic epithelial cells compared with normal cells. This was supported by a high expression of pSmad 3 and SARA in cells derived from individuals with asthma compared with normal subjects. Cycline-dependent kinase inhibitors were highly expressed in asthmatic compared with normal cells. Inhibition of TGF-beta(1) signaling in asthmatic epithelial cells restored EGFR, ERK1/2 phosphorylation, and cell proliferation induced by EGF. Our results suggest that TGF-beta restrains EGFR phosphorylation and downstream signaling in bronchial

  9. Uterine epithelial cell proliferation and endometrial hyperplasia: evidence from a mouse model.

    PubMed

    Gao, Yang; Li, Shu; Li, Qinglei

    2014-08-01

    In the uterus, epithelial cell proliferation changes during the estrous cycle and pregnancy. Uncontrolled epithelial cell proliferation results in implantation failure and/or cancer development. Transforming growth factor-β (TGF-β) signaling is a fundamental regulator of diverse biological processes and is indispensable for multiple reproductive functions. However, the in vivo role of TGF-β signaling in uterine epithelial cells remains poorly defined. We have shown that in the uterus, conditional deletion of the Type 1 receptor for TGF-β (Tgfbr1) using anti-Müllerian hormone receptor type 2 (Amhr2) Cre leads to myometrial defects. Here, we describe enhanced epithelial cell proliferation by immunostaining of Ki67 in the uteri of these mice. The aberration culminated in endometrial hyperplasia in aged females. To exclude the potential influence of ovarian steroid hormones, the proliferative status of uterine epithelial cells was assessed following ovariectomy. Increased uterine epithelial cell proliferation was also revealed in ovariectomized Tgfbr1 Amhr2-Cre conditional knockout mice. We further demonstrated that transcript levels for fibroblast growth factor 10 (Fgf10) were markedly up-regulated in Tgfbr1 Amhr2-Cre conditional knockout uteri. Consistently, treatment of primary uterine stromal cells with TGF-β1 significantly reduced Fgf10 mRNA expression. Thus, our findings suggest a potential involvement of TGFBR1-mediated signaling in the regulation of uterine epithelial cell proliferation, and provide genetic evidence supporting the role of uterine epithelial cell proliferation in the pathogenesis of endometrial hyperplasia.

  10. Activated alveolar epithelial cells initiate fibrosis through autocrine and paracrine secretion of connective tissue growth factor.

    PubMed

    Yang, Jibing; Velikoff, Miranda; Canalis, Ernesto; Horowitz, Jeffrey C; Kim, Kevin K

    2014-04-15

    Fibrogenesis involves a pathological accumulation of activated fibroblasts and extensive matrix remodeling. Profibrotic cytokines, such as TGF-β, stimulate fibroblasts to overexpress fibrotic matrix proteins and induce further expression of profibrotic cytokines, resulting in progressive fibrosis. Connective tissue growth factor (CTGF) is a profibrotic cytokine that is indicative of fibroblast activation. Epithelial cells are abundant in the normal lung, but their contribution to fibrogenesis remains poorly defined. Profibrotic cytokines may activate epithelial cells with protein expression and functions that overlap with the functions of active fibroblasts. We found that alveolar epithelial cells undergoing TGF-β-mediated mesenchymal transition in vitro were also capable of activating lung fibroblasts through production of CTGF. Alveolar epithelial cell expression of CTGF was dramatically reduced by inhibition of Rho signaling. CTGF reporter mice demonstrated increased CTGF promoter activity by lung epithelial cells acutely after bleomycin in vivo. Furthermore, mice with lung epithelial cell-specific deletion of CTGF had an attenuated fibrotic response to bleomycin. These studies provide direct evidence that epithelial cell activation initiates a cycle of fibrogenic effector cell activation during progressive fibrosis. Therapy targeted at epithelial cell production of CTGF offers a novel pathway for abrogating this progressive cycle and limiting tissue fibrosis.

  11. Epithelialization of mouse ovarian tumor cells originating in the fallopian tube stroma

    PubMed Central

    Hua, Yuanyuan; Choi, Pui-Wah; Trachtenberg, Alexander J.; Ng, Allen C.; Kuo, Winston P.; Ng, Shu-Kay; Dinulescu, Daniela M.; Matzuk, Martin M.; Berkowitz, Ross S.; Ng, Shu-Wing

    2016-01-01

    Epithelial ovarian carcinoma accounts for 90% of all ovarian cancer and is the most deadly gynecologic malignancy. Recent studies have suggested that fallopian tube fimbriae can be the origin of cells for high-grade serous subtype of epithelial ovarian carcinoma (HGSOC). A mouse HGSOC model with conditional Dicer-Pten double knockout (Dicer-Pten DKO) developed primary tumors, intriguingly, from the fallopian tube stroma. We examined the growth and epithelial phenotypes of the Dicer-Pten DKO mouse tumor cells contributable by each gene knockout. Unlike human ovarian epithelial cancer cells that expressed full-length E-cadherin, the Dicer-Pten DKO stromal tumor cells expressed cleaved E-cadherin fragments and metalloproteinase 2, a mixture of epithelial and mesenchymal markers. Although the Dicer-Pten DKO tumor cells lost the expression of mature microRNAs as expected, they showed high levels of tRNA fragment expression and enhanced AKT activation due to the loss of PTEN function. Introduction of a Dicer1-expressing construct into the DKO mouse tumor cells significantly reduced DNA synthesis and the cell growth rate, with concurrent diminished adhesion and ZO1 epithelial staining. Hence, it is likely that the loss of Dicer promoted mesenchymal-epithelial transition in fallopian tube stromal cells, and in conjunction with Pten loss, further promoted cell proliferation and epithelial-like tumorigenesis. PMID:27602775

  12. Establishment and Characterization of a Buffalo (Bubalus bubalis) Mammary Epithelial Cell Line

    PubMed Central

    Anand, Vijay; Dogra, Nilambra; Singh, Surender; Kumar, Sudarshan N.; Jena, Manoj K.; Malakar, Dhruba; Dang, Ajay K.; Mishra, Bishnu P.; Mukhopadhyay, Tapas K.; Kaushik, Jai K.; Mohanty, Ashok K.

    2012-01-01

    Background The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions. Methodology Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot. Principal Findings The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence. Conclusions We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions. PMID:22792341

  13. A physical method for separating spermatozoa from epithelial cells in sexual assault evidence.

    PubMed

    Chen, J; Kobilinsky, L; Wolosin, D; Shaler, R; Baum, H

    1998-01-01

    The analysis of genetic markers for the purpose of individualization of semen specimens is extremely important in cases of sexual abuse and assault. The serological analysis of sexual assault evidence can sometimes be complicated because stains are often composed of a mixture of spermatozoa, vaginal epithelial cells and white and red blood cells. A filtration method has been developed to cleanly separate spermatozoa from epithelial cells based upon differences in size and shape. Nylon mesh filters of the appropriate pore size can be used to separate the smaller oval shaped spermatozoal cells from the larger and flatter epithelial cells. The former pass freely through the membrane while the latter are retained on the filter. In this study, cell separation was demonstrated by (a) microscopic observation of stained cells, (b) amplified fragment length polymorphism analysis of DNA obtained from separated cells. The results of these analyses indicate that: (1) Approximately 70% of spermatozoa in the mixed cell sample will penetrate the 10 microns pore size filter, (2) Only about 1-2% of intact epithelial cells will do so, and (3) A small number of nuclei from spontaneously lysed epithelial cells will cross the filter. Experimental results using mixtures of spermatozoa and vaginal epithelial cells prepared in different ratios support the conclusion that the filtration process is an efficient and reliable method to separate spermatozoa from epithelial cells in casework specimens for subsequent DNA analysis.

  14. Examination of the role of galectins in cell migration and re-epithelialization of wounds.

    PubMed

    Cao, Zhiyi; Saravanan, Chandrassegar; Chen, Wei-Sheng; Panjwani, Noorjahan

    2015-01-01

    Re-epithelialization is a crucial step for wound healing. As galectins play important roles in re-epithelialization, we describe here protocols for in vivo, ex vivo and in vitro examination of the role of galectins in cell migration and in re-epithelialization of wounds. For in vivo models, mouse corneas are wounded by a variety of techniques and the rate of re-epithelialization is quantified. For ex vivo organ culture models, mouse corneas are wounded in situ, the eyes are enucleated, the eyeballs are cultured in the presence or absence of galectins and the rate of re-epithelialization is quantified. For cell cultured-based in vitro assays, we examine formation of lamellipodia and activation of focal adhesion kinase in various epithelial cells.

  15. Snail1 is required for the maintenance of the pancreatic acinar phenotype

    PubMed Central

    Loubat-Casanovas, Jordina; Peña, Raúl; Gonzàlez, Núria; Alba-Castellón, Lorena; Rosell, Santi; Francí, Clara; Navarro, Pilar; de Herreros, Antonio García

    2016-01-01

    The Snail1 transcriptional factor is required for correct embryonic development, yet its expression in adult animals is very limited and its functional roles are not evident. We have now conditionally inactivated Snail1 in adult mice and analyzed the phenotype of these animals. Snail1 ablation rapidly altered pancreas structure: one month after Snail1 depletion, acinar cells were markedly depleted, and pancreas accumulated adipose tissue. Snail1 expression was not detected in the epithelium but was in pancreatic mesenchymal cells (PMCs). Snail1 ablation in cultured PMCs downregulated the expression of several β-catenin/Tcf-4 target genes, modified the secretome of these cells and decreased their ability to maintain acinar markers in cultured pancreas cells. Finally, Snail1 deficiency modified the phenotype of pancreatic tumors generated in transgenic mice expressing c-myc under the control of the elastase promoter. Specifically, Snail1 depletion did not significantly alter the size of the tumors but accelerated acinar-ductal metaplasia. These results demonstrate that Snail1 is expressed in PMCs and plays a pivotal role in maintaining acinar cells within the pancreas in normal and pathological conditions. PMID:26735179

  16. Characterization of protamine uptake by opossum kidney epithelial cells.

    PubMed

    Nagai, Junya; Komeda, Takuji; Katagiri, Yuki; Yumoto, Ryoko; Takano, Mikihisa

    2013-01-01

    Protamine, a mixture of polypeptides that is rich in arginine, has been used clinically as an antidote to heparin overdoses and a complexing agent in a long-acting insulin preparation. When protamine is administered intravenously, its abundant accumulation in the kidneys has been reported. However, the renal uptake mechanism for protamine is not clear. In this study, we examined the transport mechanism for protamine in opossum kidney (OK) cells, a suitable in vitro model for renal proximal tubular epithelial cells. Flow cytometric analysis revealed that the association of fluorescein isothiocyanate (FITC)-labeled protamine from salmon (FITC-protamine) by OK cells was inhibited by unlabeled protamine in a concentration-dependent manner. The association of FITC-protamine was temperature- and energy-dependent. Confocal microscopy analysis showed that the fluorescence was localized in the cytoplasm and nucleus of OK cells. In addition, FITC-protamine association was inhibited by cationic drugs such as polycationic gentamicin and polymixin B, but it was increased by a basic amin