Clonogenic Assay: Adherent Cells

PubMed Central

Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T.; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C.

2011-01-01

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation. PMID:21445039

DNA BINDING AND ADDUCT FORMATION OF AFLATOXIN B1 IN CULTURED HUMAN AND ANIMAL TRACHEOBRONCHIAL AND BLADDER TISSUES

EPA Science Inventory

DNA binding and adduct formation of aflatoxin B1 (AFB1) was studied in cultured bladder and tracheobronchial explants from human, monkey, dog, hamster and rat. Explants were exposed to (3H)AFB1 (1 micrometer final concentration) in PFHR-4 medium (pH 7.4) without serum for 24 h, a...

Pistacia lentiscus fruit oil reduces oxidative stress in human skin explants caused by hydrogen peroxide.

PubMed

Ben Khedir, S; Moalla, D; Jardak, N; Mzid, M; Sahnoun, Z; Rebai, T

2016-10-01

We investigated the efficacy of Pistacia lentiscus fruit oil (PLFO) for protecting human skin from damage due to oxidative stress. PLFO contains natural antioxidants including polyphenols, sterols and tocopherols. We compared the antioxidant potential of PLFO with extra virgin olive oil (EVOO). Explants of healthy adult human skin were grown in culture with either PLFO or EVOO before adding hydrogen peroxide (H 2 O 2 ). We also used cultured skin explants to investigate the effects of PLFO on lipid oxidation and depletion of endogenous antioxidant defense enzymes including glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) one day after 2 h exposure to H 2 O 2 . We found that PLFO scavenged radicals and protected skin against oxidative injury. PLFO exhibited greater antioxidant and free radical scavenging activity than EVOO. Skin explants treated with PLFO inhibited H 2 O 2 induced MDA formation by inhibition of lipid oxidation. In addition, the oil inhibited H 2 O 2 induced depletion of antioxidant defense enzymes including GPx, SOD and CAT. We found that treatment with PLFO repaired skin damage owing to its antioxidant properties.

RAD-ADAPT: Software for modelling clonogenic assay data in radiation biology.

PubMed

Zhang, Yaping; Hu, Kaiqiang; Beumer, Jan H; Bakkenist, Christopher J; D'Argenio, David Z

2017-04-01

We present a comprehensive software program, RAD-ADAPT, for the quantitative analysis of clonogenic assays in radiation biology. Two commonly used models for clonogenic assay analysis, the linear-quadratic model and single-hit multi-target model, are included in the software. RAD-ADAPT uses maximum likelihood estimation method to obtain parameter estimates with the assumption that cell colony count data follow a Poisson distribution. The program has an intuitive interface, generates model prediction plots, tabulates model parameter estimates, and allows automatic statistical comparison of parameters between different groups. The RAD-ADAPT interface is written using the statistical software R and the underlying computations are accomplished by the ADAPT software system for pharmacokinetic/pharmacodynamic systems analysis. The use of RAD-ADAPT is demonstrated using an example that examines the impact of pharmacologic ATM and ATR kinase inhibition on human lung cancer cell line A549 after ionizing radiation. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers.

PubMed Central

Sutherland, H J; Lansdorp, P M; Henkelman, D H; Eaves, A C; Eaves, C J

1990-01-01

A major goal of current hematopoiesis research is to develop in vitro methods suitable for the measurement and characterization of stem cells with long-term in vivo repopulating potential. Previous studies from several centers have suggested the presence in normal human or murine marrow of a population of very primitive cells that are biologically, physically, and pharmacologically different from cells detectable by short-term colony assays and that can give rise to the latter in long-term cultures (LTCs) containing a competent stromal cell layer. In this report, we show that such cultures can be used to provide a quantitative assay for human "LTC-initiating cells" based on an assessment of the number of clonogenic cells present after 5-8 weeks. Production of derivative clonogenic cells is shown to be absolutely dependent on the presence of a stromal cell feeder. When this requirement is met, the clonogenic cell output (determined by assessment of 5-week-old cultures) is linearly related to the input cell number over a wide range of cell concentrations. Using limiting dilution analysis techniques, we have established the frequency of LTC-initiating cells in normal human marrow to be approximately 1 per 2 X 10(4) cells and in a highly purified CD34-positive subpopulation to be approximately 1 per 50-100 cells. The proliferative capacity exhibited by individual LTC-initiating cells cultured under apparently identical culture conditions was found to be highly variable. Values for the number of clonogenic cells per LTC-initiating cell in 5-week-old cultures ranged from 1 to 30 (the average being 4) with similar levels being detected in positive 8-week-old cultures. Some LTC-initiating cells are multipotent as evidenced by their generation of erythroid as well as granulopoietic progeny. The availability of a system for quantitative analysis of the proliferative and differentiative behavior of this newly defined compartment of primitive human hematopoietic cells should facilitate future studies of specific genetic or microenvironmental parameters involved in the regulation of these cells. Images PMID:2333304

Curcumin inhibits pro-inflammatory mediators and metalloproteinase-3 production by chondrocytes.

PubMed

Mathy-Hartert, M; Jacquemond-Collet, I; Priem, F; Sanchez, C; Lambert, C; Henrotin, Y

2009-12-01

This study aims to investigate the effects of curcumin (Cur) on the extracellular matrix protein metabolism of articular chondrocytes and on their production of inflammatory mediators. Human chondrocytes in alginate beads and human cartilage explants were cultured in the absence or in the presence of interleukin (IL)-1beta (10(-11) M) and with or without Cur (5-20 microM). Nitric oxide (NO) synthesis was measured by the Griess spectrophotometric method; prostaglandin (PG) E(2) by a specific radioimmunoassay; and IL-6, IL-8, aggrecan (Agg), matrix metalloproteinase (MMP)-3, and tissue inhibitor of metalloproteinase (TIMP)-1 by specific enzyme-amplified immunoassays. Proteoglycan degradation was evaluated by the release of (35)S-glycosaminoglycans (GAG) from human cartilage explants. In alginate beads and cartilage explant models, Cur inhibited the basal and the IL-1beta-stimulated NO, PGE(2), IL-6, IL-8, and MMP-3 production by human chondrocytes in a concentration-dependent manner. The TIMP-1 and the Agg productions were not modified. In the basal condition, (35)S-GAG release from cartilage explants was decreased by Cur. Curcumin was a potent inhibitor of the production of inflammatory and catabolic mediators by chondrocytes, suggesting that this natural compound could be efficient in the treatment of osteoarthritis.

Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model

PubMed Central

Swift, Brenna E.; Williams, Brent A.; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A.; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

2012-01-01

Background Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. Design and Methods The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Results Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89–99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. Conclusions This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted. PMID:22271890

Inhibition of human lung cancer cell proliferation and survival by wine

PubMed Central

2014-01-01

Background Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Wine contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt and extracellular signal-regulated kinase (Erk), and the tumor suppressor p53 are key modulators of cancer cell growth and survival. In this study, we examined the effects of wine on proliferation and survival of human Non-small cell lung cancer (NSCLC) cells and its effects on signaling events. Methods Human NSCLC adenocarcinoma A549 and H1299 cells were used. Cell proliferation was assessed by thymidine incorporation. Clonogenic assays were used to assess cell survival. Immunoblotting was used to examine total and phosphorylated levels of Akt, Erk and p53. Results In A549 cells red wine inhibited cell proliferation and reduced clonogenic survival at doses as low as 0.02%. Red wine significantly reduced basal and EGF-stimulated Akt and Erk phosphorylation while it increased the levels of total and phosphorylated p53 (Ser15). Control experiments indicated that the anti-proliferative effects of wine were not mediated by the associated contents of ethanol or the polyphenol resveratrol and were independent of glucose transport into cancer cells. White wine also inhibited clonogenic survival, albeit at a higher doses (0.5-2%), and reduced Akt phosphorylation. The effects of both red and white wine on Akt phosphorylation were also verified in H1299 cells. Conclusions Red wine inhibits proliferation of lung cancer cells and blocks clonogenic survival at low concentrations. This is associated with inhibition of basal and EGF-stimulated Akt and Erk signals and enhancement of total and phosphorylated levels of p53. White wine mediates similar effects albeit at higher concentrations. Our data suggest that wine may have considerable anti-tumour and chemoprevention properties in lung cancer and deserves further systematic investigation in animal models of lung cancer. PMID:24456610

Human low density lipoprotein as a substrate for in vitro steroidogenesis assays with fathead minnow ovary explants

EPA Science Inventory

Gonad explant in vitro steroidogenesis assays are used as part of a multifaceted strategy to detect endocrine active chemicals capable of altering steroid hormone synthesis. An in vitro steroidogenesis assay used in our laboratory involves exposing fathead minnow (FHM) gonad exp...

An in vitro human skin test for assessing sensitization potential.

PubMed

Ahmed, S S; Wang, X N; Fielding, M; Kerry, A; Dickinson, I; Munuswamy, R; Kimber, I; Dickinson, A M

2016-05-01

Sensitization to chemicals resulting in an allergy is an important health issue. The current gold-standard method for identification and characterization of skin-sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in-vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro-haptens, respiratory sensitizers, non-sensitizing chemicals (including skin-irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non-sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in-vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process. Copyright © 2015 John Wiley & Sons, Ltd.

Analysis of tamoxifen-DNA adducts in endometrial explants by MS and 32P-postlabeling.

PubMed

Beland, Frederick A; Churchwell, Mona I; Hewer, Alan; Phillips, David H; Gamboa da Costa, Gonçalo; Marques, M Matilde

2004-07-23

The nonsteroidal antiestrogen tamoxifen increases the risk of endometrial cancer; however, the mechanism for the induction of these tumors is not known. Recently, Sharma et al. [Biochem. Biophys. Res. Commun. 307 (2003) 157], using high performance liquid chromatography (HPLC) with online postcolumn photochemical activation and fluorescence detection, reported the presence of (E)-alpha-(deoxyguanosin- N2-yl)tamoxifen in DNA from human endometrial explants incubated with tamoxifen. Inasmuch as the methodology used by these investigators does not allow unambiguous characterization of tamoxifen-DNA adducts, we have used two additional techniques (HPLC coupled with electrospray ionization tandem mass spectrometry and 32P-postlabeling analyses) to assay for the presence of tamoxifen-DNA adducts in the human endometrial explant DNA. Tamoxifen-DNA adducts were not detected by either method.

An ex vivo human skin model for studying skin barrier repair.

PubMed

Danso, Mogbekeloluwa O; Berkers, Tineke; Mieremet, Arnout; Hausil, Farzia; Bouwstra, Joke A

2015-01-01

In the studies described in this study, we introduce a novel ex vivo human skin barrier repair model. To develop this, we removed the upper layer of the skin, the stratum corneum (SC) by a reproducible cyanoacrylate stripping technique. After stripping the explants, they were cultured in vitro to allow the regeneration of the SC. We selected two culture temperatures 32 °C and 37 °C and a period of either 4 or 8 days. After 8 days of culture, the explant generated SC at a similar thickness compared to native human SC. At 37 °C, the early and late epidermal differentiation programmes were executed comparably to native human skin with the exception of the barrier protein involucrin. At 32 °C, early differentiation was delayed, but the terminal differentiation proteins were expressed as in stripped explants cultured at 37 °C. Regarding the barrier properties, the SC lateral lipid organization was mainly hexagonal in the regenerated SC, whereas the lipids in native human SC adopt a more dense orthorhombic organization. In addition, the ceramide levels were higher in the cultured explants at 32 °C and 37 °C than in native human SC. In conclusion, we selected the stripped ex vivo skin model cultured at 37 °C as a candidate model to study skin barrier repair because epidermal and SC characteristics mimic more closely the native human skin than the ex vivo skin model cultured at 32 °C. Potentially, this model can be used for testing formulations for skin barrier repair. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Assessing Anticalcification Treatments in Bioprosthetic Tissue by Using the New Zealand Rabbit Intramuscular Model

PubMed Central

Wright, Gregory A; Faught, Joelle M; Olin, Jane M

2009-01-01

The objective of this work was to demonstrate that the New Zealand White (NZW) rabbit intramuscular model can be used for detecting calcification in bioprosthetic tissue and to compare the calcification in the rabbit to that of native human valves. The rabbit model was compared with the commonly used Sprague–Dawley rat subcutaneous model. Eighteen rabbits and 18 rats were used to assess calcification in bioprosthetic tissue over time (7, 14, 30, and 90 d). The explanted rabbit and rat tissue discs were measured for calcium by using atomic absorption and Raman spectroscopy. Calcium deposits on the human valve explants were assessed by using Raman spectroscopy. The results showed that the NZW rabbit model is robust for detecting calcification in a shorter duration (14 d), with less infection complications, more space to implant tissue groups (thereby reducing animal use numbers), and a more metabolically and mechanically dynamic environment than the rat subcutaneous model . The human explanted valves and rabbit explanted tissue both showed Raman peaks at 960 cm−1 which is representative of hydroxyapatite. Hydroxyapatite is the final calcium and phosphate species in the calcification of bioprosthetic heart valves and rabbit intramuscular implants. The NZW rabbit intramuscular model is an effective model for assessing calcification in bioprosthetic tissue. PMID:19619417

Human colonic myocytes are involved in postischemic inflammation through ADAM17-dependent TNFα production

PubMed Central

Jarry, Anne; Bach-Ngohou, Kalyane; Masson, Damien; Dejoie, Thomas; Lehur, Paul-Antoine; Mosnier, Jean-François; Denis, Marc G; Laboisse, Christian L

2005-01-01

The aim of this study was to identify human colonic resident cells able to initiate an inflammatory response in postischemic injury. Postischemic colonic injury, a condition relevant to various clinical settings, involves an inflammatory cascade in intestinal tissues through the recruitment of circulating inflammatory cells. However, there is no information on the nature of resident cells of the different intestinal layers able to initiate a postischemic inflammatory response. It is however an important issue in the context of a pharmacological approach of the early phase of intestinal ischemia. We reasoned that maintaining the different colonic layers as explant cultures in an oxygenated medium immediately after colonic resection, that is, after an ischemic period, would allow one to identify the resident cells able to initiate an inflammatory cascade, without interference of recruited inflammatory/immune cells. To this end, we designed an explant culture system that operationally defines three compartments in surgical specimens of the human colon, based on the microdissected layers, that is, mucosa, submucosa (containing muscularis mucosae) and muscularis propria. To validate the results obtained in explant cultures in the clinical setting of ischemic colitis, eight cases of sigmoid volvulus were examined. Only the myocytes-containing explants produced tumor necrosis factor alpha (TNFα), via an ADAM17 (a disintegrin and metalloproteinase-17)-dependent pathway, as shown by the abrogation of TNFα production by the inhibitor Tapi-2. Immunofluorescence studies identified nonvascular and vascular myocytes as resident cells coexpressing TNFα and ADAM17, both in our postischemic explant system and in surgical specimens from ischemic colitis patients. Finally, time-course experiments on explanted tissues showed that TNFα production by myocytes was an early event triggered by a postischemic oxidative stress involving nuclear factor kappa B (NF-κB). In conclusion, this study identifies human intestinal myocytes as resident cells able to initiate an inflammatory reaction through TNFα production in postischemic conditions, and delineates two points of control in TNFα production, NF-κB and ADAM17, which can be targeted by pharmacological manipulation. PMID:16273118

Endocrine Disruption in Human Fetal Testis Explants by Individual and Combined Exposures to Selected Pharmaceuticals, Pesticides, and Environmental Pollutants

PubMed Central

Gaudriault, Pierre; Mazaud-Guittot, Séverine; Lavoué, Vincent; Coiffec, Isabelle; Lesné, Laurianne; Dejucq-Rainsford, Nathalie; Scholze, Martin; Kortenkamp, Andreas

2017-01-01

Background: Numerous chemicals are capable of disrupting androgen production, but the possibility that they might act together to produce effects greater than those of the most effective component in the mixture has not been studied directly in human tissues. Suppression of androgen synthesis in fetal life has been associated with testis maldescent, malformations of the genitalia at birth, and poor semen quality later in life. Objectives: Our aim was to investigate whether chemicals can act together to disrupt androgen production in human fetal testis explants and to evaluate the importance of mixture effects when characterizing the hazard of individual chemicals. Methods: We used an organotypic culture system of human fetal testes explants called FEtal Gonad Assay (FEGA) with tissue obtained at 10 and 12 gestational wk (GW 10–12), to screen 27 chemicals individually for their possible anti-androgenic effect. Based on the results of the screen, we selected 11 compounds and tested them as mixtures. Results: We evaluated mixtures composed of four and eight antiandrogens that contained the pharmaceuticals ketoconazole and theophylline and several previously untested chemicals, such as the pesticides imazalil and propiconazole. Mixtures of antiandrogens can suppress testosterone synthesis in human fetal testicular explants to an extent greater than that seen with individual chemicals. This revealed itself as a shift towards lower doses in the dose–response curves of individual antiandrogens that became more pronounced as the number of components increased from four to eight. Conclusions: Our results with the FEGA provide the foundations of a predictive human mixture risk assessment approach for anti-androgenic exposures in fetal life. https://doi.org/10.1289/EHP1014 PMID:28796631

Endocrine Disruption in Human Fetal Testis Explants by Individual and Combined Exposures to Selected Pharmaceuticals, Pesticides, and Environmental Pollutants.

PubMed

Gaudriault, Pierre; Mazaud-Guittot, Séverine; Lavoué, Vincent; Coiffec, Isabelle; Lesné, Laurianne; Dejucq-Rainsford, Nathalie; Scholze, Martin; Kortenkamp, Andreas; Jégou, Bernard

2017-08-04

Numerous chemicals are capable of disrupting androgen production, but the possibility that they might act together to produce effects greater than those of the most effective component in the mixture has not been studied directly in human tissues. Suppression of androgen synthesis in fetal life has been associated with testis maldescent, malformations of the genitalia at birth, and poor semen quality later in life. Our aim was to investigate whether chemicals can act together to disrupt androgen production in human fetal testis explants and to evaluate the importance of mixture effects when characterizing the hazard of individual chemicals. We used an organotypic culture system of human fetal testes explants called FEtal Gonad Assay (FEGA) with tissue obtained at 10 and 12 gestational wk (GW 10-12), to screen 27 chemicals individually for their possible anti-androgenic effect. Based on the results of the screen, we selected 11 compounds and tested them as mixtures. We evaluated mixtures composed of four and eight antiandrogens that contained the pharmaceuticals ketoconazole and theophylline and several previously untested chemicals, such as the pesticides imazalil and propiconazole. Mixtures of antiandrogens can suppress testosterone synthesis in human fetal testicular explants to an extent greater than that seen with individual chemicals. This revealed itself as a shift towards lower doses in the dose-response curves of individual antiandrogens that became more pronounced as the number of components increased from four to eight. Our results with the FEGA provide the foundations of a predictive human mixture risk assessment approach for anti-androgenic exposures in fetal life. https://doi.org/10.1289/EHP1014.

Comparative phenotypic and functional analysis of migratory dendritic cell subsets from human oral mucosa and skin

PubMed Central

van de Ven, Rieneke; Thon, Maria; Gibbs, Susan; de Gruijl, Tanja D.

2017-01-01

Antigen exposure to oral mucosa is generally thought to lead to immune tolerance induction. However, very little is known about the subset composition and function of dendritic cells (DC) migrating from human oral mucosa. Here we show that migratory DC from healthy human gingival explants consist of the same phenotypic subsets in the same frequency distribution as DC migrating from human skin. The gingival CD1a+ Langerhans cell and interstitial DC subsets lacked CXCR4 expression in contrast to their cutaneous counterparts, pointing to different migration mechanisms, consistent with previous observations in constructed skin and gingival equivalents. Remarkably, without any exogenous conditioning, gingival explants released higher levels of inflammatory cytokines than human skin explants, resulting in higher DC migration rates and a superior ability of migrated DC to prime allogeneic T cells and to induce type-1 effector T cell differentiation. From these observations we conclude that rather than an intrinsic ability to induce T cell tolerance, DC migrating from oral mucosa may have a propensity to induce effector T cell immunity and maintain a high state of alert against possible pathogenic intruders in the steady state. These findings may have implications for oral immunization strategies. PMID:28704477

Effect of ultrasound sonication on clonogenic survival and mitochondria of ovarian cancer cells in the presence of methylene blue.

PubMed

Xiang, Junyan; Leung, Albert Wingnang; Xu, Chuanshan

2014-10-01

This study aimed to investigate the effect of ultrasound sonication in the presence of methylene blue on clonogenic survival and mitochondria of ovarian cancer cells. Human ovarian cancer HO-8910 cells, which were incubated with different concentrations of methylene blue for 1 hour, were exposed to an ultrasonic wave for 5 seconds with intensity of 0.46 W/cm(2). Clonogenic survival of HO-8910 cells after ultrasound sonication was measured by a colony-forming unit assay. Mitochondrial structural changes were observed on transmission electron microscopy, and the mitochondrial membrane potential was evaluated by confocal laser-scanning microscopy with rhodamine 123 staining. The colony-forming units of HO-8910 cells decreased considerably after ultrasound sonication in the presence of methylene blue. Transmission electron microscopy showed slightly enlarged mitochondria in the ultrasound-treated cells in the absence of methylene blue; however, seriously damaged mitochondria, even with almost complete disappearance of cristae, were found in the cells treated by ultrasound sonication in the presence of methylene blue. The mitochondrial membrane potential collapsed significantly when HO-8910 cells were treated by ultrasound sonication in the presence of methylene blue (P < .05). Ultrasound sonication in the presence of methylene blue markedly damaged mitochondrial structure and function and decreased clonogenic survival of HO-8910 cells. © 2014 by the American Institute of Ultrasound in Medicine.

Reduction of friction by recombinant human proteoglycan 4 in IL-1α stimulated bovine cartilage explants.

PubMed

Larson, Katherine M; Zhang, Ling; Elsaid, Khaled A; Schmidt, Tannin A; Fleming, Braden C; Badger, Gary J; Jay, Gregory D

2017-03-01

A boundary lubricant attaches and protects sliding bearing surfaces by preventing interlocking asperity-asperity contact. Proteoglycan-4 (PRG4) is a boundary lubricant found in the synovial fluid that provides chondroprotection to articular surfaces. Inflammation of the diarthrodial joint modulates local PRG4 concentration. Thus, we measured the effects of inflammation, with Interleukin-1α (IL-1α) incubation, upon boundary lubrication and PRG4 expression in bovine cartilage explants. We further aimed to determine whether the addition of exogenous human recombinant PRG4 (rhPRG4) could mitigate the effects of inflammation on boundary lubrication and PRG4 expression in vitro. Cartilage explants, following a 7 day incubation with IL-1α, were tested in a disc-on-disc configuration using either rhPRG4 or saline (PBS control) as a lubricant. Following mechanical testing, explants were studied immunohistochemically or underwent RNA extraction for real-time polymerase chain reaction (RT-PCR). We found that static coefficient of friction (COF) significantly decreased to 0.14 ± 0.065 from 0.21 ± 0.059 (p = 0.014) in IL-1α stimulated explants lubricated with rhPRG4, as compared to PBS. PRG4 expression was significantly up regulated from 30.8 ± 19 copies in control explants lubricated with PBS to 3330 ± 1760 copies in control explants lubricated with rhPRG4 (p < 0.001). Explants stimulated with IL-1α displayed no increase in PRG4 expression upon lubrication with rhPRG4, but with PBS as the lubricant, IL-1α stimulation significantly increased PRG4 expression compared to the control condition from 30.8 ± 19 copies to 401 ± 340 copies (p = 0.015). Overall, these data suggest that exogenous rhPRG4 may provide a therapeutic option for reducing friction in transient inflammatory conditions and increasing PRG4 expression. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:580-589, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Expression of varicella-zoster virus and herpes simplex virus in normal human trigeminal ganglia

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

Vafai, A.; Wellish, M.; Devlin, M.

1988-04-01

Lysates of radiolabeled explants from four human trigeminal ganglia were immunoprecipitated with antibodies to varicella-zoster virus (VZV) and to herpes simplex virus. Both herpes simplex virus- and VZV-specific proteins were detected in lysates of all four ganglia. Absence of reactivity in ganglion explants with monoclonal antibodies suggested that herpes simplex virus and VZV were not reactivated during the culture period. In situ hybridization studies demonstrated the presence of RNA transcripts from the VZV immediate early gene 63. This approach to the detection of herpes simplex virus and VZV expression in human ganglia should facilitate analysis of viral RNA and proteinsmore » in human sensory ganglia.« less

Critical role of zinc finger protein 521 in the control of growth, clonogenicity and tumorigenic potential of medulloblastoma cells

PubMed Central

Iaccino, Enrico; Scicchitano, Stefania; Lupia, Michela; Chiarella, Emanuela; Mega, Tiziana; Bernaudo, Francesca; Pelaggi, Daniela; Mesuraca, Maria; Pazzaglia, Simonetta; Semenkow, Samantha; Bar, Eli E.; Kool, Marcel; Pfister, Stefan; Bond, Heather M.; Eberhart, Charles G.; Steinkühler, Christian; Morrone, Giovanni

2013-01-01

The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of hematopoietic, osteo-adipogenic and neural progenitor cells. ZNF521 is highly expressed in cerebellum and in particular in the neonatal external granule layer that contains candidate medulloblastoma cells-of-origin, and in the majority of human medulloblastomas. Here we have explored its involvement in the control of human and murine medulloblastoma cells. The effect of ZNF521 on growth and tumorigenic potential of human medulloblastoma cell lines as well as primary Ptc1−/+ mouse medulloblastoma cells was investigated in a variety of in vitro and in vivo assays, by modulating its expression using lentiviral vectors carrying the ZNF521 cDNA, or shRNAs that silence its expression. Enforced overexpression of ZNF521 in DAOY medulloblastoma cells significantly increased their proliferation, growth as spheroids and ability to generate clones in single-cell cultures and semisolid media, and enhanced their migratory ability in wound-healing assays. Importantly, ZNF521-expressing cells displayed a greatly enhanced tumorigenic potential in nude mice. All these activities required the ZNF521 N-terminal motif that recruits the nucleosome remodeling and histone deacetylase complex, which might therefore represent an appealing therapeutic target. Conversely, silencing of ZNF521 in human UW228 medulloblastoma cells that display high baseline expression decreased their proliferation, clonogenicity, sphere formation and wound-healing ability. Similarly, Zfp521 silencing in mouse Ptc1−/+ medulloblastoma cells drastically reduced their growth and tumorigenic potential. Our data strongly support the notion that ZNF521, through the recruitment of the NuRD complex, contributes to the clonogenic growth, migration and tumorigenicity of medulloblastoma cells. PMID:23907569

Critical role of zinc finger protein 521 in the control of growth, clonogenicity and tumorigenic potential of medulloblastoma cells.

PubMed

Spina, Raffaella; Filocamo, Gessica; Iaccino, Enrico; Scicchitano, Stefania; Lupia, Michela; Chiarella, Emanuela; Mega, Tiziana; Bernaudo, Francesca; Pelaggi, Daniela; Mesuraca, Maria; Pazzaglia, Simonetta; Semenkow, Samantha; Bar, Eli E; Kool, Marcel; Pfister, Stefan; Bond, Heather M; Eberhart, Charles G; Steinkühler, Christian; Morrone, Giovanni

2013-08-01

The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of hematopoietic, osteo-adipogenic and neural progenitor cells. ZNF521 is highly expressed in cerebellum and in particular in the neonatal external granule layer that contains candidate medulloblastoma cells-of-origin, and in the majority of human medulloblastomas. Here we have explored its involvement in the control of human and murine medulloblastoma cells. The effect of ZNF521 on growth and tumorigenic potential of human medulloblastoma cell lines as well as primary Ptc1-/+ mouse medulloblastoma cells was investigated in a variety of in vitro and in vivo assays, by modulating its expression using lentiviral vectors carrying the ZNF521 cDNA, or shRNAs that silence its expression. Enforced overexpression of ZNF521 in DAOY medulloblastoma cells significantly increased their proliferation, growth as spheroids and ability to generate clones in single-cell cultures and semisolid media, and enhanced their migratory ability in wound-healing assays. Importantly, ZNF521-expressing cells displayed a greatly enhanced tumorigenic potential in nude mice. All these activities required the ZNF521 N-terminal motif that recruits the nucleosome remodeling and histone deacetylase complex, which might therefore represent an appealing therapeutic target. Conversely, silencing of ZNF521 in human UW228 medulloblastoma cells that display high baseline expression decreased their proliferation, clonogenicity, sphere formation and wound-healing ability. Similarly, Zfp521 silencing in mouse Ptc1-/+ medulloblastoma cells drastically reduced their growth and tumorigenic potential. Our data strongly support the notion that ZNF521, through the recruitment of the NuRD complex, contributes to the clonogenic growth, migration and tumorigenicity of medulloblastoma cells.

Multisite Comparison of Anti-Human Immunodeficiency Virus Microbicide Activity in Explant Assays Using a Novel Endpoint Analysis ▿ †

PubMed Central

Richardson-Harman, Nicola; Lackman-Smith, Carol; Fletcher, Patricia S.; Anton, Peter A.; Bremer, James W.; Dezzutti, Charlene S.; Elliott, Julie; Grivel, Jean-Charles; Guenthner, Patricia; Gupta, Phalguni; Jones, Maureen; Lurain, Nell S.; Margolis, Leonid B.; Mohan, Swarna; Ratner, Deena; Reichelderfer, Patricia; Roberts, Paula; Shattock, Robin J.; Cummins, James E.

2009-01-01

Microbicide candidates with promising in vitro activity are often advanced for evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of human immunodeficiency virus type 1 (HIV-1), such as tonsil, cervical, or rectal tissue. To compare virus growth or the anti-HIV-1 efficacies of candidate microbicides in tissue explants, a novel soft-endpoint method was evaluated to provide a single, objective measurement of virus growth. The applicability of the soft endpoint is shown across several different ex vivo tissue types, with the method performed in different laboratories, and for a candidate microbicide (PRO 2000). The soft-endpoint method was compared to several other endpoint methods, including (i) the growth of virus on specific days after infection, (ii) the area under the virus growth curve, and (iii) the slope of the virus growth curve. Virus growth at the assay soft endpoint was compared between laboratories, methods, and experimental conditions, using nonparametric statistical analyses. Intra-assay variability determinations using the coefficient of variation demonstrated higher variability for virus growth in rectal explants. Significant virus inhibition by PRO 2000 and significant differences in the growth of certain primary HIV-1 isolates were observed by the majority of laboratories. These studies indicate that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy when (i) the soft endpoint or another standardized endpoint is used, (ii) drugs and/or virus reagents are centrally sourced, and (iii) the same explant tissue type and method are used. Application of the soft-endpoint method reduces the inherent variability in comparisons of preclinical assays used for microbicide development. PMID:19726602

Exposure of a tendon extracellular matrix to synovial fluid triggers endogenous and engrafted cell death: A mechanism for failed healing of intrathecal tendon injuries.

PubMed

Garvican, Elaine R; Salavati, Mazdak; Smith, Roger K W; Dudhia, Jayesh

2017-09-01

The purpose of this study was to investigate the effect of normal synovial fluid (SF) on exposed endogenous tendon-derived cells (TDCs) and engrafted mesenchymal stem cells (MSCs) within the tendon extracellular matrix. Explants from equine superficial digital flexor (extra-synovial) and deep digital flexor tendons (DDFTs) from the compressed, intra-synovial and the tensile, extra-synovial regions were cultured in allogeneic or autologous SF-media. Human hamstring explants were cultured in allogeneic SF. Explant viability was assessed by staining. Proliferation of equine monolayer MSCs and TDCs in SF-media and co-culture with DDFT explants was determined by alamarblue®. Non-viable Native Tendon matrices (NNTs) were re-populated with MSCs or TDCs and cultured in SF-media. Immunohistochemical staining of tendon sections for the apoptotic proteins caspase-3, -8, and -9 was performed. Contact with autologous or allogeneic SF resulted in rapid death of resident tenocytes in equine and human tendon. SF did not affect the viability of equine epitenon cells, or of MSCs and TDCs in the monolayer or indirect explant co-culture. MSCs and TDCs, engrafted into NNTs, died when cultured in SF. Caspase-3, -8, and -9 expression was the greatest in SDFT explants exposed to allogeneic SF. The efficacy of cells administered intra-synovially for tendon lesion repair is likely to be limited, since once incorporated into the matrix, cells become vlnerable to the adverse effects of SF. These observations could account for the poor success rate of intra-synovial tendon healing following damage to the epitenon and contact with SF, common with most soft tissue intra-synovial pathologies.

Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues.

PubMed

Hendijani, Fatemeh

2017-04-01

Mesenchymal stem cell (MSC) research progressively moves towards clinical phases. Accordingly, a wide range of different procedures were presented in the literature for MSC isolation from human tissues; however, there is not yet any close focus on the details to offer precise information for best method selection. Choosing a proper isolation method is a critical step in obtaining cells with optimal quality and yield in companion with clinical and economical considerations. In this concern, current review widely discusses advantages of omitting proteolysis step in isolation process and presence of tissue pieces in primary culture of MSCs, including removal of lytic stress on cells, reduction of in vivo to in vitro transition stress for migrated/isolated cells, reduction of price, processing time and labour, removal of viral contamination risk, and addition of supporting functions of extracellular matrix and released growth factors from tissue explant. In next sections, it provides an overall report of technical highlights and molecular events of explant culture method for isolation of MSCs from human tissues including adipose tissue, bone marrow, dental pulp, hair follicle, cornea, umbilical cord and placenta. Focusing on informative collection of molecular and methodological data about explant methods can make it easy for researchers to choose an optimal method for their experiments/clinical studies and also stimulate them to investigate and optimize more efficient procedures according to clinical and economical benefits. © 2017 John Wiley & Sons Ltd.

Hematopoietic Stem Cells from Ts65Dn Mice Are Deficient in the Repair of DNA Double-Strand Breaks.

PubMed

Wang, Yingying; Chang, Jianhui; Shao, Lijian; Feng, Wei; Luo, Yi; Chow, Marie; Du, Wei; Meng, Aimin; Zhou, Daohong

2016-06-01

Down syndrome (DS) is a genetic disorder caused by the presence of an extra partial or whole copy of chromosome 21. In addition to musculoskeletal and neurodevelopmental abnormalities, children with DS exhibit various hematologic disorders and have an increased risk of developing acute lymphoblastic leukemia and acute megakaryocytic leukemia. Using the Ts65Dn mouse model, we investigated bone marrow defects caused by trisomy for 132 orthologs of the genes on human chromosome 21. The results showed that, although the total bone marrow cellularity as well as the frequency of hematopoietic progenitor cells (HPCs) was comparable between Ts65Dn mice and their age-matched euploid wild-type (WT) control littermates, human chromosome 21 trisomy led to a significant reduction in hematopoietic stem cell (HSC) numbers and clonogenic function in Ts65Dn mice. We also found that spontaneous DNA double-strand breaks (DSBs) were significantly increased in HSCs from the Ts65Dn mice, which was correlated with the significant reduction in HSC clonogenic activity compared to those from WT controls. Moreover, analysis of the repair kinetics of radiation-induced DSBs revealed that HSCs from Ts65Dn mice were less proficient in DSB repair than the cells from WT controls. This deficiency was associated with a higher sensitivity of Ts65Dn HSCs to radiation-induced suppression of HSC clonogenic activity than that of euploid HSCs. These findings suggest that an additional copy of genes on human chromosome 21 may selectively impair the ability of HSCs to repair DSBs, which may contribute to DS-associated hematological abnormalities and malignancies.

Efficient and safe gene delivery to human corneal endothelium using magnetic nanoparticles.

PubMed

Czugala, Marta; Mykhaylyk, Olga; Böhler, Philip; Onderka, Jasmine; Stork, Björn; Wesselborg, Sebastian; Kruse, Friedrich E; Plank, Christian; Singer, Bernhard B; Fuchsluger, Thomas A

2016-07-01

To develop a safe and efficient method for targeted, anti-apoptotic gene therapy of corneal endothelial cells (CECs). Magnetofection (MF), a combination of lipofection with magnetic nanoparticles (MNPs; PEI-Mag2, SO-Mag5, PalD1-Mag1), was tested in human CECs and in explanted human corneas. Effects on cell viability and function were investigated. Immunocompatibility was assessed in human peripheral blood mononuclear cells. Silica iron-oxide MNPs (SO-Mag5) combined with X-tremeGENE-HP achieved high transfection efficiency in human CECs and explanted human corneas, without altering cell viability or function. Magnetofection caused no immunomodulatory effects in human peripheral blood mononuclear cells. Magnetofection with anti-apoptotic P35 gene effectively blocked apoptosis in CECs. Magnetofection is a promising tool for gene therapy of corneal endothelial cells with potential for targeted on-site delivery.

Insulin regulates the novel adipokine adipolin/CTRP12: in vivo and ex vivo effects.

PubMed

Tan, Bee K; Lewandowski, Krzysztof C; O'Hare, Joseph Paul; Randeva, Harpal S

2014-04-01

There has been intense interest in the adipokines of the C1q complement/TNF-related protein (CTRP) superfamily. Adipolin (CTRP12) has been described as a novel adipokine, abundantly expressed in adipose tissue with insulin-sensitising and anti-inflammatory effects. We wanted to investigate the effects of acute and chronic hyperinsulinaemia on circulating adipolin concentrations (ELISA) via a prolonged insulin-glucose infusion in humans. We also examined the effects of insulin and the insulin sensitiser, rosiglitazone, on adipolin concentrations (western blotting) in human adipose tissue explants. We found that hyperinsulinaemic induction in healthy lean human subjects significantly increased circulating levels of adipolin (P<0.05 and P<0.01). Furthermore, in subcutaneous adipose tissue explants, insulin significantly increased adipolin protein expression and secretion (P<0.05 and P<0.01). This effect was attenuated by the phosphatidylinositol 3-kinase inhibitor, LY294002 (P<0.05). Moreover, the insulin-sensitising peroxisome proliferator-activated receptor γ (PPARγ) agonist, rosiglitazone, significantly increased adipolin protein expression and secretion in subcutaneous adipose tissue explants (P<0.05 and P<0.01). This effect was inhibited by the PPARγ antagonist, GW9662 (P<0.05). Our data provide novel insights into adipolin physiology in human subjects.

George Papanicolaou's Efforts to Develop Novel Cytologic Methods for the Early Diagnosis of Endometrial Carcinoma.

PubMed

Austin, R Marshall

2017-01-01

Toward the end of his career, Dr. George Papanicolaou became interested in human endometrial explants placed into tissue culture. The initial focus of his studies was on phagocytic cells emanating from endometrial explants and their role in cleansing the uterine cavity after each menstrual cycle and in sterilizing the uterine cavity in the face of infection. Papanicolaou also observed that growth rates of explanted normal and pathologic endometrial tissues differed considerably. Explants of endometrial malignancies exhibited not only increased growth rates but also visible proliferation of cells with readily identifiable cytologic features of malignancy. Acknowledging that cytologic screening for early diagnosis of intrauterine malignancies had up to that point not proven to be reliable as screening for cervical cancer, he hoped that the tissue culture explant technique could prove to be a new adjunctive diagnostic method for the diagnosis of endometrial and other female genital tract malignancies not readily detectible by other diagnostic procedures. Papanicolaou's untimely death in 1962 cut short his progress in this area of study. © 2017 S. Karger AG, Basel.

Corneal endothelial autocrine trophic factor VIP in a mechanism-based strategy to enhance human donor cornea preservation for transplantation.

PubMed

Koh, Shay-Whey Margaret

2012-02-01

Vasoactive intestinal peptide (VIP) and ciliary neurotrophic factor (CNTF) are identified as autocrines of human corneal endothelial (CE) cells working in concert to maintain the differentiated state and promote the survival of the corneal endothelium. From VIP gene knockdown study, endogenous VIP is shown to maintain the level of the differentiation marker, the adhesion molecule N-cadherin, CE cell size, shape, and retention, in situ in the human donor corneoscleral explants. Exogenous VIP protects the corneal endothelium against the killing effect of oxidative stress, in part by upholding ATP levels in CE cells dying of oxidative stress-induced injury, allowing them to die of an apoptotic death instead of an acute necrotic one. The switch from the acute necrosis to the programmed cell death (apoptosis) may have allowed the injured CE cell to be rescued by the VIP-upregulated pathways, including those of Bcl-2 and N-cadherin, and resulted in long-term CE cell survival. The endogenous VIP in CE cells is upregulated by CNTF, which is released by CE cells surviving the oxidative stress. The CNTF receptor (CNTFRα) is expressed in CE cells in human donor corneoscleral explant and gradually becomes lost during corneal storage. VIP treatment (10(-8) M, 37 °C, 30 min) prior to storage of freshly dissected human donor corneoscleral explants increases their CE cell CNTFRα level and responsiveness to CNTF in upregulating the gap junctional protein connexin-43 expression. VIP treatment of both fresh and preserved corneoscleral explants reduces CE damage in the corneoscleral explants and in the corneal buttons trephined from them. CE cell loss is a critical risk factor in corneal graft failure at any time in the life of the graft, which can be as late as 5-10 years after an initially successful transplant. A new procedure, Descemet's stripping automated endothelial keratoplasty (DSAEK), which is superior to the traditional full thickness transplantation in many aspects, nevertheless subjects the corneal endothelium to extensive mechanical forces, resulting in even more pronounced CE cell loss than the traditional technique. Whereas it is known that cells transduce mechanical stress through N-cadherin, stimulation of the N-cadherin pathway increases the anti-apoptotic protein Bcl-2 expression. Since N-cadherin and Bcl-2 in the corneal endothelium are both upregulated by VIP, we aim to strengthen the CE sheet by VIP treatments of the corneoscleral explants for full thickness traditional corneal transplantation and pre-cut corneas for DSAEK. Copyright © 2011 Elsevier Ltd. All rights reserved.

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells.

PubMed

Jayakumar, Sundarraj; Bhilwade, Hari N; Pandey, Badri N; Sandur, Santosh K; Chaubey, Ramesh C

2012-10-09

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation. © 2012 Elsevier B.V. All rights reserved.

Sacha Inchi Oil (Plukenetia volubilis L.), effect on adherence of Staphylococus aureus to human skin explant and keratinocytes in vitro.

PubMed

Gonzalez-Aspajo, German; Belkhelfa, Haouaria; Haddioui-Hbabi, Laïla; Bourdy, Geneviève; Deharo, Eric

2015-08-02

Plukenetia volubilis L. (Euphorbiaceae) is a domesticated vine distributed from the high-altitude Andean rain forest to the lowlands of the Peruvian Amazon. Oil from the cold-pressed seeds, sold under the commercial name of Sacha Inchi Oil (SIO) is actually much in favour because it contains a high percentage of omega 3 and omega 6, and is hence used as a dietary supplement. SIO is also used traditionally for skin care, in order to maintain skin softness, and for the treatment of wounds, insect bites and skin infections, in a tropical context where the skin is frequently damaged. This study was designed in order to verify whether the traditional use of SIO for skin care would have any impact on Staphylococcus aureus growth and skin adherence, as S. aureus is involved in many skin pathologies (impetigo, folliculitis, furuncles and subcutaneous abscesses) being one if the main pathogens that can be found on the skin. Therefore, our objective was to assess SIO bactericidal activity and interference with adherence to human skin explants and the keratinocyte cell line. Cytotoxicity on that cells was also determined. The activity of SIO was compared to coconut oil (CocO), which is widely used for skin care but has different unsaturated fatty acids contents. Laboratory testing with certified oil, determined antibacterial activity against radio labelled S. aureus. Cytotoxic effects were measured with XTT on keratinocyte cells and with neutral red on human skin explants; phenol was used as cytotoxic control. Adherence assays were carried out by mixing H3-labelled S. aureus bacteria with keratinocyte cells and human skin explants, incubated with oils 2h before (to determine the inhibition of adherence, assimilated to a preventive effect) or 2h after the contact of the biological material with S. aureus (to assess the detachment of the bacteria, assimilated to a curative effect). Residual radioactivity measured after washings made it possible to determine the adherence intensity. Bactericidal effect was determined by colony counting on trypticase soy agar. Laboratory assays showed that SIO and CocO, tested undiluted, were not cytotoxic on keratinocytes nor human explants and were not bactericidal neither. SIO was more active as antiadherent (preventive) than CocO on keratinocytes. There was no significant difference between detachment effects (curative) of both oils on keratinocytes but SIO was almost 5 times more active on the detachment of S. aureus from human skin explants. From that study it can be concluded that the use of SIO on dermal cells is safe and efficient in the inhibition of S. aureus adherence. Our results tend to support the traditional use of undiluted SIO in skin care. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Comprehensive Profiling of Radiosensitive Human Cell Lines with DNA Damage Response Assays Identifies the Neutral Comet Assay as a Potential Surrogate for Clonogenic Survival

PubMed Central

Nahas, Shareef A.; Davies, Robert; Fike, Francesca; Nakamura, Kotoka; Du, Liutao; Kayali, Refik; Martin, Nathan T.; Concannon, Patrick; Gatti, Richard A.

2015-01-01

In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We characterized a unique panel of 19 “radiosensitive” human lymphoblastoid cell lines from individuals with undiagnosed diseases suggestive of a DNA repair disorder. Radiosensitivity was defined by reduced cellular survival using a clonogenic survival assay. Each assay identified cell lines with defects in DNA damage response functions. The highest concordance rate observed, 89% (17/19), was between an abnormal neutral comet assay and reduced survival by the colony survival assay. Our data also suggested that the neutral comet assay would be a more rapid surrogate for analyzing DNA repair/processing disorders. PMID:21962002

Expression and function of human hemokinin-1 in human and guinea pig airways.

PubMed

Grassin-Delyle, Stanislas; Naline, Emmanuel; Buenestado, Amparo; Risse, Paul-André; Sage, Edouard; Advenier, Charles; Devillier, Philippe

2010-10-07

Human hemokinin-1 (hHK-1) and endokinins are peptides of the tachykinin family encoded by the TAC4 gene. TAC4 and hHK-1 expression as well as effects of hHK-1 in the lung and airways remain however unknown and were explored in this study. RT-PCR analysis was performed on human bronchi to assess expression of tachykinin and tachykinin receptors genes. Enzyme immunoassay was used to quantify hHK-1, and effects of hHK-1 and endokinins on contraction of human and guinea pig airways were then evaluated, as well as the role of hHK-1 on cytokines production by human lung parenchyma or bronchi explants and by lung macrophages. In human bronchi, expression of the genes that encode for hHK-1, tachykinin NK1-and NK2-receptors was demonstrated. hHK-1 protein was found in supernatants from explants of human bronchi, lung parenchyma and lung macrophages. Exogenous hHK-1 caused a contractile response in human bronchi mainly through the activation of NK2-receptors, which blockade unmasked a NK1-receptor involvement, subject to a rapid desensitization. In the guinea pig trachea, hHK-1 caused a concentration-dependant contraction mainly mediated through the activation of NK1-receptors. Endokinin A/B exerted similar effects to hHK-1 on both human bronchi and guinea pig trachea, whereas endokinins C and D were inactive. hHK-1 had no impact on the production of cytokines by explants of human bronchi or lung parenchyma, or by human lung macrophages. We demonstrate endogenous expression of TAC4 in human bronchi, the encoded peptide hHK-1 being expressed and involved in contraction of human and guinea pig airways.

Hematopoietic Stem Cells from Ts65Dn Mice Are Deficient in the Repair of DNA Double-Strand Breaks

PubMed Central

Wang, Yingying; Chang, Jianhui; Shao, Lijian; Feng, Wei; Luo, Yi; Chow, Marie; Du, Wei; Meng, Aimin; Zhou, Daohong

2016-01-01

Down syndrome (DS) is a genetic disorder caused by the presence of an extra partial or whole copy of chromosome 21. In addition to musculoskeletal and neurodevelopmental abnormalities, children with DS exhibit various hematologic disorders and have an increased risk of developing acute lymphoblastic leukemia and acute megakaryocytic leukemia. Using the Ts65Dn mouse model, we investigated bone marrow defects caused by trisomy for 132 orthologs of the genes on human chromosome 21. The results showed that, although the total bone marrow cellularity as well as the frequency of hematopoietic progenitor cells (HPCs) was comparable between Ts65Dn mice and their age-matched euploid wild-type (WT) control littermates, human chromosome 21 trisomy led to a significant reduction in hematopoietic stem cell (HSC) numbers and clonogenic function in Ts65Dn mice. We also found that spontaneous DNA double-strand breaks (DSBs) were significantly increased in HSCs from the Ts65Dn mice, which was correlated with the significant reduction in HSC clonogenic activity compared to those from WT controls. Moreover, analysis of the repair kinetics of radiation-induced DSBs revealed that HSCs from Ts65Dn mice were less proficient in DSB repair than the cells from WT controls. This deficiency was associated with a higher sensitivity of Ts65Dn HSCs to radiation-induced suppression of HSC clonogenic activity than that of euploid HSCs. These findings suggest that an additional copy of genes on human chromosome 21 may selectively impair the ability of HSCs to repair DSBs, which may contribute to DS-associated hematological abnormalities and malignancies. PMID:27243896

Candidate Microbicides Block HIV-1 Infection of Human Immature Langerhans Cells within Epithelial Tissue Explants

PubMed Central

Kawamura, Tatsuyoshi; Cohen, Sandra S.; Borris, Debra L.; Aquilino, Elisabeth A.; Glushakova, Svetlana; Margolis, Leonid B.; Orenstein, Jan M.; Offord, Robin E.; Neurath, A. Robert; Blauvelt, Andrew

2000-01-01

Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1Ba-L infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08–4.77%). HIV-1–infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1–1 μg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1Ba-L (an R5 HIV-1 strain) more efficiently infected LC–T cell cocultures when compared with HIV-1IIIB (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1. PMID:11085750

Heterogeneity of clonogenic cells in acute myeloblastic leukemia.

PubMed Central

Sabbath, K D; Ball, E D; Larcom, P; Davis, R B; Griffin, J D

1985-01-01

The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells. PMID:3855866

Heterogeneity of clonogenic cells in acute myeloblastic leukemia.

PubMed

Sabbath, K D; Ball, E D; Larcom, P; Davis, R B; Griffin, J D

1985-02-01

The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells.

Viral Infection of Human Lung Macrophages Increases PDL1 Expression via IFNβ

PubMed Central

Staples, Karl J.; Nicholas, Ben; McKendry, Richard T.; Spalluto, C. Mirella; Wallington, Joshua C.; Bragg, Craig W.; Robinson, Emily C.; Martin, Kirstin; Djukanović, Ratko; Wilkinson, Tom M. A.

2015-01-01

Lung macrophages are an important defence against respiratory viral infection and recent work has demonstrated that influenza-induced macrophage PDL1 expression in the murine lung leads to rapid modulation of CD8+ T cell responses via the PD1 receptor. This PD1/PDL1 pathway may downregulate acute inflammatory responses to prevent tissue damage. The aim of this study was to investigate the mechanisms of PDL1 regulation by human macrophages in response to viral infection. Ex-vivo viral infection models using influenza and RSV were established in human lung explants, isolated lung macrophages and monocyte-derived macrophages (MDM) and analysed by flow cytometry and RT-PCR. Incubation of lung explants, lung macrophages and MDM with X31 resulted in mean cellular infection rates of 18%, 18% and 29% respectively. Viral infection significantly increased cell surface expression of PDL1 on explant macrophages, lung macrophages and MDM but not explant epithelial cells. Infected MDM induced IFNγ release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFNβ mRNA and protein release by MDM in response to influenza infection. Knockdown of IFNβ by siRNA, resulted in a 37.5% reduction in IFNβ gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFNβ, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFNγ release in response to virus and that this expression is regulated by autologous IFNβ production. PMID:25775126

Viral infection of human lung macrophages increases PDL1 expression via IFNβ.

PubMed

Staples, Karl J; Nicholas, Ben; McKendry, Richard T; Spalluto, C Mirella; Wallington, Joshua C; Bragg, Craig W; Robinson, Emily C; Martin, Kirstin; Djukanović, Ratko; Wilkinson, Tom M A

2015-01-01

Lung macrophages are an important defence against respiratory viral infection and recent work has demonstrated that influenza-induced macrophage PDL1 expression in the murine lung leads to rapid modulation of CD8+ T cell responses via the PD1 receptor. This PD1/PDL1 pathway may downregulate acute inflammatory responses to prevent tissue damage. The aim of this study was to investigate the mechanisms of PDL1 regulation by human macrophages in response to viral infection. Ex-vivo viral infection models using influenza and RSV were established in human lung explants, isolated lung macrophages and monocyte-derived macrophages (MDM) and analysed by flow cytometry and RT-PCR. Incubation of lung explants, lung macrophages and MDM with X31 resulted in mean cellular infection rates of 18%, 18% and 29% respectively. Viral infection significantly increased cell surface expression of PDL1 on explant macrophages, lung macrophages and MDM but not explant epithelial cells. Infected MDM induced IFNγ release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFNβ mRNA and protein release by MDM in response to influenza infection. Knockdown of IFNβ by siRNA, resulted in a 37.5% reduction in IFNβ gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFNβ, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFNγ release in response to virus and that this expression is regulated by autologous IFNβ production.

Limbal explants from cryopreserved cadaver human corneas. Immunofluorescence and light microscopy of epithelial cells growing in culture.

PubMed

Bratanov, M; Neronov, A; Nikolova, E

2009-01-01

The aim of the present study was to determine whether human cadaver corneas, that were subject to cryopreservation, would be a source of migrating epithelial cells in vitro and what kind of morphological features these cells possess. Limbal explant culture was used for expanding the epithelial cells. Non-quantitative light microscopical examinations of the cultures within a period of 28 days were carried out. The phenotype of cultured cells, particularly of the presumed adult stem cell population, was examined by indirect fluorescent immunostaining using antibodies against corneal stem cell associated markers p63 and vimentin. The effectiveness of the freezing-thawing protocol was confirmed by cultivation of limbal explants taken from non-cryopreserved cadaver corneoscleral rims. The result clearly showed that limbal tissue, subjected to cryopreservation and long lasting (up to 12 months) storage in liquid nitrogen, retains the capacity to be source of migrating and proliferating epithelial cells in vitro including the presumed adult stem cells and transient amplifying cells.

Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy

PubMed Central

da Silva, Rafaela J.; Gomes, Angelica O.; Franco, Priscila S.; Pereira, Ariane S.; Milian, Iliana C. B.; Ribeiro, Mayara; Fiorenzani, Paolo; dos Santos, Maria C.; Mineo, José R.; da Silva, Neide M.; Ferro, Eloisa A. V.; de Freitas Barbosa, Bellisa

2017-01-01

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure. PMID:28798905

Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy.

PubMed

da Silva, Rafaela J; Gomes, Angelica O; Franco, Priscila S; Pereira, Ariane S; Milian, Iliana C B; Ribeiro, Mayara; Fiorenzani, Paolo; Dos Santos, Maria C; Mineo, José R; da Silva, Neide M; Ferro, Eloisa A V; de Freitas Barbosa, Bellisa

2017-01-01

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure.

Somatic embryogenesis and massive shoot regeneration from immature embryo explants of tef.

PubMed

Gugsa, Likyelesh; Kumlehn, Jochen

2011-01-01

Tef (Eragrostis tef) provides a major source of human nutrition in the Horn of Africa, but biotechnology has had little impact on its improvement to date. Here, we report the elaboration of an in vitro regeneration protocol, based on the use of immature zygotic embryos as explant. Explant size was an important determinant of in vitro regeneration efficiency, as was the formulation of the culture medium. Optimal results were obtained by culturing 0.2-0.35 mm embryo explants on a medium containing KBP minerals, 9.2-13.8 μM 2,4-dichlorophenoxyacetic acid, 6 mM glutamine, and 0.5% Phytagel. Although this protocol was effective for both the improved cultivar "DZ-01-196" and the landrace "Fesho", the former produced consistently more embryogenic tissue and a higher number of regenerants. An average of more than 2,800 shoots could be obtained from each "DZ-01-196" explant after 12 weeks of in vitro culture. These shoots readily formed roots, and plantlets transferred to soil were able to develop into morphologically normal, fertile plants. This regeneration and multiplication system should allow for the application of a range of biotechnological methods to tef.

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target

PubMed Central

Asim, Mohammad; Massie, Charles E.; Orafidiya, Folake; Pértega-Gomes, Nelma; Warren, Anne Y.; Esmaeili, Mohsen; Selth, Luke A.; Zecchini, Heather I.; Luko, Katarina; Qureshi, Arham; Baridi, Ajoeb; Menon, Suraj; Madhu, Basetti; Escriu, Carlos; Lyons, Scott; Vowler, Sarah L.; Zecchini, Vincent R.; Shaw, Greg; Hessenkemper, Wiebke; Russell, Roslin; Mohammed, Hisham; Stefanos, Niki; Lynch, Andy G.; Grigorenko, Elena; D’Santos, Clive; Taylor, Chris; Lamb, Alastair; Sriranjan, Rouchelle; Yang, Jiali; Stark, Rory; Dehm, Scott M.; Rennie, Paul S.; Carroll, Jason S.; Griffiths, John R.; Tavaré, Simon; Mills, Ian G.; McEwan, Iain J.; Baniahmad, Aria; Tilley, Wayne D.; Neal, David E.

2016-01-01

Background: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. Methods: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ2 tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. Results: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. Conclusions: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa. PMID:26657335

Human Bone Marrow-Derived Mesenchymal Stem Cells Display Enhanced Clonogenicity but Impaired Differentiation With Hypoxic Preconditioning

PubMed Central

Boyette, Lisa B.; Creasey, Olivia A.; Guzik, Lynda; Lozito, Thomas

2014-01-01

Stem cells are promising candidate cells for regenerative applications because they possess high proliferative capacity and the potential to differentiate into other cell types. Mesenchymal stem cells (MSCs) are easily sourced but do not retain their proliferative and multilineage differentiative capabilities after prolonged ex vivo propagation. We investigated the use of hypoxia as a preconditioning agent and in differentiating cultures to enhance MSC function. Culture in 5% ambient O2 consistently enhanced clonogenic potential of primary MSCs from all donors tested. We determined that enhanced clonogenicity was attributable to increased proliferation, increased vascular endothelial growth factor secretion, and increased matrix turnover. Hypoxia did not impact the incidence of cell death. Application of hypoxia to osteogenic cultures resulted in enhanced total mineral deposition, although this effect was detected only in MSCs preconditioned in normoxic conditions. Osteogenesis-associated genes were upregulated in hypoxia, and alkaline phosphatase activity was enhanced. Adipogenic differentiation was inhibited by exposure to hypoxia during differentiation. Chondrogenesis in three-dimensional pellet cultures was inhibited by preconditioning with hypoxia. However, in cultures expanded under normoxia, hypoxia applied during subsequent pellet culture enhanced chondrogenesis. Whereas hypoxic preconditioning appears to be an excellent way to expand a highly clonogenic progenitor pool, our findings suggest that it may blunt the differentiation potential of MSCs, compromising their utility for regenerative tissue engineering. Exposure to hypoxia during differentiation (post-normoxic expansion), however, appears to result in a greater quantity of functional osteoblasts and chondrocytes and ultimately a larger quantity of high-quality differentiated tissue. PMID:24436440

Generation and characterisation of human umbilical cord derived mesenchymal stem cells by explant method.

PubMed

Yusoff, Z; Maqbool, M; George, E; Hassan, R; Ramasamy, R

2016-06-01

Mesenchymal stem cells (MSCs) derived from human umbilical cord (UC) have been considered as an important tool for treating various malignancies, tissue repair and organ regeneration. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are better alternative to MSCs that derived from bone marrow (BM-MSCs) as they are regarded as medical waste with little ethical concern for research and easily culture-expanded. In this present study, the foetal distal end of human UC was utilised to generate MSC by explant method. Upon in vitro culture, adherent cells with fibroblastic morphology were generated with rapid growth kinetics. Under the respective inductive conditions, these cells were capable of differentiating into adipocytes and osteocytes; express an array of standard MSC's surface markers CD29, CD73, CD90, CD106 and MHC-class I. Further assessment of immunosuppression activity revealed that MSCs generated from UC had profoundly inhibited the proliferation of mitogen-activated T lymphocytes in a dosedependent manner. The current laboratory findings have reinforced the application of explant method to generate UCMSCs thus, exploring an ideal platform to fulfil the increasing demand of MSCs for research and potential clinical use.

1,25D3 enhances antitumor activity of gemcitabine and cisplatin in human bladder cancer models

PubMed Central

Ma, Yingyu; Yu, Wei-Dong; Trump, Donald L.; Johnson, Candace S.

2010-01-01

Background 1,25 dihydroxyvitamin D3 (1,25D3) potentiates the cytotoxic effects of several common chemotherapeutic agents. The combination of gemcitabine and cisplatin (GC) is a current standard chemotherapy regimen for bladder cancer. We investigated whether 1,25D3 could enhance the antitumor activity of GC in bladder cancer model systems. Methods Human bladder cancer T24 and UMUC3 cells were pretreated with 1,25D3 followed by GC. Apoptosis were assessed by annexin V staining. Caspase activation was examined by immunoblot analysis and substrate-based caspase activity assay. The cytotoxic effects were examined using MTT and in vitro clonogenic assay. p73 protein levels were assessed by immunoblot analysis. Knockdown of p73 was achieved by siRNA. The in vivo antitumor activity was assessed by in vivo excision clonogenic assay and tumor regrowth delay in the T24 xenograft model. Results 1,25D3 pretreatment enhanced GC-induced apoptosis and the activities of caspases- 8, 9 and 3 in T24 and UMUC3 cells. 1,25D3 synergistically reduced GC-suppressed surviving fraction in T24 cells. 1,25D3, gemcitabine, or cisplatin induced p73 accumulation, which was enhanced by GC or 1,25D3 and GC. p73 expression was lower in human primary bladder tumor tissue compared with adjacent normal tissue. Knockdown of p73 increased clonogenic capacity of T24 cells treated with 1,25D3, GC or 1,25D3 and GC. 1,25D3 and GC combination enhanced tumor regression compared with 1,25D3 or GC alone. Conclusions 1,25D3 potentiates GC-mediated growth inhibition in human bladder cancer models in vitro and in vivo, which involves p73 induction and apoptosis. PMID:20564622

Part 2. Comparison of emergency washing solutions in 70% hydrofluoric acid-burned human skin in an established ex vivo explants model

PubMed Central

Burgher, François; Mathieu, Laurence; Lati, Elian; Gasser, Philippe; Peno-Mazzarino, Laurent; Blomet, Joël; Hall, Alan H; Maibach, Howard I

2011-01-01

Background: Hydrofluoric acid (HF) is a small and partially dissociated acid (pKa 3.2), able to deeply penetrate into human skin in addition to the corrosiveness of the hydrogen ion (H+) and the toxicity of the fluoride ion (F-). However, there has been a lack of experimental studies to objectively characterize the results of human HF skin exposure decontamination. Methodology/principal findings: A previously established experimental method using a human skin explants ex vivo model (Part 1. Experimental 70% hydrofluoric acid (HF) burns: Histological observations in an established human skin explants ex vivo model) described the lesions that appeared following 70% HF penetration. Within 5min, 70% HF penetrates to the dermis. Using the same experimental conditions, a comparison study of two different washing protocols was performed: water + topical calcium gluconate (CaG) versus Hexafluorine®. In these conditions, washing for 15min with running tap water followed by topical CaG ointment only delayed burn onset, while severe tissue damage appeared later. In contrast, after washing with Hexafluorine® over 10 min, no histological lesions developed. These results are in accordance with the results of accidental human industrial case reports. Conclusion/significance: Amphoteric and hypertonic Hexafluorine® can deactivate H+ and chelate F- ions. Based on these results, it should be considered as a promising first-aid decontamination solution to prevent or minimize significant local and systemic consequences of concentrated HF skin exposures. PMID:21083510

Somatic Embryogenesis and Massive Shoot Regeneration from Immature Embryo Explants of Tef

PubMed Central

Gugsa, Likyelesh; Kumlehn, Jochen

2011-01-01

Tef (Eragrostis tef) provides a major source of human nutrition in the Horn of Africa, but biotechnology has had little impact on its improvement to date. Here, we report the elaboration of an in vitro regeneration protocol, based on the use of immature zygotic embryos as explant. Explant size was an important determinant of in vitro regeneration efficiency, as was the formulation of the culture medium. Optimal results were obtained by culturing 0.2–0.35 mm embryo explants on a medium containing KBP minerals, 9.2–13.8 μM 2,4-dichlorophenoxyacetic acid, 6 mM glutamine, and 0.5% Phytagel. Although this protocol was effective for both the improved cultivar “DZ-01-196” and the landrace “Fesho”, the former produced consistently more embryogenic tissue and a higher number of regenerants. An average of more than 2,800 shoots could be obtained from each “DZ-01-196” explant after 12 weeks of in vitro culture. These shoots readily formed roots, and plantlets transferred to soil were able to develop into morphologically normal, fertile plants. This regeneration and multiplication system should allow for the application of a range of biotechnological methods to tef. PMID:22028975

Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures.

PubMed

Meller, D; Pires, R T F; Tseng, S C G

2002-04-01

Amniotic membrane (AM) transplantation effectively expands the remaining limbal epithelial stem cells in patients with partial limbal stem cell deficiency. The authors investigated whether this action could be produced ex vivo. The outgrowth rate on AM was compared among explants derived from human limbus, peripheral cornea, and central cornea. For outgrowth of human limbal epithelial cells (HLEC), cell cycle kinetics were measured by BrdU labelling for 1 or 7 days, of which the latter was also chased in primary cultures, secondary 3T3 fibroblast cultures, and in athymic Balb/c mice following a brief treatment with a phorbol ester. Epithelial morphology was studied by histology and transmission electron microscopy, and phenotype was defined by immunostaining with monoclonal antibodies to keratins and mucins. Outgrowth rate was 0/22 (0%) and 2/24 (8.3%) for central and peripheral corneal explants, respectively, but was 77/80 (96.2%) for limbal explants (p <0.0001). 24 hour BrdU labelling showed a uniformly low (that is, less than 5%) labelling index in 65% of the limbal explants, but a mixed pattern with areas showing a high (that is, more than 40%) labelling index in 35% of limbal explants, and in all (100%) peripheral corneal explants. Continuous BrdU labelling for 7 days detected a high labelling index in 61.5% of the limbal explants with the remainder still retaining a low labelling index. A number of label retaining cells were noted after 7 day labelling followed by 14 days of chase in primary culture or by 21 days of chase after transplantation to 3T3 fibroblast feeder layers. After exposure to phorbol 12-myristate 13-acetate for 24 hours and 7 day labelling, HLEC transplanted in athymic mice still showed a number of label retaining basal cells after 9 days of chase. HLEC cultured on AM were strongly positive for K14 keratin and MUC4 and slightly positive in suprabasal cells for K3 keratin but negative for K12 keratin, AMEM2, and MUC5AC. After subcutaneous implantation in athymic mice, the resultant epithelium was markedly stratified and the basal epithelial cells were strongly positive for K14 keratin, while the suprabasal epithelial cells were strongly positive for K3 keratin and MUC4, and the entire epithelium was negative for K12 keratin and MUC5A/C. These data support the notion that AM cultures preferentially preserve and expand limbal epithelial stem cells that retain their in vivo properties of slow cycling, label retaining, and undifferentiation. This finding supports the feasibility of ex vivo expansion of limbal epithelial stem cells for treating patients with total limbal stem cell deficiency using a small amount of donor limbal tissue.

Chondroprotective effects of a proanthocyanidin rich Amazonian genonutrient reflects direct inhibition of matrix metalloproteinases and upregulation of IGF-1 production by human chondrocytes

PubMed Central

Miller, Mark JS; Bobrowski, Paul; Shukla, Meenakshi; Gupta, Kalpana; Haqqi, Tariq M

2007-01-01

Background The Amazonian medicinal plant Sangre de grado (Croton palanostigma) has traditional applications for the treatment of wound healing and inflammation. We sought to characterize two extracts (progrado and zangrado) in terms of safety and oligomeric proanthocyanidin chain length. Additionally progrado was evaluated for antioxidant activity and possible chondroprotective actions. Methods Acute oral safety and toxicity was tested in rats according under OECD protocol number 420. The profile of proanthocyanidin oligomers was determined by HPLC and progrado's antioxidant activity quantified by the ORAC, NORAC and HORAC assays. Human cartilage explants, obtained from surgical specimens, were used to assess chondroproteciton with activity related to direct inhibitory effects on human matrix metalloproteinase (MMP, gelatinolytic) activity using synovial fluid and chondrocytes activated with IL-1β (10 ng/ml). Additionally, progrado (2–10 μg/ml) was tested for its ability to maintain optimal IGF-1 transcription and translation in cartilage explants and cultured chondrocytes. Results Both progrado and zangrado at doses up to 2000 mg/kg (po) displayed no evidence of toxicity. Oligomeric proanthocyanidin content was high for both progrado (158 mg/kg) and zangrado (124 mg/kg), with zangrado almost entirely composed of short oligomers (<6 mer), whereas the majority of oligomers in progrado exceeded 10 mers. Progrado was a remarkably potent antioxidant in the standardized tests ORAC, NORAC and HORAC. Progrado was exceptionally effective in reducing both basal and IL-1β induced glycosaminoglycan release from human cartilage explants at concentrations that also directly blocked the gelatinolytic activity of MMP-2 and MMP-9. Progrado prevented IL-1β induced suppression of IGF-1 production from human cartilage explants as well as stimulating basal IGF-1 production (P < 0.05). Comparable changes in IGF-1 gene expression were noted in cultured human chondrocytes. Conclusion Progrado has a promising safety profile, significant chondroprotective and antioxidant actions, directly inhibits MMP activity and promotes the production of the cartilage repair factor, IGF-1. This suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation. PMID:17697350

Novel histone deacetylase inhibitor CG200745 induces clonogenic cell death by modulating acetylation of p53 in cancer cells.

PubMed

Oh, Eun-Taex; Park, Moon-Taek; Choi, Bo-Hwa; Ro, Seonggu; Choi, Eun-Kyung; Jeong, Seong-Yun; Park, Heon Joo

2012-04-01

Histone deacetylase (HDAC) plays an important role in cancer onset and progression. Therefore, inhibition of HDAC offers potential as an effective cancer treatment regimen. CG200745, (E)-N(1)-(3-(dimethylamino)propyl)-N(8)-hydroxy-2-((naphthalene-1-loxy)methyl)oct-2-enediamide, is a novel HDAC inhibitor presently undergoing a phase I clinical trial. Enhancement of p53 acetylation by HDAC inhibitors induces cell cycle arrest, differentiation, and apoptosis in cancer cells. The purpose of the present study was to investigate the role of p53 acetylation in the cancer cell death caused by CG200745. CG200745-induced clonogenic cell death was 2-fold greater in RKO cells expressing wild-type p53 than in p53-deficient RC10.1 cells. CG200745 treatment was also cytotoxic to PC-3 human prostate cancer cells, which express wild-type p53. CG200745 increased acetylation of p53 lysine residues K320, K373, and K382. CG200745 induced the accumulation of p53, promoted p53-dependent transactivation, and enhanced the expression of MDM2 and p21(Waf1/Cip1) proteins, which are encoded by p53 target genes. An examination of CG200745 effects on p53 acetylation using cells transfected with various p53 mutants showed that cells expressing p53 K382R mutants were significantly resistant to CG200745-induced clonogenic cell death compared with wild-type p53 cells. Moreover, p53 transactivation in response to CG200745 was suppressed in all cells carrying mutant forms of p53, especially K382R. Taken together, these results suggest that acetylation of p53 at K382 plays an important role in CG200745-induced p53 transactivation and clonogenic cell death.

Surgical Marking Pen Dye Inhibits Saphenous Vein Cell Proliferation and Migration in Saphenous Vein Graft Tissue

PubMed Central

Kikuchi, Shinsuke; Kenagy, Richard D; Gao, Lu; Wight, Thomas N; Azuma, Nobuyoshi; Sobel, Michael; Clowes, Alexander W

2014-01-01

Objective Markers containing dyes such as crystal violet (CAS 548-62-9) are routinely used on the adventitia of vein bypass grafts to avoid twisting during placement. Since little is known about how these dyes affect vein graft healing and function, we determined the effect of crystal violet on cell migration and proliferation, which are responses to injury after grafting. Methods Fresh human saphenous veins were obtained as residual specimens from leg bypass surgeries. Portions of the vein that had been surgically marked with crystal violet were analyzed separately from those that had no dye marking. In the laboratory, they were split into easily dissected inner and outer layers after removal of endothelium. This f cleavage plane was within the circular muscle layer of the media. Cell migration from explants was measured daily as either 1) % migration positive explants, which exclusively measures migration, or 2) the number of cells on the plastic surrounding each explant, which measures migration plus proliferation. Cell proliferation and apoptosis (Ki67 and TUNEL staining, respectively) were determined in dye-marked and unmarked areas of cultured vein rings. The dose-dependent effects of crystal violet were measured for cell migration from explants as well as proliferation, migration, and death of cultured outer layer cells. Dye was extracted from explants with ethanol and quantified by spectrophotometry. Results There was significantly less cell migration from visibly blue, compared to unstained, outer layer explants by both methods. There was no significant difference in migration from inner layer explants adjacent to blue-stained or unstained sections of vein, because dye did not penetrate to the inner layer. Ki67 staining of vein in organ culture, which is a measure of proliferation, progressively increased up to 6 days in non-blue outer layer and was abolished in the blue outer layer. Evidence of apoptosis (TUNEL staining) was present throughout the wall and not different in blue-stained and unstained vein wall segments. Blue outer layer explants had 65.9±8.0 ng dye/explant compared to 2.1±1.3 for non-blue outer layer explants. Dye applied in vitro to either outer or inner layer explants dose-dependently inhibited migration (IC50=8.5 ng/explant). The IC50s of crystal violet for outer layer cell proliferation and migration were 0.1 and 1.2 μg/ml, while the EC50 for death was between 1 and 10 μg/ml. Conclusion Crystal violet inhibits venous cell migration and proliferation indicating that alternative methods should be considered for marking vein grafts. PMID:25935273

Effects of Monobutyl and Di(n-butyl) Phthalate in Vitro on Steroidogenesis and Leydig Cell Aggregation in Fetal Testis Explants from the Rat: Comparison with Effects in Vivo in the Fetal Rat and Neonatal Marmoset and in Vitro in the Human

PubMed Central

Hallmark, Nina; Walker, Marion; McKinnell, Chris; Mahood, I. Kim; Scott, Hayley; Bayne, Rosemary; Coutts, Shiona; Anderson, Richard A.; Greig, Irene; Morris, Keith; Sharpe, Richard M.

2007-01-01

Background Certain phthalates can impair Leydig cell distribution and steroidogenesis in the fetal rat in utero, but it is unknown whether similar effects might occur in the human. Objectives Our aim in this study was to investigate the effects of di(n-butyl) phthalate (DBP), or its metabolite monobutyl phthalate (MBP), on testosterone production and Leydig cell aggregation (LCA) in fetal testis explants from the rat and human, and to compare the results with in vivo findings for DBP-exposed rats. We also wanted to determine if DBP/MBP affects testosterone production in vivo in the neonatal male marmoset. Methods Fetal testis explants obtained from the rat [gestation day (GD)19.5] and from the human (15–19 weeks of gestation) were cultured for 24–48 hr with or without human chorionic gonadotropin (hCG) or 22R-hydroxycholesterol (22R-OH), and with or without DBP/MBP. Pregnant rats and neonatal male marmosets were dosed with 500 mg/kg/day DBP or MBP. Results Exposure of rats in utero to DBP (500 mg/kg/day) for 48 hr before GD21.5 induced major suppression of intratesticular testosterone levels and cytochrome P450 side chain cleavage enzyme (P450scc) expression; this short-term treatment induced LCA, but was less marked than longer term (GD13.5–20.5) DBP treatment. In vitro, MBP (10−3 M) did not affect basal or 22R-OH-stimulated testosterone production by fetal rat testis explants but slightly attenuated hCG-stimulated steroidogenesis; MBP induced minor LCA in vitro. None of these parameters were affected in human fetal testis explants cultured with 10−3 M MBP for up to 48 hr. Because the in vivo effects of DBP/MBP were not reproduced in vitro in the rat, the absence of MBP effects in vitro on fetal human testes is inconclusive. In newborn (Day 2–7) marmosets, administration of a single dose of 500 mg/kg MBP significantly (p = 0.019) suppressed blood testosterone levels 5 hr later. Similar treatment of newborn co-twin male marmosets for 14 days resulted in increased Leydig cell volume per testis (p = 0.011), compared with co-twin controls; this is consistent with MBP-induced inhibition of steroidogenesis followed by compensatory Leydig cell hyperplasia/hypertrophy. Conclusions These findings suggest that MBP/DBP suppresses steroidogenesis by fetal-type Leydig cells in primates as in rodents, but this cannot be studied in vitro. PMID:17431488

Glucocorticoids Affect 24 h Clock Genes Expression in Human Adipose Tissue Explant Cultures

PubMed Central

Gómez-Abellán, Purificación; Díez-Noguera, Antoni; Madrid, Juan A.; Luján, Juan A.; Ordovás, José M.; Garaulet, Marta

2012-01-01

Aims to examine firstly whether CLOCK exhibits a circadian expression in human visceral (V) and subcutaneous (S) adipose tissue (AT) in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX) on positive and negative clock genes expression. Subjects and Methods VAT and SAT biopsies were obtained from morbid obese women (body mass index≥40 kg/m2) (n = 6). In order to investigate rhythmic expression pattern of clock genes and the effect of DEX on CLOCK, PER2 and BMAL1 expression, control AT (without DEX) and AT explants treated with DEX (2 hours) were cultured during 24 h and gene expression was analyzed at the following times: 10:00 h, 14:00 h, 18:00 h, 22:00 h, 02:00 h and 06:00 h, using qRT-PCR. Results CLOCK, BMAL1 and PER2 expression exhibited circadian patterns in both VAT and SAT explants that were adjusted to a typical 24 h sinusoidal curve. PER2 expression (negative element) was in antiphase with respect to CLOCK and in phase with BMAL1 expression (both positive elements) in the SAT (situation not present in VAT). A marked effect of DEX exposure on both positive and negative clock genes expression patterns was observed. Indeed, DEX treatment modified the rhythmicity pattern towards altered patterns with a period lower than 24 hours in all genes and in both tissues. Conclusions 24 h patterns in CLOCK and BMAL1 (positive clock elements) and PER2 (negative element) mRNA levels were observed in human adipose explants. These patterns were altered by dexamethasone exposure. PMID:23251369

Elucidation of the EP defect in Diamond-Blackfan anemia by characterization and prospective isolation of human EPs.

PubMed

Iskander, Deena; Psaila, Bethan; Gerrard, Gareth; Chaidos, Aristeidis; En Foong, Hui; Harrington, Yvonne; Karnik, Leena C; Roberts, Irene; de la Fuente, Josu; Karadimitris, Anastasios

2015-04-16

Diamond-Blackfan anemia (DBA) is a disorder characterized by a selective defect in erythropoiesis. Delineation of the precise defect is hampered by a lack of markers that define cells giving rise to erythroid burst- and erythroid colony-forming unit (BFU-E and CFU-E) colonies, the clonogenic assays that quantify early and late erythroid progenitor (EEP and LEP) potential, respectively. By combining flow cytometry, cell-sorting, and single-cell clonogenic assays, we identified Lin(-)CD34(+)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(-)CD36(-) bone marrow cells as EEP giving rise to BFU-E, and Lin(-)CD34(+/-)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(+)CD36(+) cells as LEP giving rise to CFU-E, in a hierarchical fashion. We then applied these definitions to DBA and identified that, compared with controls, frequency, and clonogenicity of DBA, EEP and LEP are significantly decreased in transfusion-dependent but restored in corticosteroid-responsive patients. Thus, both quantitative and qualitative defects in erythroid progenitor (EP) contribute to defective erythropoiesis in DBA. Prospective isolation of defined EPs will facilitate more incisive study of normal and aberrant erythropoiesis. © 2015 by The American Society of Hematology.

Inhibition of Human Immunodeficiency Virus Type 1 Infection by the Candidate Microbicide Dapivirine, a Nonnucleoside Reverse Transcriptase Inhibitor▿

PubMed Central

Fletcher, P.; Harman, S.; Azijn, H.; Armanasco, N.; Manlow, P.; Perumal, D.; de Bethune, M.-P.; Nuttall, J.; Romano, J.; Shattock, R.

2009-01-01

Heterosexual transmission of human immunodeficiency virus (HIV) remains the major route of infection worldwide; thus, there is an urgent need for additional prevention strategies, particularly strategies that could be controlled by women, such as topical microbicides. Potential microbicide candidates must be both safe and effective. Using cellular and tissue explant models, we have evaluated the activity of the nonnucleoside reverse transcriptase inhibitor (NNRTI) dapivirine as a vaginal microbicide. In tissue compatibility studies, dapivirine was well tolerated by epithelial cells, T cells, macrophages, and cervical tissue explants. Dapivirine demonstrated potent dose-dependent inhibitory effects against a broad panel of HIV type 1 isolates from different clades. Furthermore, dapivirine demonstrated potent activity against a wide range of NNRTI-resistant isolates. In human cervical explant cultures, dapivirine was able not only to inhibit direct infection of mucosal tissue but also to prevent the dissemination of the virus by migratory cells. Activity was retained in the presence of semen or a cervical mucus simulant. Furthermore, dapivirine demonstrated prolonged inhibitory effects: it was able to prevent both localized and disseminated infection for as long as 6 days posttreatment. The prolonged protection observed following pretreatment of genital tissue and the lack of observable toxicity suggest that dapivirine has considerable promise as a potential microbicide candidate. PMID:19029331

Inhibition of human immunodeficiency virus type 1 infection by the candidate microbicide dapivirine, a nonnucleoside reverse transcriptase inhibitor.

PubMed

Fletcher, P; Harman, S; Azijn, H; Armanasco, N; Manlow, P; Perumal, D; de Bethune, M-P; Nuttall, J; Romano, J; Shattock, R

2009-02-01

Heterosexual transmission of human immunodeficiency virus (HIV) remains the major route of infection worldwide; thus, there is an urgent need for additional prevention strategies, particularly strategies that could be controlled by women, such as topical microbicides. Potential microbicide candidates must be both safe and effective. Using cellular and tissue explant models, we have evaluated the activity of the nonnucleoside reverse transcriptase inhibitor (NNRTI) dapivirine as a vaginal microbicide. In tissue compatibility studies, dapivirine was well tolerated by epithelial cells, T cells, macrophages, and cervical tissue explants. Dapivirine demonstrated potent dose-dependent inhibitory effects against a broad panel of HIV type 1 isolates from different clades. Furthermore, dapivirine demonstrated potent activity against a wide range of NNRTI-resistant isolates. In human cervical explant cultures, dapivirine was able not only to inhibit direct infection of mucosal tissue but also to prevent the dissemination of the virus by migratory cells. Activity was retained in the presence of semen or a cervical mucus simulant. Furthermore, dapivirine demonstrated prolonged inhibitory effects: it was able to prevent both localized and disseminated infection for as long as 6 days posttreatment. The prolonged protection observed following pretreatment of genital tissue and the lack of observable toxicity suggest that dapivirine has considerable promise as a potential microbicide candidate.

Clonogenicity of human leukemic cells protected from cell-lethal agents by heat shock protein 70

PubMed Central

Bases, Robert

2005-01-01

Pretreatment of human leukemia THP-1 cells with heat shock protein Hsp70 (Hsp70) protected them from the cell-lethal effects of the topoisomerase II inhibitor, lucanthone and from ionizing radiation. Cell viability was scored in clonogenic assays of single cells grown in liquid medium containing 0.5% methyl cellulose. Colonies were observed and rapidly scored after staining with the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide. The frequency of abasic sites in the deoxyribonucleic acid (DNA) of THP-1 cells was reduced when these cells were treated with Hsp70. Hsp70 is presumed to have protected the cells by promoting repair of cell DNA, in agreement with previous studies that showed that Hsp70 enhanced base excision repair by purified enzymes. The shoulders of radiation dose-response curves were enhanced by pretreatment of cells with Hsp70 and, importantly, were reduced when cells were transfected with ribonucleic acid designed to silence Hsp70. Hsp70 influenced repair of sublethal damage after radiation. PMID:15832946

Ca(2+)-mediated prostaglandin E2 induction reduces haematoporphyrin-derivative-induced cytotoxicity of T24 human bladder transitional carcinoma cells in vitro.

PubMed Central

Penning, L C; Keirse, M J; VanSteveninck, J; Dubbelman, T M

1993-01-01

The effects of haematoporphyrin-derivative-mediated photodynamic treatment on arachidonic acid metabolism and its relation to clonogenicity have been studied in human bladder-tumour cells. Photodynamic treatment resulted in a transient release of arachidonic acid-derived compounds; prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) especially were strongly increased. This release was reduced by chelation of intracellular Ca2+ with Quin-2 or by lowering the extracellular Ca2+ concentration in the medium with EGTA, presumably resulting in inhibition of phospholipase A2. A similar reduction was obtained when indomethacin, an inhibitor of the cyclo-oxygenase pathway, was added prior to light exposure. These three treatments enhanced the photosensitivity, as revealed by the clonogenicity assay. Incubation with PGE2 prior to light exposure, but not with TXB2, protected against reproductive-cell death. The results of these experiments suggest that Ca(2+)-mediated activation of cyclo-oxygenase, resulting in increased levels of PGE2, participates in a cellular-defence mechanism against photodynamic cell killing. PMID:8503851

The effects of monosodium urate monohydrate crystals on chondrocyte viability and function: implications for development of cartilage damage in gout.

PubMed

Chhana, Ashika; Callon, Karen E; Pool, Bregina; Naot, Dorit; Gamble, Gregory D; Dray, Michael; Pitto, Rocco; Bentley, Jarome; McQueen, Fiona M; Cornish, Jillian; Dalbeth, Nicola

2013-12-01

Cartilage damage is frequently observed in advanced destructive gout. The aim of our study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on chondrocyte viability and function. The alamarBlue assay and flow cytometry were used to assess the viability of primary human chondrocytes and cartilage explants following culture with MSU crystals. The number of dead chondrocytes in cartilage explants cultured with MSU crystals was quantified. Real-time PCR was used to determine changes in the relative mRNA expression levels of chondrocytic genes. The histological appearance of cartilage in joints affected by gout was also examined. MSU crystals rapidly reduced primary human chondrocyte and cartilage explant viability in a dose-dependent manner (p < 0.01 for both). Cartilage explants cultured with MSU crystals had a greater percentage of dead chondrocytes at the articular surface compared to untreated cartilage (p = 0.004). Relative mRNA expression of type II collagen and the cartilage matrix proteins aggrecan and versican was decreased in chondrocytes following culture with MSU crystals (p < 0.05 for all). However, expression of the degradative enzymes ADAMTS4 and ADAMTS5 was increased (p < 0.05 for both). In joints affected by gout, normal cartilage architecture was lost, with empty chondrocyte lacunae observed. MSU crystals have profound inhibitory effects on chondrocyte viability and function. Interactions between MSU crystals and chondrocytes may contribute to cartilage damage in gout through reduction of chondrocyte viability and promotion of a catabolic state.

Microbial biotransformation of DON: molecular basis for reduced toxicity

PubMed Central

Pierron, Alix; Mimoun, Sabria; Murate, Leticia S.; Loiseau, Nicolas; Lippi, Yannick; Bracarense, Ana-Paula F. L.; Schatzmayr, Gerd; He, Jian Wei; Zhou, Ting; Moll, Wulf-Dieter; Oswald, Isabelle P.

2016-01-01

Bacteria are able to de-epoxidize or epimerize deoxynivalenol (DON), a mycotoxin, to deepoxy-deoxynivalenol (deepoxy-DON or DOM-1) or 3-epi-deoxynivalenol (3-epi-DON), respectively. Using different approaches, the intestinal toxicity of 3 molecules was compared and the molecular basis for the reduced toxicity investigated. In human intestinal epithelial cells, deepoxy-DON and 3-epi-DON were not cytotoxic, did not change the oxygen consumption or impair the barrier function. In intestinal explants, exposure for 4 hours to 10 μM DON induced intestinal lesions not seen in explants treated with deepoxy-DON and 3-epi-DON. A pan-genomic transcriptomic analysis was performed on intestinal explants. 747 probes, representing 323 genes, were differentially expressed, between DON-treated and control explants. By contrast, no differentially expressed genes were observed between control, deepoxy-DON and 3-epi-DON treated explants. Both DON and its biotransformation products were able to fit into the pockets of the A-site of the ribosome peptidyl transferase center. DON forms three hydrogen bonds with the A site and activates MAPKinases (mitogen-activated protein kinases). By contrast deepoxy-DON and 3-epi-DON only form two hydrogen bonds and do not activate MAPKinases. Our data demonstrate that bacterial de-epoxidation or epimerization of DON altered their interaction with the ribosome, leading to an absence of MAPKinase activation and a reduced toxicity. PMID:27381510

Microbial biotransformation of DON: molecular basis for reduced toxicity

NASA Astrophysics Data System (ADS)

Pierron, Alix; Mimoun, Sabria; Murate, Leticia S.; Loiseau, Nicolas; Lippi, Yannick; Bracarense, Ana-Paula F. L.; Schatzmayr, Gerd; He, Jian Wei; Zhou, Ting; Moll, Wulf-Dieter; Oswald, Isabelle P.

2016-07-01

Bacteria are able to de-epoxidize or epimerize deoxynivalenol (DON), a mycotoxin, to deepoxy-deoxynivalenol (deepoxy-DON or DOM-1) or 3-epi-deoxynivalenol (3-epi-DON), respectively. Using different approaches, the intestinal toxicity of 3 molecules was compared and the molecular basis for the reduced toxicity investigated. In human intestinal epithelial cells, deepoxy-DON and 3-epi-DON were not cytotoxic, did not change the oxygen consumption or impair the barrier function. In intestinal explants, exposure for 4 hours to 10 μM DON induced intestinal lesions not seen in explants treated with deepoxy-DON and 3-epi-DON. A pan-genomic transcriptomic analysis was performed on intestinal explants. 747 probes, representing 323 genes, were differentially expressed, between DON-treated and control explants. By contrast, no differentially expressed genes were observed between control, deepoxy-DON and 3-epi-DON treated explants. Both DON and its biotransformation products were able to fit into the pockets of the A-site of the ribosome peptidyl transferase center. DON forms three hydrogen bonds with the A site and activates MAPKinases (mitogen-activated protein kinases). By contrast deepoxy-DON and 3-epi-DON only form two hydrogen bonds and do not activate MAPKinases. Our data demonstrate that bacterial de-epoxidation or epimerization of DON altered their interaction with the ribosome, leading to an absence of MAPKinase activation and a reduced toxicity.

Three-dimensional Invasion of Human Glioblastoma Cells Remains Unchanged by X-ray and Carbon Ion Irradiation In Vitro

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

Eke, Iris; Storch, Katja; Kaestner, Ina

Purpose: Cell invasion represents one of the major determinants that treatment has failed for patients suffering from glioblastoma. Contrary findings have been reported for cell migration upon exposure to ionizing radiation. Here, the migration and invasion capability of glioblastoma cells on and in collagen type I were evaluated upon irradiation with X-rays or carbon ions. Methods and Materials: Migration on and invasion in collagen type I were evaluated in four established human glioblastoma cell lines exposed to either X-rays or carbon ions. Furthermore, clonogenic radiation survival, proliferation (5-bromo-2-deoxyuridine positivity), DNA double-strand breaks ({gamma}H2AX/53BP1-positive foci), and expression of invasion-relevant proteins (eg,more » {beta}1 integrin, FAK, MMP2, and MMP9) were explored. Migration and invasion assays for primary glioblastoma cells also were carried out with X-ray irradiation. Results: Neither X-ray nor carbon ion irradiation affected glioblastoma cell migration and invasion, a finding similarly observed in primary glioblastoma cells. Intriguingly, irradiated cells migrated unhampered, despite DNA double-strand breaks and reduced proliferation. Clonogenic radiation survival was increased when cells had contact with extracellular matrix. Specific inhibition of the {beta}1 integrin or proliferation-associated signaling molecules revealed a critical function of JNK, PI3K, and p38 MAPK in glioblastoma cell invasion. Conclusions: These findings indicate that X-rays and carbon ion irradiation effectively reduce proliferation and clonogenic survival without modifying the migration and invasion ability of glioblastoma cells in a collagen type I environment. Addition of targeted agents against members of the MAPK and PI3K signaling axis to conventional chemoradiation therapy seems potentially useful to optimize glioblastoma therapy.« less

Comparison of interleukin 10 homologs on dermal wound healing using a novel human skin ex vivo organ culture model

PubMed Central

Balaji, Swathi; Moles, Chad M.; Bhattacharya, Sukanta S.; LeSaint, Maria; Dhamija, Yashu; Le, Louis D.; King, Alice; Kidd, Mykia; Bouso, Muhammad F.; Shaaban, Aimen; Crombleholme, Timothy M.; Bollyky, Paul; Keswani, Sundeep G.

2015-01-01

Background Anti-inflammatory cytokine interleukin (IL)-10 has been shown to induce regenerative healing in postnatal wounds. A viral homolog of IL-10 produced by human cytomegalovirus (CMV IL-10) similarly generates potent immunoregulatory effects, but its effects on wound healing have not been investigated. Currently, there are limited cost-effective methods of screening vulnerary therapeutics. Taken together, we aim to develop and validate a novel human ex vivo dermal wound model and hypothesize that CMV IL-10 will enhance dermal wound healing. Methods Full-thickness circular (6-mm) explants were taken from surgical skin samples and 3-mm full-thickness wounds were created. Explants were embedded in collagen I matrix and maintained in specially formulated media with the epidermis at air–liquid interface, and treated with human IL-10 or CMV IL-10 (200 ng/mL). The viability of cultured explants was validated by histology and lactate dehydrogenase (LDH) activity. Epithelial gap, epithelial height, basal keratinocyte migration, vascular endothelial growth factor levels, and neovascularization were measured at days 3 and 7 to determine IL-10 effects on wound healing. Results Culture explants at day 7 appeared similar to fresh skin in morphology, cell, and vessel density. By day 14, the epidermis separated from the dermis and the cell density diminished. Day 7 wounds appeared viable with advancing epithelial and basal keratinocyte migration with no evidence of necrosis. Cytotoxicity analysis via the quantification of LDH revealed no differences between controls and treated groups. There was a slight increase in the quantity of LDH in media at day 3; however, this decreased at day 5 and continued to decline up to day 21. CMV IL-10 treatment resulted in a significant decrease in the epithelial gap and an increase in epithelial height. There were no differences in the rates of basal keratinocyte migration at day 7 between treated and control groups. Interestingly, human IL-10 increased vascular endothelial growth factor expression and neovascularization compared with controls. Conclusions The human ex vivo wound model provides a simple and viable design to study dermal wound healing. Both IL-10 homologs demonstrate vulnerary effects. The viral homolog demonstrates enhanced effects on wound closure compared with human IL-10. These data represent a novel tool that can be used to screen therapeutics, such as CMV IL-10, before preclinical studies. PMID:24814764

Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.

PubMed Central

Sanderson, I R; Ezzell, R M; Kedinger, M; Erlanger, M; Xu, Z X; Pringault, E; Leon-Robine, S; Louvard, D; Walker, W A

1996-01-01

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8755542

Ionizing Radiation-Induced Responses in Human Cells with Differing TP53 Status

PubMed Central

Mirzayans, Razmik; Andrais, Bonnie; Scott, April; Wang, Ying W.; Murray, David

2013-01-01

Ionizing radiation triggers diverse responses in human cells encompassing apoptosis, necrosis, stress-induced premature senescence (SIPS), autophagy, and endopolyploidy (e.g., multinucleation). Most of these responses result in loss of colony-forming ability in the clonogenic survival assay. However, not all modes of so-called clonogenic cell “death” are necessarily advantageous for therapeutic outcome in cancer radiotherapy. For example, the crosstalk between SIPS and autophagy is considered to influence the capacity of the tumor cells to maintain a prolonged state of growth inhibition that unfortunately can be succeeded by tumor regrowth and disease recurrence. Likewise, endopolyploid giant cells are able to segregate into near diploid descendants that continue mitotic activities. Herein we review the current knowledge on the roles that the p53 and p21WAF1 tumor suppressors play in determining the fate of human fibroblasts (normal and Li-Fraumeni syndrome) and solid tumor-derived cells after exposure to ionizing radiation. In addition, we discuss the important role of WIP1, a p53-regulated oncogene, in the temporal regulation of the DNA damage response and its contribution to p53 dynamics post-irradiation. This article highlights the complexity of the DNA damage response and provides an impetus for rethinking the nature of cancer cell resistance to therapeutic agents. PMID:24232458

The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals

PubMed Central

Shanmuganatham, Karthik K; Jones, Jeremy C; Marathe, Bindumadhav M; Feeroz, Mohammed M; Jones-Engel, Lisa; Walker, David; Turner, Jasmine; Rabiul Alam, S M; Kamrul Hasan, M; Akhtar, Sharmin; Seiler, Patrick; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

2016-01-01

H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans. PMID:27094903

The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals.

PubMed

Shanmuganatham, Karthik K; Jones, Jeremy C; Marathe, Bindumadhav M; Feeroz, Mohammed M; Jones-Engel, Lisa; Walker, David; Turner, Jasmine; Rabiul Alam, S M; Kamrul Hasan, M; Akhtar, Sharmin; Seiler, Patrick; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

2016-04-20

H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans.

[A method for the primary culture of fibroblasts isolated from human airway granulation tissues].

PubMed

Chen, Nan; Zhang, Jie; Xu, Min; Wang, Yu-ling; Pei, Ying-hua

2013-04-01

To establish a feasible method to culture primary fibroblasts isolated from human airway granulation tissues, and therefore to provide experimental data for the investigation of the pathogenesis of benign airway stenosis. The granulation tissues were collected from 6 patients during routine bronchoscopy at our department of Beijing Tiantan Hospital from April to June 2011. Primary fibroblasts were obtained by culturing the explanted tissues. Cell growth was observed under inverted microscope. All of these 6 primary cultures were successful. Fibroblast-like cells were observed to migrate from the tissue pieces 3 d after inoculation. After 9-11 d of culture, cells reached to 90% confluence and could be sub-cultured. After passage, the cells were still in a typical elongated spindle-shape and grew well. The cells could be sub-cultured further when they formed a monolayer. Explant culture is a reliable method for culturing primary fibroblasts from human airway granulation tissues.

A MIV-150/zinc acetate gel inhibits SHIV-RT infection in macaque vaginal explants.

PubMed

Barnable, Patrick; Calenda, Giulia; Ouattara, Louise; Gettie, Agegnehu; Blanchard, James; Jean-Pierre, Ninochka; Kizima, Larisa; Rodríguez, Aixa; Abraham, Ciby; Menon, Radhika; Seidor, Samantha; Cooney, Michael L; Roberts, Kevin D; Sperling, Rhoda; Piatak, Michael; Lifson, Jeffrey D; Fernandez-Romero, Jose A; Zydowsky, Thomas M; Robbiani, Melissa; Teleshova, Natalia

2014-01-01

To extend our observations that single or repeated application of a gel containing the NNRTI MIV-150 (M) and zinc acetate dihydrate (ZA) in carrageenan (CG) (MZC) inhibits vaginal transmission of simian/human immunodeficiency virus (SHIV)-RT in macaques, we evaluated safety and anti-SHIV-RT activity of MZC and related gel formulations ex vivo in macaque mucosal explants. In addition, safety was further evaluated in human ectocervical explants. The gels did not induce mucosal toxicity. A single ex vivo exposure to diluted MZC (1∶30, 1∶100) and MC (1∶30, the only dilution tested), but not to ZC gel, up to 4 days prior to viral challenge, significantly inhibited SHIV-RT infection in macaque vaginal mucosa. MZC's activity was not affected by seminal plasma. The antiviral activity of unformulated MIV-150 was not enhanced in the presence of ZA, suggesting that the antiviral activity of MZC was mediated predominantly by MIV-150. In vivo administration of MZC and CG significantly inhibited ex vivo SHIV-RT infection (51-62% inhibition relative to baselines) of vaginal (but not cervical) mucosa collected 24 h post last gel exposure, indicating barrier effect of CG. Although the inhibitory effect of MZC (65-74%) did not significantly differ from CG (32-45%), it was within the range of protection (∼75%) against vaginal SHIV-RT challenge 24 h after gel dosing. Overall, the data suggest that evaluation of candidate microbicides in macaque explants can inform macaque efficacy and clinical studies design. The data support advancing MZC gel for clinical evaluation.

Comparison of the Effects of the Selective Estrogen Receptor Modulators Ospemifene, Raloxifene, and Tamoxifen on Breast Tissue in Ex Vivo Culture.

PubMed

Eigeliene, Natalija; Erkkola, Risto; Härkönen, Pirkko

2016-01-01

Explant tissue culture provides a model for studying the direct effects of steroid hormones, their analogs, and novel hormonally active compounds on normal freshly isolated human breast tissues (HBTs). For this purpose, pre- and postmenopausal HBTs can be maintained in this culture system. The results demonstrate that the morphological integrity of HBT explants can be maintained in tissue culture up to 2 weeks and expression of differentiation markers, steroid hormone receptors, proliferation and apoptosis ratios can be evaluated as a response to hormonal stimulation. This chapter describes an ex vivo culture model that we have applied to study the effects of various hormonally active substances, including 17β-estradiol and selective estrogen receptor modulators (SERMs), on normal human breast tissues.

Oxygen and tissue culture affect placental gene expression.

PubMed

Brew, O; Sullivan, M H F

2017-07-01

Placental explant culture is an important model for studying placental development and functions. We investigated the differences in placental gene expression in response to tissue culture, atmospheric and physiologic oxygen concentrations. Placental explants were collected from normal term (38-39 weeks of gestation) placentae with no previous uterine contractile activity. Placental transcriptomic expressions were evaluated with GeneChip ® Human Genome U133 Plus 2.0 arrays (Affymetrix). We uncovered sub-sets of genes that regulate response to stress, induction of apoptosis programmed cell death, mis-regulation of cell growth, proliferation, cell morphogenesis, tissue viability, and protection from apoptosis in cultured placental explants. We also identified a sub-set of genes with highly unstable pattern of expression after exposure to tissue culture. Tissue culture irrespective of oxygen concentration induced dichotomous increase in significant gene expression and increased enrichment of significant pathways and transcription factor targets (TFTs) including HIF1A. The effect was exacerbated by culture at atmospheric oxygen concentration, where further up-regulation of TFTs including PPARA, CEBPD, HOXA9 and down-regulated TFTs such as JUND/FOS suggest intrinsic heightened key biological and metabolic mechanisms such as glucose use, lipid biosynthesis, protein metabolism; apoptosis, inflammatory responses; and diminished trophoblast proliferation, differentiation, invasion, regeneration, and viability. These findings demonstrate that gene expression patterns differ between pre-culture and cultured explants, and the gene expression of explants cultured at atmospheric oxygen concentration favours stressed, pro-inflammatory and increased apoptotic transcriptomic response. Copyright © 2017 Elsevier Ltd. All rights reserved.

Human Platelet Lysate as a Replacement for Fetal Bovine Serum in Limbal Stem Cell Therapy.

PubMed

Suri, Kunal; Gong, Hwee K; Yuan, Ching; Kaufman, Stephen C

2016-10-01

To evaluate the use of human platelet lysate (HPL) as an alternative supplement for limbal explant culture. Culture media were prepared using either 10% pooled HPL (PHPL), single donor HPL, or fetal bovine serum (FBS). Limbal tissues, obtained from the Minnesota Lions Eye Bank, were cultured in each medium on plastic plates or on denuded amniotic membrane (AM). Immunofluorescence staining was performed for ABCG2, tumor protein p63α, and cytokeratin 3 (K3). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of ABCG2 and p63. Limbal explants grown in each medium were labeled with bromodeoxyuridine (BrdU) to assess the proliferative capacity in each medium. Concentration of growth factors including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet derived growth factor (PDGF) in HPL and PHPL was compared to that in human serum (HS). Immunofluorescence staining on AM showed prominent expression of ABCG2, p63α but sparse expression of K3 in HPL and PHPL supplemented medium. Real time-PCR showed 1.7 fold higher expression of ABCG2 in PHPL supplemented medium (p = 0.03), and similar expression of p63 in HPL and PHPL supplemented medium compared to FBS medium. The proliferation assay showed that LSCs retained their proliferative potential in HPL supplemented medium. Higher concentration of growth factors were found in HPL, compared to HS. Human platelet lysate has higher concentration of grown factors and is effective in maintaining growth and stem cell phenotype of corneal limbal explant cultures.

The regulatory role of the tetrapeptide AcSDKP in skin and hair physiology and the prevention of ageing effects in these tissues--a potential cosmetic role.

PubMed

Hajem, N; Chapelle, A; Bignon, J; Pinault, A; Liu, J-M; Salah-Mohellibi, N; Lati, E; Wdzieczak-Bakala, J

2013-06-01

The naturally occurring tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) recognized as a potent angiogenic factor was shown recently to contribute to the repair of cutaneous injuries. In the current article, we report the ability of AcSDKP to exert a beneficial effect on normal healthy skin and scalp and to compensate for the ageing process. In vitro AcSDKP at 10⁻¹¹-10⁻⁷ M significantly stimulates the growth of human keratinocytes, fibroblasts and follicle dermal papilla cells. Moreover, it enhances the growth of human epidermal keratinocyte progenitor and stem cells as shown in a clonogenic survival assay. Topical treatment of ex vivo cultured skin explants with 10⁻⁵ M AcSDKP increases the thickness of the epidermis and upregulates the synthesis of keratins 14 and 19, fibronectin, collagen III and IV as well as the glycoaminoglycans (GAGs). In the ex vivo-cultured hair follicles, AcSDKP promotes hair shaft elongation and induces morphological and molecular modifications matching the criteria of hair growth. Furthermore, AcSDKP at 10⁻¹¹-10⁻⁷ M was shown to improve epidermal barrier, stimulating expression of three protein components of tight junctions (claudin-1, occludin, ZO-1) playing an important role in connecting neighbouring cells. This tetrapeptide exercises also activation of SIRT1 implicated in the control of cell longevity. Indeed, a two-fold increase in the synthesis of SIRT1 by cultured keratinocytes was observed in the presence of 10⁻¹¹-10⁻⁷ M AcSDKP. In conclusion, these findings provide convincing evidence of the regulatory role of AcSDKP in skin and hair physiology and suggest a cosmetic use of this natural tetrapeptide to prevent skin ageing and hair loss and to promote the cutaneous regeneration and hair growth. © 2013 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Comparison of interleukin 10 homologs on dermal wound healing using a novel human skin ex vivo organ culture model.

PubMed

Balaji, Swathi; Moles, Chad M; Bhattacharya, Sukanta S; LeSaint, Maria; Dhamija, Yashu; Le, Louis D; King, Alice; Kidd, Mykia; Bouso, Muhammad F; Shaaban, Aimen; Crombleholme, Timothy M; Bollyky, Paul; Keswani, Sundeep G

2014-07-01

Anti-inflammatory cytokine interleukin (IL)-10 has been shown to induce regenerative healing in postnatal wounds. A viral homolog of IL-10 produced by human cytomegalovirus (CMV IL-10) similarly generates potent immunoregulatory effects, but its effects on wound healing have not been investigated. Currently, there are limited cost-effective methods of screening vulnerary therapeutics. Taken together, we aim to develop and validate a novel human ex vivo dermal wound model and hypothesize that CMV IL-10 will enhance dermal wound healing. Full-thickness circular (6-mm) explants were taken from surgical skin samples and 3-mm full-thickness wounds were created. Explants were embedded in collagen I matrix and maintained in specially formulated media with the epidermis at air-liquid interface, and treated with human IL-10 or CMV IL-10 (200 ng/mL). The viability of cultured explants was validated by histology and lactate dehydrogenase (LDH) activity. Epithelial gap, epithelial height, basal keratinocyte migration, vascular endothelial growth factor levels, and neovascularization were measured at days 3 and 7 to determine IL-10 effects on wound healing. Culture explants at day 7 appeared similar to fresh skin in morphology, cell, and vessel density. By day 14, the epidermis separated from the dermis and the cell density diminished. Day 7 wounds appeared viable with advancing epithelial and basal keratinocyte migration with no evidence of necrosis. Cytotoxicity analysis via the quantification of LDH revealed no differences between controls and treated groups. There was a slight increase in the quantity of LDH in media at day 3; however, this decreased at day 5 and continued to decline up to day 21. CMV IL-10 treatment resulted in a significant decrease in the epithelial gap and an increase in epithelial height. There were no differences in the rates of basal keratinocyte migration at day 7 between treated and control groups. Interestingly, human IL-10 increased vascular endothelial growth factor expression and neovascularization compared with controls. The human ex vivo wound model provides a simple and viable design to study dermal wound healing. Both IL-10 homologs demonstrate vulnerary effects. The viral homolog demonstrates enhanced effects on wound closure compared with human IL-10. These data represent a novel tool that can be used to screen therapeutics, such as CMV IL-10, before preclinical studies. Copyright © 2014 Elsevier Inc. All rights reserved.

Metabolic Response of Human Osteoarthritic Cartilage to Biochemically Characterized Collagen Hydrolysates

PubMed Central

Schadow, Saskia; Simons, Viktor S.; Lochnit, Guenter; Kordelle, Jens; Gazova, Zuzana; Siebert, Hans-Christian; Steinmeyer, Juergen

2017-01-01

The most frequent disease of the locomotor system is osteoarthritis (OA), which, as a chronic joint disease, might benefit more from nutrition than acute illnesses. Collagen hydrolysates (CHs) are peptidic mixtures that are often used as nutraceuticals for OA. Three CHs were characterized biochemically and pharmacologically. Our biophysical (MALDI-TOF-MS, NMR, AFM) and fluorescence assays revealed marked differences between CHs of fish (Peptan® F 5000, Peptan® F 2000) and porcine (Mobiforte®) origin with respect to the total number of peptides and common peptides between them. Using a novel dual radiolabeling procedure, no CH modulated collagen biosynthesis in human knee cartilage explants. Peptan® F 2000 enhanced the activities of the aggrecanase ADMATS4 and ADMATS5 in vitro without loss of proteoglycan from cartilage explants; the opposite effect was observed with Mobiforte®. Interleukin (IL)-6, matrix metalloproteinase (MMP)-1, -3 and -13 levels were elevated in explants that were treated with Mobiforte® and Peptan® F 5000, but not with Peptan® F 2000. In conclusion, the heterogeneous peptide composition and disparate pharmacological effects between CHs suggest that the effect of a CH preparation cannot be extrapolated to other formulations. Thus, the declaration of a CH as a safe and effective nutraceutical requires a thorough examination of its pleiotropic effects. PMID:28117674

Metabolic Response of Human Osteoarthritic Cartilage to Biochemically Characterized Collagen Hydrolysates.

PubMed

Schadow, Saskia; Simons, Viktor S; Lochnit, Guenter; Kordelle, Jens; Gazova, Zuzana; Siebert, Hans-Christian; Steinmeyer, Juergen

2017-01-20

The most frequent disease of the locomotor system is osteoarthritis (OA), which, as a chronic joint disease, might benefit more from nutrition than acute illnesses. Collagen hydrolysates (CHs) are peptidic mixtures that are often used as nutraceuticals for OA. Three CHs were characterized biochemically and pharmacologically. Our biophysical (MALDI-TOF-MS, NMR, AFM) and fluorescence assays revealed marked differences between CHs of fish (Peptan ® F 5000, Peptan ® F 2000) and porcine (Mobiforte ® ) origin with respect to the total number of peptides and common peptides between them. Using a novel dual radiolabeling procedure, no CH modulated collagen biosynthesis in human knee cartilage explants. Peptan ® F 2000 enhanced the activities of the aggrecanase ADMATS4 and ADMATS5 in vitro without loss of proteoglycan from cartilage explants; the opposite effect was observed with Mobiforte ® . Interleukin (IL)-6, matrix metalloproteinase (MMP)-1, -3 and -13 levels were elevated in explants that were treated with Mobiforte ® and Peptan ® F 5000, but not with Peptan ® F 2000. In conclusion, the heterogeneous peptide composition and disparate pharmacological effects between CHs suggest that the effect of a CH preparation cannot be extrapolated to other formulations. Thus, the declaration of a CH as a safe and effective nutraceutical requires a thorough examination of its pleiotropic effects.

Obatoclax, a Pan-BCL-2 Inhibitor, Targets Cyclin D1 for Degradation to Induce Antiproliferation in Human Colorectal Carcinoma Cells.

PubMed

Or, Chi-Hung R; Chang, Yachu; Lin, Wei-Cheng; Lee, Wee-Chyan; Su, Hong-Lin; Cheung, Muk-Wing; Huang, Chang-Po; Ho, Cheesang; Chang, Chia-Che

2016-12-27

Colorectal cancer is the third most common cancer worldwide. Aberrant overexpression of antiapoptotic BCL-2 (B-cell lymphoma 2) family proteins is closely linked to tumorigenesis and poor prognosis in colorectal cancer. Obatoclax is an inhibitor targeting all antiapoptotic BCL-2 proteins. A previous study has described the antiproliferative action of obatoclax in one human colorectal cancer cell line without elucidating the underlying mechanisms. We herein reported that, in a panel of human colorectal cancer cell lines, obatoclax inhibits cell proliferation, suppresses clonogenicity, and induces G₁-phase cell cycle arrest, along with cyclin D1 downregulation. Notably, ectopic cyclin D1 overexpression abrogated clonogenicity suppression but also G₁-phase arrest elicited by obatoclax. Mechanistically, pre-treatment with the proteasome inhibitor MG-132 restored cyclin D1 levels in all obatoclax-treated cell lines. Cycloheximide chase analyses further revealed an evident reduction in the half-life of cyclin D1 protein by obatoclax, confirming that obatoclax downregulates cyclin D1 through induction of cyclin D1 proteasomal degradation. Lastly, threonine 286 phosphorylation of cyclin D1, which is essential for initiating cyclin D1 proteasomal degradation, was induced by obatoclax in one cell line but not others. Collectively, we reveal a novel anticancer mechanism of obatoclax by validating that obatoclax targets cyclin D1 for proteasomal degradation to downregulate cyclin D1 for inducing antiproliferation.

Inflammatory Response of Human Gestational Membranes to Ureaplasma parvum Using a Novel Dual-Chamber Tissue Explant System.

PubMed

Potts, Lauren C; Feng, Liping; Seed, Patrick C; Jayes, Friederike L; Kuchibhatla, Maragatha; Antczak, Brian; Nazzal, Matthew K; Murtha, Amy P

2016-05-01

Preterm premature rupture of membranes (PPROM) is often associated with intra-amniotic inflammation and infection. Current understanding of the pathogenesis of PPROM includes activation of pro-inflammatory cytokines and proteolytic enzymes leading to compromise of membrane integrity. The impact of exposure to bacterial pathogens, including Ureaplasma parvum, on gestational membranes is poorly understood. Our objective was to develop a dual-chamber system to characterize the inflammatory response of gestational membranes to U. parvum in a directional nature. Full-thickness human gestational membrane explants, with either choriodecidua or amnion oriented superiorly, were suspended between two washers in a cylindrical device, creating two distinct compartments. Brilliant green dye was introduced into the top chamber to assess the integrity of the system. Tissue viability was evaluated after 72 h using a colorimetric cell proliferation assay. Choriodecidua or amnion was exposed to three doses of U. parvum and incubated for 24 h. Following treatment, media from each compartment were used for quantification of U. parvum (quantitative PCR), interleukin (IL)-8 (enzyme-linked immunosorbent assay), and matrix metalloproteinase (MMP)-2 and MMP-9 activity (zymography). We observed that system integrity and explant viability were maintained over 72 h. Dose-dependent increases in recovered U. parvum, IL-8 concentration, and MMP-2 activity were detected in both compartments. Significant differences in IL-8 concentration and MMP-9 activity were found between the choriodecidua and amnion. This tissue explant system can be used to investigate the inflammatory consequences of directional bacterial exposure for gestational membranes and provides insight into the pathogenesis of PPROM and infectious complications of pregnancy. © 2016 by the Society for the Study of Reproduction, Inc.

Testicular cells exhibit similar molecular responses to cigarette smoke condensate ex vivo and in vivo.

PubMed

Esakky, Prabagaran; Hansen, Deborah A; Drury, Andrea M; Felder, Paul; Cusumano, Andrew; Moley, Kelle H

2018-01-01

Male exposure to cigarette smoke is associated with seminal defects and with congenital anomalies and childhood cancers in offspring. In mice, paternal exposure to cigarette smoke condensate (CSC) causes molecular defects in germ cells and phenotypic effects in their offspring. Here we used an ex vivo testicular explant model and in vivo exposure to determine the concentration at which CSC impairs spermatogenesis and offspring development. We explanted testis tissue at postnatal day (P)5.5 and cultured it until P11.5. Assessment of growth parameters by analyzing expression of cell-specific markers revealed that the explant system maintained structural and functional integrity. We exposed the P5.5 to -11.5 explants to various concentrations (40-160 µg/ml) of CSC and confirmed that nicotine in the CSC was metabolized to cotinine. We assessed various growth and differentiation parameters, as well as testosterone production, and observed that many spermatogenesis features were impaired at 160 µg/ml CSC. The same parameters were impaired by a similar CSC concentration in vivo Finally, females mated to males that were exposed to 160 µg/ml CSC neonatally had increased rates of pup resorption. We conclude that male exposure to CSC impairs offspring development and that the concentration at which CSC impairs spermatogenesis is similar in vivo and ex vivo. Given that the concentrations of CSC we used contained similar doses of nicotine as human smokers are exposed to, we argue that our model mimics human male reproductive effects of smoking.-Esakky, P., Hansen, D. A., Drury, A. M., Felder, P., Cusumano, A., Moley, K. H. Testicular cells exhibit similar molecular responses to cigarette smoke condensate ex vivo and in vivo . © FASEB.

Genetic transformation of carnation (Dianthus caryophylus L.).

PubMed

Nontaswatsri, Chalermsri; Fukai, Seiichi

2010-01-01

This chapter describes a rapid and efficient protocol for explant preparation and genetic transformation of carnation. Node explants from greenhouse-grown plants and leaf explants from in vitro plants are infected with Agrobacterium tumefaciens AGL0 harboring pKT3 plasmid, consisting of GUS and NPTII genes. Explant preparation is an important factor to obtain the transformed plants. The GUS-staining area was located only on the cut end of explants and only explants with a cut end close to the connecting area between node and leaf, produced transformed shoots. The cocultivation medium is also an important factor for the successful genetic transformation of carnation node and leaf explants. High genetic transformation efficiency of node and leaf explants cocultured with Agrobacterium tumefaciens was achieved when the explants were cocultivated on a filter paper soaked with water or water and acetosyringone mixture (AS).

No adaptive response is induced by chronic low-dose radiation from Ra-226 in the CHSE/F fish embryonic cell line and the HaCaT human epithelial cell line

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

Shi, Xiaopei, E-mail: shix22@mcmaster.ca; Mothersi

Purpose: To determine whether chronic low-dose α-particle radiation from Ra-226 over multiple cell generations can lead to an adaptive response in CHSE/F fish embryonic cells or HaCaT human epithelial cells receiving subsequent acute high-dose γ-ray radiation. Methods: CHSE/F and HaCaT cells were exposed to very low doses of Ra-226 in medium for multiple generations prior to being challenged by a higher dose γ-ray radiation. The clonogenic assay was used to test the clonogenic survival of cells with or without being pretreated by radiation from Ra-226. Results: In general, pretreatment with chronic radiation has no significant influence on the reaction ofmore » cells to the subsequent challenge radiation. Compared to unprimed cells, the change in clonogenic survival of primed cells after receiving challenge radiation is mainly due to the influence of the chronic exposure, and there's little adaptive response induced. However at several dose points, pretreatment of CHSE/F fish cells with chronic radiation resulted in a radiosensitive response to a challenge dose of γ-ray radiation, and pretreatment of HaCaT cells resulted in no effect except for a slightly radioresistant response to the challenge radiation which was not significant. Conclusion: The results suggest that chronic low-dose radiation is not effective enough to induce adaptive response. There was a difference between human and fish cells and it may be important to consider results from multiple species before making conclusions about effects of chronic or low doses of radiation in the environment. The term “radiosensitive” or “adaptive” make no judgment about whether such responses are ultimately beneficial or harmful. - Highlights: • No obvious adaptive response is induced by chronic low-dose radiation from Ra-226. • Priming radiation from Ra-226 sensitized CHSE/F cells to the challenge radiation. • Linear model is inconsistent with current work using chronic low-dose radiation.« less

Placental transport and in vitro effects of Bisphenol A.

PubMed

Mørck, Thit J; Sorda, Giuseppina; Bechi, Nicoletta; Rasmussen, Brian S; Nielsen, Jesper B; Ietta, Francesca; Rytting, Erik; Mathiesen, Line; Paulesu, Luana; Knudsen, Lisbeth E

2010-08-01

Bisphenol A (BPA), an estrogen-like chemical, leaches from consumer products potentially causing human exposure. To examine the effects of BPA exposure during pregnancy, we performed studies using the BeWo trophoblast cell line, placental explant cultures, placental perfusions and skin diffusion models, all of human origin. Results showed BPA cytotoxicity in BeWo cells with an apparent EC50 at 100-125 microM. BPA exposure significantly increased beta-hCG secretion and caspase-3 expression in placental explants at an environmentally relevant concentration of 1 nM. In the transport studies, a rapid transfer of BPA was observed across the term placentae and the BeWo cell monolayer. Further, transdermal transport of BPA was observed. These results indicate that fetal BPA exposure through placental exchange occurs with potential adverse implications for placental and fetal development. This battery of test systems within the realm of human implantation and fetal development represents important elements in risk assessment of reproductive toxicity. Copyright 2010 Elsevier Inc. All rights reserved.

Glucocorticoids affect 24 h clock genes expression in human adipose tissue explant cultures

USDA-ARS?s Scientific Manuscript database

To examine firstly whether CLOCK exhibits a circadian expression in human visceral (V) and subcutaneous (S) adipose tissue (AT) in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX) on positive and negative clock ...

Notch3 marks clonogenic mammary luminal progenitor cells in vivo.

PubMed

Lafkas, Daniel; Rodilla, Veronica; Huyghe, Mathilde; Mourao, Larissa; Kiaris, Hippokratis; Fre, Silvia

2013-10-14

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive "triple negative" human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2(SAT) transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells.

Notch3 marks clonogenic mammary luminal progenitor cells in vivo

PubMed Central

Lafkas, Daniel; Rodilla, Veronica; Huyghe, Mathilde; Mourao, Larissa; Kiaris, Hippokratis

2013-01-01

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive “triple negative” human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2SAT transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells. PMID:24100291

Prolonged viability of human organotypic skin explant in culture method (hOSEC)*

PubMed Central

Frade, Marco Andrey Cipriani; de Andrade, Thiago Antônio Moretti; Aguiar, Andréia Fernanda Carvalho Leone; Guedes, Flávia Araújo; Leite, Marcel Nani; Passos, Williane Rodrigues; Coelho, Eduardo Barbosa; Das, Pranab Kummar

2015-01-01

BACKGROUND: Currently, the cosmetic industry is overwhelmed in keeping up with the safety assessment of the increasing number of new products entering the market. To meet such demand, research centers have explored alternative methods to animal testing and also the large number of volunteers necessary for preclinical and clinical tests. OBJECTIVES: This work describes the human skin ex-vivo model (hOSEC: Human Organotypic Skin Explant Culture) as an alternative to test the effectiveness of cosmetics and demonstrate its viability through cutaneous keratinocytes' proliferative capacity up to 75 days in culture. METHODS: The skin explants obtained from surgeries were cultured in CO2-humid incubator. After 1, 7, 30 and 75 days in culture, skin fragments were harvested for analysis with histomorphological exam (HE staining) on all days of follow-up and immunohistochemistry for Ck5/6, Ck10 and Ki-67 only on the 75th day. RESULTS: On the 7th day, the epidermis was perfect in the dermoepidermal junction, showing the viability of the model. On the 30th day, the epidermis was thicker, with fewer layers on the stratum corneum, although the cutaneous structure was unaltered. On the 75th day, the skin became thinner but the dermoepidermal junctions were preserved and epidermal proliferation was maintained. After the 75th day on culture, the skin was similar to normal skin, expressing keratinocytes with Ck5/6 on supra-basal layers; Ck10 on differentiated layers; and viability could be assessed by the positivity of basal cells by Ki-67. CONCLUSION: The hOSEC model seems a good alternative to animal testing; it can be used as a preclinical test analogous to clinical human skin test with similar effectiveness and viability proven by immunohistological analyses. PMID:26131864

A synthetic PPAR-γ agonist triterpenoid ameliorates experimental fibrosis: PPAR-γ-independent suppression of fibrotic responses.

PubMed

Wei, Jun; Zhu, Hongyan; Komura, Kazuhiro; Lord, Gabriel; Tomcik, Michal; Wang, Wenxia; Doniparthi, Sruthi; Tamaki, Zenshiro; Hinchcliff, Monique; Distler, Joerg H W; Varga, John

2014-02-01

Persistent fibroblast activation initiated by transforming growth factor β (TGF-β) is a fundamental event in the pathogenesis of systemic sclerosis, and its pharmacological inhibition represents a potential therapeutic strategy. The nuclear receptor, peroxisome proliferator-activated receptor γ (PPAR-γ), exerts potent fibrotic activity. The synthetic oleanane triterpenoid, 2-cyano-3,12-dioxo-olean-1,9-dien-28-oic acid (CDDO), is a PPAR-γ agonist with potential effects on TGF-β signalling and dermal fibrosis. To examine the modulation of fibrogenesis by CDDO in explanted fibroblasts, skin organ cultures and murine models of scleroderma. The effects of CDDO on experimental fibrosis induced by bleomycin injection or by overexpression of constitutively active type I TGF-β receptor (TgfbR1ca) were evaluated. Modulation of fibrotic gene expression was examined in human skin organ cultures. To delineate the mechanisms underlying the antifibrotic effects of CDDO, explanted skin fibroblasts cultured in two-dimensional monolayers or in three-dimensional full-thickness human skin equivalents were studied. CDDO significantly ameliorated dermal fibrosis in two complementary mouse models of scleroderma, as well as in human skin organ cultures and in three-dimensional human skin equivalents. In two-dimensional monolayer cultures of explanted normal fibroblasts, CDDO abrogated fibrogenic responses induced by TGF-β. These CDDO effects occurred via disruption of Smad-dependent transcription and were associated with inhibition of Akt activation. In scleroderma fibroblasts, CDDO attenuated the elevated synthesis of collagen. Remarkably, the in vitro antifibrotic effects of CDDO were independent of PPAR-γ. The PPAR-γ agonist triterpenoid CDDO attenuates fibrogenesis by antagonistically targeting canonical TGF-β/Smad and Akt signalling in a PPAR-γ-independent manner. These findings identify this synthetic triterpenoid as a potential new therapy for the control of fibrosis.

Antimicrobial peptides and pro-inflammatory cytokines are differentially regulated across epidermal layers following bacterial stimuli.

PubMed

Percoco, Giuseppe; Merle, Chloé; Jaouen, Thomas; Ramdani, Yasmina; Bénard, Magalie; Hillion, Mélanie; Mijouin, Lily; Lati, Elian; Feuilloley, Marc; Lefeuvre, Luc; Driouich, Azeddine; Follet-Gueye, Marie-Laure

2013-12-01

The skin is a natural barrier between the body and the environment and is colonised by a large number of microorganisms. Here, we report a complete analysis of the response of human skin explants to microbial stimuli. Using this ex vivo model, we analysed at both the gene and protein level the response of epidermal cells to Staphylococcus epidermidis (S. epidermidis) and Pseudomonas fluorescens (P. fluorescens), which are present in the cutaneous microbiota. We showed that both bacterial species affect the structure of skin explants without penetrating the living epidermis. We showed by real-time quantitative polymerase chain reaction (qPCR) that S. epidermidis and P. fluorescens increased the levels of transcripts that encode antimicrobial peptides (AMPs), including human β defensin (hBD)2 and hBD3, and the pro-inflammatory cytokines interleukin (IL)-1α and (IL)-1-β, as well as IL-6. In addition, we analysed the effects of bacterial stimuli on the expression profiles of genes related to innate immunity and the inflammatory response across the epidermal layers, using laser capture microdissection (LCM) coupled to qPCR. We showed that AMP transcripts were principally upregulated in suprabasal keratinocytes. Conversely, the expression of pro-inflammatory cytokines was upregulated in the lower epidermis. These findings were confirmed by protein localisation using specific antibodies coupled to optical or electron microscopy. This work underscores the potential value of further studies that use LCM on human skin explants model to study the roles and effects of the epidermal microbiota on human skin physiology. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Prolonged viability of human organotypic skin explant in culture method (hOSEC).

PubMed

Frade, Marco Andrey Cipriani; Andrade, Thiago Antônio Moretti de; Aguiar, Andréia Fernanda Carvalho Leone; Guedes, Flávia Araújo; Leite, Marcel Nani; Passos, Williane Rodrigues; Coelho, Eduardo Barbosa; Das, Pranab Kummar

2015-01-01

Currently, the cosmetic industry is overwhelmed in keeping up with the safety assessment of the increasing number of new products entering the market. To meet such demand, research centers have explored alternative methods to animal testing and also the large number of volunteers necessary for preclinical and clinical tests. This work describes the human skin ex-vivo model (hOSEC: Human Organotypic Skin Explant Culture) as an alternative to test the effectiveness of cosmetics and demonstrate its viability through cutaneous keratinocytes' proliferative capacity up to 75 days in culture. The skin explants obtained from surgeries were cultured in CO2-humid incubator. After 1, 7, 30 and 75 days in culture, skin fragments were harvested for analysis with histomorphological exam (HE staining) on all days of follow-up and immunohistochemistry for Ck5/6, Ck10 and Ki-67 only on the 75th day. On the 7th day, the epidermis was perfect in the dermoepidermal junction, showing the viability of the model. On the 30th day, the epidermis was thicker, with fewer layers on the stratum corneum, although the cutaneous structure was unaltered. On the 75th day, the skin became thinner but the dermoepidermal junctions were preserved and epidermal proliferation was maintained. After the 75th day on culture, the skin was similar to normal skin, expressing keratinocytes with Ck5/6 on supra-basal layers; Ck10 on differentiated layers; and viability could be assessed by the positivity of basal cells by Ki-67. The hOSEC model seems a good alternative to animal testing; it can be used as a preclinical test analogous to clinical human skin test with similar effectiveness and viability proven by immunohistological analyses.

Radiosensitivity of Patient-Derived Glioma Stem Cell 3-Dimensional Cultures to Photon, Proton, and Carbon Irradiation

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

Chiblak, Sara; Tang, Zili; Molecular and Translational Radiation Oncology, Heidelberg Ion Therapy Center, Heidelberg Institute of Radiation Oncology, University of Heidelberg Medical School and National Center for Tumor Diseases, German Cancer Research Center, Heidelberg

Purpose: To investigate the radiosensitivity of primary glioma stem cell (GSC) cultures with different CD133 status in a 3-dimensional (3D) model after photon versus proton versus carbon irradiation. Methods and Materials: Human primary GSC spheroid cultures were established from tumor specimens of six consented glioblastoma patients. Human U87MG was used as a classical glioblastoma radioresistant cell line. Cell suspensions were generated by mechanical dissociation of GSC spheroids and embedded in a semi-solid 3D matrix before irradiation. Spheroid-like colonies were manually counted by microscopy. Cells were also recovered and quantified by fluorescence. CD133 expression and DNA damage were evaluated by flow cytometry.more » Results: The fraction of CD133{sup +} cells varied between 0.014% and 96% in the six GSC cultures and showed a nonsignificant correlation with plating efficiency and survival fractions. The 4 most photon-radioresistant GSC cultures were NCH644, NCH421k, NCH441, and NCH636. Clonogenic survival for proton irradiation revealed relative biologic effectiveness (RBE) in the range of 0.7-1.20. However, carbon irradiation rendered the photon-resistant GSC cultures sensitive, with average RBE of 1.87-3.44. This effect was partly attributed to impaired capability of GSC to repair carbon ion–induced DNA double-strand breaks as determined by residual DNA repair foci. Interestingly, radiosensitivity of U87 cells was comparable to GSC cultures using clonogenic survival as the standard readout. Conclusions: Carbon irradiation is effective in GSC eradication with similar RBE ranges approximately 2-3 as compared with non-stem GSC cultures (U87). Our data strongly suggest further exploration of GSC using classic radiobiology endpoints such as the here-used 3D clonogenic survival assay and integration of additional GSC-specific markers.« less

Miniaturization of the Clonogenic Assay Using Confluence Measurement

PubMed Central

Mayr, Christian; Beyreis, Marlena; Dobias, Heidemarie; Gaisberger, Martin; Pichler, Martin; Ritter, Markus; Jakab, Martin; Neureiter, Daniel; Kiesslich, Tobias

2018-01-01

The clonogenic assay is a widely used method to study the ability of cells to ‘infinitely’ produce progeny and is, therefore, used as a tool in tumor biology to measure tumor-initiating capacity and stem cell status. However, the standard protocol of using 6-well plates has several disadvantages. By miniaturizing the assay to a 96-well microplate format, as well as by utilizing the confluence detection function of a multimode reader, we here describe a new and modified protocol that allows comprehensive experimental setups and a non-endpoint, label-free semi-automatic analysis. Comparison of bright field images with confluence images demonstrated robust and reproducible detection of clones by the confluence detection function. Moreover, time-resolved non-endpoint confluence measurement of the same well showed that semi-automatic analysis was suitable for determining the mean size and colony number. By treating cells with an inhibitor of clonogenic growth (PTC-209), we show that our modified protocol is suitable for comprehensive (broad concentration range, addition of technical replicates) concentration- and time-resolved analysis of the effect of substances or treatments on clonogenic growth. In summary, this protocol represents a time- and cost-effective alternative to the commonly used 6-well protocol (with endpoint staining) and also provides additional information about the kinetics of clonogenic growth. PMID:29510509

Bacterial stimulation of adventitious rooting on in vitro cultured slash pine (Pinus elliottii Engelm.) seedling explants.

PubMed

Burns, J A; Schwarz, O J

1996-02-01

A bacterium has been isolated that initiates adventitious rooting when co-cultured under in vitro conditions with seedling-produced hypocotylary explants of slash pine (Pinus elliottii). Rooting efficiencies produced through bacterial-explant co-culture range from approximately 15% to greater than 90% over non-treated controls. Explant exposure to the root inducing bacterium has produced no obvious pathology in the regenerated plantlets. Seedling explants rooted by bacterial-explant co-culture have been successfully transitioned to ambient greenhouse conditions.

Evidence for CB2 receptor involvement in LPS-induced reduction of cAMP intracellular levels in uterine explants from pregnant mice: pathophysiological implications.

PubMed

Salazar, Ana Inés; Carozzo, Alejandro; Correa, Fernando; Davio, Carlos; Franchi, Ana María

2017-07-01

What is the role of the endocannabinoid system (eCS) on the lipopolysaccharide (LPS) effects on uterine explants from 7-day pregnant mice in a murine model of endotoxin-induced miscarriage? We found evidence for cannabinoid receptor type2 (CB2) involvement in LPS-induced increased prostaglandin-F2α (PGF2α) synthesis and diminished cyclic adenosine monophosphate (cAMP) intracellular content in uterine explants from early pregnant mice. Genital tract infections by Gram-negative bacteria are a common complication of human pregnancy that results in an increased risk of pregnancy loss. LPS, the main component of the Gram-negative bacterial wall, elicits a strong maternal inflammatory response that results in embryotoxicity and embryo resorption in a murine model endotoxin-induced early pregnancy loss. We have previously shown that the eCS mediates the embryotoxic effects of LPS, mainly via CB1 receptor activation. An in vitro study of mice uterine explants was performed to investigate the eCS in mediating the effects of LPS on PGF2α production and cAMP intracellular content. Eight to 12-week-old virgin female BALB/c or CD1 (wild-type [WT] or CB1-knockout [CB1-KO]) mice were paired with 8- to 12-week-old BALB/c or CD1 (WT or CB1-KO) males, respectively. On day 7 of pregnancy, BALB/c, CD1 WT or CD1 CB1-KO mice were euthanized, the uteri were excised, implantation sites were removed and the uterine tissues were separated from decidual and embryo tissues. Uterine explants were cultured and exposed for an appropriate amount of time to different pharmacological treatments. The tissues were then collected for cAMP assay and PGF2α content determination by radioimmunoassay. In vitro treatment of uteri explants from 7-day pregnant BALB/c or CD1 (WT or CB1-KO) mice with LPS induced an increased production of PGF2α (P < 0.05) and a reduction of the tissue content of cAMP (P < 0.05). These effects were mediated by CB2 receptors since exposure to AM630 (a specific CB2 receptor antagonist) prevented these LPS-induced effects (P < 0.05). Collectively, our results suggest a role for the eCS mediating LPS-induced deleterious effects on reproductive tissues. Since our experimental design involves in vitro experiments of uterine explants, the extrapolation of the results presented here to humans is limited. Our findings provide evidence for the role of CB2 receptors in reproductive events as well as their participation as a mediator of LPS deleterious effects on reproductive tissues. None. Dr Ana María Franchi was funded by Agencia Nacional para la Promoción Científica y Tecnológica (PICT 2010/0813 and PICT 2013/0097) and by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2012/0061). Dr Carlos Davio was funded by Agencia Nacional para la Promoción Científica y Tecnológica (PICT 2013/2050). The authors have no competing interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Damnacanthal, a noni anthraquinone, inhibits c-Met and is a potent antitumor compound against Hep G2 human hepatocellular carcinoma cells.

PubMed

García-Vilas, Javier A; Quesada, Ana R; Medina, Miguel A

2015-01-26

Damnacanthal, an anthraquinone present in noni plants, targets several tyrosine kinases and has antitumoral effects. This study aims at getting additional insight on the potential of damnacanthal as a natural antitumor compound. The direct effect of damnacanthal on c-Met was tested by in vitro activity assays. Additionally, Western blots of c-Met phosphorylation in human hepatocellular carcinoma Hep G2 cells were performed. The antitumor effects of damnacanthal were tested by using cell growth, soft agar clonogenic, migration and invasion assays. Their mechanisms were studied by Western blot, and cell cycle, apoptosis and zymographic assays. Results show that damnacanthal targets c-Met both in vitro and in cell culture. On the other hand, damnacanthal also decreases the phosphorylation levels of Akt and targets matrix metalloproteinase-2 secretion in Hep G2 cells. These molecular effects are accompanied by inhibition of the growth and clonogenic potential of Hep G2 hepatocellular carcinoma cells, as well as induction of Hep G2 apoptosis. Since c-Met has been identified as a new potential therapeutical target for personalized treatment of hepatocellular carcinoma, damnacanthal and noni extract supplements containing it could be potentially interesting for the treatment and/or chemoprevention of hepatocellular carcinoma through its inhibitory effects on the HGF/c-Met axis.

Mechanisms involved in p53 downregulation by leptin in trophoblastic cells.

PubMed

Toro, Ayelén Rayen; Pérez-Pérez, Antonio; Corrales Gutiérrez, Isabel; Sánchez-Margalet, Víctor; Varone, Cecilia Laura

2015-11-01

Leptin, a 16-kDa polypeptide hormone, is produced by the adipocyte and can also be synthesized by placenta. We previously demonstrated that leptin promotes proliferation and survival in placenta, in part mediated by the p53 pathway. In this work, we investigated the mechanisms involved in leptin down-regulation of p53 level. The human first trimester cytotrophoblastic Swan-71 cell line and human placental explants at term were used. In order to study the late phase of apoptosis, triggered by serum deprivation, experiments of DNA fragmentation were carried out. Exogenous leptin added to human placental explants, showed a decrease on DNA ladder formation and MAPK pathway is involved in this leptin effect. We also found that under serum deprivation condition, leptin decreases p53 levels and the inhibitory leptin effect is lost when cells were pretreated with 50 μM PD98059 or 10 μM LY29004; or were transfected with dominant negative mutants of intermediates of these pathways, suggesting that MAPK and PI3K signaling pathways are necessaries for leptin action. Additionally, leptin diminished Ser-46 p53 phosphorylation and this effect in placental explants was mediated by the activation of MAPK and PI3K pathways. Finally, in order to assess leptin effect on p53 half-life experiments with cycloheximide were performed and MDM-2 expression was analyzed. Leptin diminished p53 half-life and up-regulated MDM-2 expression. In summary, we provided evidence suggesting that leptin anti-apoptotic effect is mediated by MAPK and PI3K pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

Functional activation of PPARγ in human upper aerodigestive cancer cell lines.

PubMed

Wright, Simon K; Wuertz, Beverly R; Harris, George; Abu Ghazallah, Raed; Miller, Wendy A; Gaffney, Patrick M; Ondrey, Frank G

2017-01-01

Upper aerodigestive cancer is an aggressive malignancy with relatively stagnant long-term survival rates over 20 yr. Recent studies have demonstrated that exploitation of PPARγ pathways may be a novel therapy for cancer and its prevention. We tested whether PPARγ is expressed and inducible in aerodigestive carcinoma cells and whether it is present in human upper aerodigestive tumors. Human oral cancer CA-9-22 and NA cell lines were treated with the PPAR activators eicosatetraynoic acid (ETYA), 15-deoxy-δ- 12,14-prostaglandin J2 (PG-J2), and the thiazolidinedione, ciglitazone, and evaluated for their ability to functionally activate PPARγ luciferase reporter gene constructs. Cellular proliferation and clonogenic potential after PPARγ ligand treatment were also evaluated. Aerodigestive cancer specimens and normal tissues were evaluated for PPARγ expression on gene expression profiling and immunoblotting. Functional activation of PPARγ reporter gene constructs and increases in PPARγ protein were confirmed in the nuclear compartment after PPARγ ligand treatment. Significant decreases in cell proliferation and clonogenic potential resulted from treatment. Lipid accumulation was induced by PPARγ activator treatment. 75% of tumor specimens and 100% of normal control tissues expressed PPARγ RNA, and PPARγ protein was confirmed in 66% of tumor specimens analyzed by immunoblotting. We conclude PPARγ can be functionally activated in upper aerodigestive cancer and that its activation downregulates several features of the neoplastic phenotype. PPARγ expression in human upper aerodigestive tract tumors and normal cells potentially legitimizes it as a novel intervention target in this disease. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Role of M Cells in Human Mucosal Immunity to Mycobacterium tuberculosis

PubMed Central

Nair, Vidhya; Khan, Haaris; Mitchell, Ron; Shiloh, Michael U

2017-01-01

Abstract Background Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a bacterial pathogen that infects roughly one-third of the worldÕs population and causes 1–2 million deaths per year. The current paradigm is that phagocytosis of Mtb by patrolling alveolar macrophages initiates Mtb infection. While this model can account for pulmonary TB, it does not adequately explain the occurrence of extrapulmonary forms of TB that manifest in the absence of obvious lung involvement, such as tuberculous cervical lymphadenitis, also known as scrofula. We hypothesized that specialized epithelial cells called microfold cells (M cells) may be an alternate portal of entry for Mtb. Previously we demonstrated that Mtb is able to transcytose across an epithelial barrier in an M cell dependent manner and that M cell mediated transcytosis is vital for Mtb pathogenesis in a mouse model of tuberculosis. Methods We used an in vitro M-cell mediated translocation assay and a Mtb mutant lacking a key virulence factor, ESAT6. We used biochemistry and genetics to identify a novel receptor for ESAT6. We also developed a novel explanted human adenoid Mtb infection model to study mucosal immunity. Results We now demonstrate that the Mtb virulence factor ESAT6 is necessary and sufficient to mediate binding and transcytosis by M cells in vitro and in vivo, and that uptake of Mtb by M cells requires a unique cell surface ESAT6 receptor. We developed a novel explanted human adenoid model of M cell biology and demonstrate rapid Mtb transcytosis by primary human tissue within 60–120 minutes. Using flow cytometry we find that Mtb is first ingested by M cells and then after transcytosis, by tissue resident antigen-presenting cells. Explanted adenoids from 10 independent donors display a wide range of Mtb uptake. Conclusion We conclude that Mtb ESAT6 is necessary for Mtb uptake by M-cells and that binding and transcytosis require a host receptor. Because explanted adenoids display a wide range of Mtb uptake, M cell mediated transcytosis may confer differential susceptibility to scrofula and disseminated disease. These findings are significant as M cells could potentially serve as the basis for novel therapeutic targets against primary Mtb infection. Disclosures All authors: No reported disclosures.

TRPM8 is required for survival and radioresistance of glioblastoma cells

PubMed Central

Klumpp, Dominik; Frank, Stephanie C.; Klumpp, Lukas; Sezgin, Efe C.; Eckert, Marita; Edalat, Lena; Bastmeyer, Martin; Zips, Daniel; Ruth, Peter; Huber, Stephan M.

2017-01-01

TRPM8 is a Ca2+-permeable nonselective cation channel belonging to the melastatin sub-group of the transient receptor potential (TRP) family. TRPM8 is aberrantly overexpressed in a variety of tumor entities including glioblastoma multiforme where it reportedly contributes to tumor invasion. The present study aimed to disclose further functions of TRPM8 in glioma biology in particular upon cell injury by ionizing radiation. To this end, TCGA data base was queried to expose the TRPM8 mRNA abundance in human glioblastoma specimens and immunoblotting was performed to analyze the TRPM8 protein abundance in primary cultures of human glioblastoma. Moreover, human glioblastoma cell lines were irradiated with 6 MV photons and TRPM8 channels were targeted pharmacologically or by RNA interference. TRPM8 abundance, Ca2+ signaling and resulting K+ channel activity, chemotaxis, cell migration, clonogenic survival, DNA repair, apoptotic cell death, and cell cycle control were determined by qRT-PCR, fura-2 Ca2+ imaging, patch-clamp recording, transfilter migration assay, wound healing assay, colony formation assay, immunohistology, flow cytometry, and immunoblotting. As a result, human glioblastoma upregulates TRPM8 channels to variable extent. TRPM8 inhibition or knockdown slowed down cell migration and chemotaxis, attenuated DNA repair and clonogenic survival, triggered apoptotic cell death, impaired cell cycle and radiosensitized glioblastoma cells. Mechanistically, ionizing radiation activated and upregulated TRPM8-mediated Ca2+ signaling that interfered with cell cycle control probably via CaMKII, cdc25C and cdc2. Combined, our data suggest that TRPM8 channels contribute to spreading, survival and radioresistance of human glioblastoma and, therefore, might represent a promising target in future anti-glioblastoma therapy. PMID:29221175

Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

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

Wei, Yan; Department of Otolaryngology, Head and Neck Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou; Li, Yuan

2011-04-15

Human skeletal muscle contains an accessible adult stem-cell compartment in which differentiated myofibers are maintained and replaced by a self-renewing stem cell pool. Previously, studies using mouse models have established a critical role for resident stem cells in skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate for extended periods of time as floating clusters of rounded cells, termed 'myospheres' or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional properties of these cells were determined usingmore » reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times, despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types.« less

Mary Jane Hogue (1883-1962): A pioneer in human brain tissue culture.

PubMed

Zottoli, Steven J; Seyfarth, Ernst-August

2018-05-16

The ability to maintain human brain explants in tissue culture was a critical step in the use of these cells for the study of central nervous system disorders. Ross G. Harrison (1870-1959) was the first to successfully maintain frog medullary tissue in culture in 1907, but it took another 38 years before successful culture of human brain tissue was accomplished. One of the pioneers in this achievement was Mary Jane Hogue (1883-1962). Hogue was born into a Quaker family in 1883 in West Chester, Pennsylvania, and received her undergraduate degree from Goucher College in Baltimore, Maryland. Research with the developmental biologist Theodor Boveri (1862-1915) in Würzburg, Germany, resulted in her Ph.D. (1909). Hogue transitioned from studying protozoa to the culture of human brain tissue in the 1940s and 1950s, when she was one of the first to culture cells from human fetal, infant, and adult brain explants. We review Hogue's pioneering contributions to the study of human brain cells in culture, her putative identification of progenitor neuroblast and/or glioblast cells, and her use of the cultures to study the cytopathogenic effects of poliovirus. We also put Hogue's work in perspective by discussing how other women pioneers in tissue culture influenced Hogue and her research.

Human extrahepatic portal vein obstruction correlates with decreased factor VII and protein C transcription but increased hepatocyte proliferation.

PubMed

Chiu, Bill; Melin-Aldana, Hector; Superina, Riccardo A

2007-10-01

A 3-year-old girl developed extrahepatic portal vein obstruction (EHPVO) after a liver transplant. She had sequelae of portal hypertension that required another transplantation. The circumstances allowed for comparison of liver-dependent coagulation factor production between the second donor liver and the explanted liver with EHPVO. Liver samples from the explanted first graft and the second transplant were obtained. Fresh tissue was used to perform reverse transcription-polymerase chain reaction with primers against factors V, VII, as well as VIII, protein C, and paraffin-embedded sections for hepatocyte proliferation using Ki-67 antibody as well as for apoptosis using TUNEL assay. The transcription of factor VII and that of protein C were decreased in the explant as compared with the newly transplanted liver (factor VII, 77% of the donor; protein C, 88% of the donor). The transcription of factor V and that of factor VIII were unchanged. The explant had a greater percentage of proliferating hepatocytes than the new organ (0.85% +/- 0.75% vs 0.11% +/- 0.21%). The percentage of apoptotic cells was similar between the 2 livers (0.09% +/- 0.13% vs 0.09% +/- 0.13%). Idiopathic EHPVO is associated with a reduction in liver-dependent coagulation factor transcription and an increase in hepatocyte proliferation. Portal blood flow deprivation alters hepatic homeostasis and initiates mechanisms that attempt to restore liver-dependent coagulation factors.

Neural transcription factors bias cleavage stage blastomeres to give rise to neural ectoderm

PubMed Central

Gaur, Shailly; Mandelbaum, Max; Herold, Mona; Majumdar, Himani Datta; Neilson, Karen M.; Maynard, Thomas M.; Mood, Kathy; Daar, Ira O.; Moody, Sally A.

2016-01-01

The decision by embryonic ectoderm to give rise to epidermal versus neural derivatives is the result of signaling events during blastula and gastrula stages. However, there also is evidence in Xenopus that cleavage stage blastomeres contain maternally derived molecules that bias them toward a neural fate. We used a blastomere explant culture assay to test whether maternally deposited transcription factors bias 16-cell blastomere precursors of epidermal or neural ectoderm to express early zygotic neural genes in the absence of gastrulation interactions or exogenously supplied signaling factors. We found that Foxd4l1, Zic2, Gmnn and Sox11 each induced explants made from ventral, epidermis-producing blastomeres to express early neural genes, and that at least some of the Foxd4l1 and Zic2 activity is required at cleavage stages. Similarly, providing extra Foxd4l1 or Zic2 to explants made from dorsal, neural plate-producing blastomeres significantly increased expression of early neural genes, whereas knocking down either significantly reduced them. These results show that maternally delivered transcription factors bias cleavage stage blastomeres to a neural fate. We demonstrate that mouse and human homologues of Foxd4l1 have similar functional domains compared to the frog protein, as well as conserved transcriptional activities when expressed in Xenopus embryos and blastomere explants. PMID:27092474

Plantlet regeneration potential from seedling explants of vitegnus (Vitex agnus castus).

PubMed

Chamandoosti, F

2007-11-15

In this research a simple and repeatable method for regeneration of a important medicinal plant (Vitex agnus castus) described. Different seedling explants such as hypocotyl, cotyledon, root and apical meristem were cultured in MS basal media with different kinds and concentrations of PGRs. Root and apical meristem explants were the only explants that have regeneration whole plantlets potential. It was interesting that regeneration whole plantlets from root and apical meristem explants have different developmental pathways. Whole plantlets from apical meristem explants regenerated by passing phase callusing whereas regeneration whole plantlets from root was direct and without phase callusing. This subject implies that we can have many manipulation possibilities in order to different objects of tissue culture by selecting different explants in vitegnus.

Chondrocytes and stem cells in 3D-bioprinted structures create human cartilage in vivo

PubMed Central

Amoroso, Matteo; Lindahl, Anders; Brantsing, Camilla; Rotter, Nicole; Gatenholm, Paul; Kölby, Lars

2017-01-01

Cartilage repair and replacement is a major challenge in plastic reconstructive surgery. The development of a process capable of creating a patient-specific cartilage framework would be a major breakthrough. Here, we described methods for creating human cartilage in vivo and quantitatively assessing the proliferative capacity and cartilage-formation ability in mono- and co-cultures of human chondrocytes and human mesenchymal stem cells in a three-dimensional (3D)-bioprinted hydrogel scaffold. The 3D-bioprinted constructs (5 × 5 × 1.2 mm) were produced using nanofibrillated cellulose and alginate in combination with human chondrocytes and human mesenchymal stem cells using a 3D-extrusion bioprinter. Immediately following bioprinting, the constructs were implanted subcutaneously on the back of 48 nude mice and explanted after 30 and 60 days, respectively, for morphological and immunohistochemical examination. During explantation, the constructs were easy to handle, and the majority had retained their macroscopic grid appearance. Constructs consisting of human nasal chondrocytes showed good proliferation ability, with 17.2% of the surface areas covered with proliferating chondrocytes after 60 days. In constructs comprising a mixture of chondrocytes and stem cells, an additional proliferative effect was observed involving chondrocyte production of glycosaminoglycans and type 2 collagen. This clinically highly relevant study revealed 3D bioprinting as a promising technology for the creation of human cartilage. PMID:29236765

Chondrocytes and stem cells in 3D-bioprinted structures create human cartilage in vivo.

PubMed

Apelgren, Peter; Amoroso, Matteo; Lindahl, Anders; Brantsing, Camilla; Rotter, Nicole; Gatenholm, Paul; Kölby, Lars

2017-01-01

Cartilage repair and replacement is a major challenge in plastic reconstructive surgery. The development of a process capable of creating a patient-specific cartilage framework would be a major breakthrough. Here, we described methods for creating human cartilage in vivo and quantitatively assessing the proliferative capacity and cartilage-formation ability in mono- and co-cultures of human chondrocytes and human mesenchymal stem cells in a three-dimensional (3D)-bioprinted hydrogel scaffold. The 3D-bioprinted constructs (5 × 5 × 1.2 mm) were produced using nanofibrillated cellulose and alginate in combination with human chondrocytes and human mesenchymal stem cells using a 3D-extrusion bioprinter. Immediately following bioprinting, the constructs were implanted subcutaneously on the back of 48 nude mice and explanted after 30 and 60 days, respectively, for morphological and immunohistochemical examination. During explantation, the constructs were easy to handle, and the majority had retained their macroscopic grid appearance. Constructs consisting of human nasal chondrocytes showed good proliferation ability, with 17.2% of the surface areas covered with proliferating chondrocytes after 60 days. In constructs comprising a mixture of chondrocytes and stem cells, an additional proliferative effect was observed involving chondrocyte production of glycosaminoglycans and type 2 collagen. This clinically highly relevant study revealed 3D bioprinting as a promising technology for the creation of human cartilage.

Supra-organization and optical anisotropies of the extracellular matrix in the amniotic membrane and limbal stroma before and after explant culture

PubMed Central

Valdetaro, Gisele P.; Aldrovani, Marcela; Padua, Ivan R. M.; Cristovam, Priscila C.; Gomes, José A. P.; Laus, José L.

2016-01-01

In this research we evaluated the supramolecular organizations and the optical anisotropical properties of the de-epithelialized human amniotic membrane and rabbit limbal stroma, before and after explant culture. Birefringence, monochromatic light spectral absorption and linear dichroism of the main extracellular matrix biopolymers, that is, the fibrillar collagens and proteoglycans, were investigated by polarized light microscopy combined with image analysis. Our results demonstrated that the culture procedure–induced stimuli altered the supra-organizational characteristics (in terms of collagens/proteoglycans spatial orientation and ordered-aggregational state) of the amniotic and limbal extracellular matrix, which led to changes in optical anisotropical properties. PMID:28018719

LDHA in neuroblastoma is associated with poor outcome and its depletion decreases neuroblastoma growth independent of aerobic glycolysis.

PubMed

Dorneburg, Carmen; Fischer, Matthias; Barth, Thomas F E; Mueller-Klieser, Wolfgang; Hero, Barbara; Gecht, Judith; Carter, Daniel R; De Preter, Katleen; Mayer, Benjamin; Christner, Lisa; Speleman, Frank; Marshall, Glenn M; Debatin, Klaus-Michael; Beltinger, Christian

2018-06-20

To investigate whether lactate dehydrogenase A (LDHA), an important component of the LDH tetramer crucial for aerobic glycolysis, is associated with patient outcome and constitutes a therapeutic target in neuroblastoma (NB). Expression of LDHA mRNA and protein was determined in 709 and 110 NB patient samples, respectively, and correlated to survival and risk factors. LDHA and LDHB were depleted in human NB cell lines by CRISPR/Cas9 and shRNA, respectively, and aerobic glycolysis, clonogenicity and tumorigenicity were determined. Expression of LDHA in relation to MYCN was measured in NB cell lines and in the TH-MYCN NB mouse model. Expression of LDHA, both on the mRNA and the protein level, was significantly and independently associated with decreased patient survival. Predominant cytoplasmic localization of LDHA protein was associated with poor outcome. Amplification and expression of MYCN did not correlate with expression of LDHA in NB cell lines or TH-MYCN mice, respectively. Knockout of LDHA inhibited clonogenicity, tumorigenicity and tumor growth without abolishing LDH activity or significantly decreasing aerobic glycolysis. Concomitant depletion of LDHA and the isoform LDHB ablated clonogenicity while not abrogating LDH activity or decreasing aerobic glycolysis. The isoform LDHC was not expressed. High expression of LDHA is independently associated with poor outcome of NB and NB cells can be inhibited by depletion of LDHA or LDHB. This inhibition appears to be unrelated to LDH activity and aerobic glycolysis. Thus, investigations of inhibitory mechanisms beyond attenuation of aerobic glycolysis are warranted, both in NB and normal cells. Copyright ©2018, American Association for Cancer Research.

Enhanced micropropagation and tiller formation in sugarcane through pretreatment of explants with thidiazuron (TDZ).

PubMed

Kumari, Kavita; Lal, Madan; Saxena, Sangeeta

2017-10-01

An efficient, simple and commercially applicable protocol for rapid micropropagation of sugarcane has been designed using variety Co 05011. Pretreatment of shoot tip explants with thidiazuron (TDZ) induced high frequency regeneration of shoot cultures with improved multiplication ratio. The highest frequency (80%) of shoot initiation in explants pretreated with 10 mg/l of TDZ was obtained during the study. Maximum 65% shoot cultures could be established from the explants pretreated with TDZ as compared to minimum 40% establishment in explants without pretreatment. The explants pretreated with 10 mg/l of TDZ required minimum 40 days for the establishment of shoot cultures as compared to untreated explants which required 60 days. The highest average number of shoots per culture (19.1) could be obtained from the explants pretreated with 10 mg/l of TDZ, indicating the highest multiplication ratio (1:6). Highest rooting (over 94%) was obtained in shoots regenerated from pretreated explants on ½ strength MS medium containing 5.0 mg/l of NAA and 50 g/l of sucrose within 15 days. Higher number of tillers/clump (15.3) could be counted in plants regenerated from pretreated explants than untreated ones (10.9 tillers/clump) in field condition, three months after transplantation. Molecular analysis using RAPD and DAMD markers suggested that the pretreatment of explants with TDZ did not adversely affect the genetic stability of regenerated plants and maintained high clonal purity.

INTERSPECIES COMPARISONS OF BENZO(A)PYRENE METABOLISM AND DNA-ADDUCT FORMATION IN CULTURED HUMAN AND ANIMAL BLADDER AND TRACHEOBRONCHIAL TISSUES

EPA Science Inventory

Cultured bladder and tracheobronchial explants from human, monkey, dog, hamster, and rat were used to study the metabolism, covalent binding to DNA, and DNA:adduct formation of (3H0benzo(a)pyrene (BP). Both organs from all species formed large amounts (40 to 70% of total 3H in th...

Synthesis and Antiproliferative Activity of 2,5-bis(3′-Indolyl)pyrroles, Analogues of the Marine Alkaloid Nortopsentin

PubMed Central

Carbone, Anna; Parrino, Barbara; Barraja, Paola; Spanò, Virginia; Cirrincione, Girolamo; Diana, Patrizia; Maier, Armin; Kelter, Gerhard; Fiebig, Heinz-Herbert

2013-01-01

2,5-bis(3′-Indolyl)pyrroles, analogues of the marine alkaloid nortopsentin, were conveniently prepared through a three step procedure in good overall yields. Derivatives 1a and 1b exhibited concentration-dependent antitumor activity towards a panel of 42 human tumor cell lines with mean IC50 values of 1.54 μM and 0.67 μM, respectively. Investigating human tumor xenografts in an ex-vivo clonogenic assay revealed selective antitumor activity, whereas sensitive tumor models were scattered among various tumor histotypes. PMID:23455514

Investigating the anti-proliferative activity of styrylazanaphthalenes and azanaphthalenediones.

PubMed

Mrozek-Wilczkiewicz, Anna; Kalinowski, Danuta S; Musiol, Robert; Finster, Jacek; Szurko, Agnieszka; Serafin, Katarzyna; Knas, Magdalena; Kamalapuram, Sishir K; Kovacevic, Zaklina; Jampilek, Josef; Ratuszna, Alicja; Rzeszowska-Wolny, Joanna; Richardson, Des R; Polanski, Jaroslaw

2010-04-01

A group of styrylazanaphthalenes and azanaphthalenediones were synthesized and tested for their anti-proliferative activity. Most of the compounds were obtained with the use of microwave-assisted synthesis. The lipophilicity of the compounds was measured by RP-HPLC and their anti-proliferative activity was assayed against the human SK-N-MC neuroepithelioma and HCT116 human colon carcinoma cell lines. Active compounds were also tested in clonogenity and comet assays. Several quinazolinone and styrylquinazoline analogues were found to have markedly greater anti-proliferative activity than desferoxamine and cis-platin. Copyright 2010 Elsevier Ltd. All rights reserved.

Pharmacological rescue of the dystrophin-glycoprotein complex in Duchenne and Becker skeletal muscle explants by proteasome inhibitor treatment.

PubMed

Assereto, Stefania; Stringara, Silvia; Sotgia, Federica; Bonuccelli, Gloria; Broccolini, Aldobrando; Pedemonte, Marina; Traverso, Monica; Biancheri, Roberta; Zara, Federico; Bruno, Claudio; Lisanti, Michael P; Minetti, Carlo

2006-02-01

In this report, we have developed a novel method to identify compounds that rescue the dystrophin-glycoprotein complex (DGC) in patients with Duchenne or Becker muscular dystrophy. Briefly, freshly isolated skeletal muscle biopsies (termed skeletal muscle explants) from patients with Duchenne or Becker muscular dystrophy were maintained under defined cell culture conditions for a 24-h period in the absence or presence of a specific candidate compound. Using this approach, we have demonstrated that treatment with a well-characterized proteasome inhibitor, MG-132, is sufficient to rescue the expression of dystrophin, beta-dystroglycan, and alpha-sarcoglycan in skeletal muscle explants from patients with Duchenne or Becker muscular dystrophy. These data are consistent with our previous findings regarding systemic treatment with MG-132 in a dystrophin-deficient mdx mouse model (Bonuccelli G, Sotgia F, Schubert W, Park D, Frank PG, Woodman SE, Insabato L, Cammer M, Minetti C, and Lisanti MP. Am J Pathol 163: 1663-1675, 2003). Our present results may have important new implications for the possible pharmacological treatment of Duchenne or Becker muscular dystrophy in humans.

Causes of Phakic Implantable Collamer Lens Explantation/Exchange at King Khaled Eye Specialist Hospital

PubMed Central

AlSabaani, Nasser A.; Behrens, Ashley; Jastanieah, Sabah; Al Malki, Salem; Al Jindan, Mohanna; Al Motowa, Saeed

2016-01-01

PURPOSE: The purpose of this study is to evaluate the causes of phakic implantable collamer lens (ICL) explantation/exchange at an eye hospital in Saudi Arabia. MATERIALS AND METHODS: A retrospective chart review was performed for patients who underwent ICL implantation from 2007 to March 2014 and data were collected on cases that underwent ICL explantation. RESULTS: Of the 787 ICL implants, 30 implants (3.8% [95% confidence interval 2.6%; 5.3%]) were explanted. The causes of explantation included incorrect lens size (22), cataract (4), high residual astigmatism (2), rhegmatogenous retinal detachment (1), and intolerable glare (1). Corrective measures mainly included an exchange with an appropriately sized lens (9), ICL explantation (11), with phacoemulsification and posterior chamber intraocular lens implantation (6), or replacement with an ICL of correct power (2). CONCLUSION: Incorrect ICL size was the most common cause of ICL explantation. More accurate sizing methods for ICL are required to reduce the explantation/exchange rate. PMID:27994391

The effect of in vitro tracheal occlusion on branching morphogenesis in fetal lung explants from the rat nitrofen model of congenital diaphragmatic hernia.

PubMed

Grushka, Jeremy R; Al-Abbad, Saleh; Baird, Robert; Puligandla, Pramod; Kaplan, Feige; Laberge, Jean-Martin

2010-05-01

Fetal tracheal occlusion (TO) has been investigated as a treatment option for lung hypoplasia secondary to congenital diaphragmatic hernia. Tracheal occlusion has been shown to accelerate lung growth, but its effect on bronchial branching is unknown. In this study, we characterize the effects of in vitro TO on bronchial branch development in fetal lung explants derived from the nitrofen rat model of congenital diaphragmatic hernia. Rat dams were gavaged nitrofen on gestational day 9.5, and fetal lungs were harvested for explant culture on gestational day 14 (term, 22 days). Four experimental groups were investigated, with TO performed ex vivo using cautery: control, control + TO, nitrofen, and nitrofen + TO. Explants were incubated for 72 hours. Representative photographs were taken at 0, 24, 48, and 72 hours from the time of culture, and the number of distal branches was counted for each explant. The Student t test was used to compare distal branch measurements. A minimum of 12 fetal lung explants were cultured for each group. By 24 hours, all explants undergoing TO had more branch iterations than explants that did not. Moreover, TO in nitrofen-exposed explants increased bronchial branching to control levels by 24 hours in culture. Our results suggest that TO at day 14 increases branching in normal and nitrofen-exposed lung explants. In addition, TO increases airway branching in nitrofen-exposed explants to control levels suggesting that early TO reverses the lung hypoplasia seen in this model. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Leptin reduces apoptosis triggered by high temperature in human placental villous explants: The role of the p53 pathway.

PubMed

Pérez-Pérez, Antonio; Toro, Ayelén R; Vilarino-Garcia, Teresa; Guadix, Pilar; Maymó, Julieta L; Dueñas, José L; Varone, Cecilia L; Sánchez-Margalet, Víctor

2016-06-01

Maternal fever is common during pregnancy and has for many years been suspected to harm the developing fetus. Whether increased maternal temperature produces exaggerated apoptosis in trophoblast cells remains unclear. Since p53 is a critical regulator of apoptosis we hypothesized that increased temperature in placenta produces abnormal expression of proteins in the p53 pathway and finally caspase-3 activation. Moreover, leptin, produced by placenta, is known to promote the proliferation and survival of trophoblastic cells. Thus, we aimed to study the possible role of leptin preventing apoptosis triggered by high temperature, as well as the molecular mechanisms underlying this effect. Fresh placental tissue was collected from normal pregnancies. Explants of placental villi were exposed to 37 °C, 40 °C and 42 °C during 3 h in the presence or absence of 10 nM leptin in DMEM-F12 medium. Western blotting and qRT-PCR was performed to analyze the expression of p53 and downstream effector, P53AIP1, Mdm2, p21, BAX and BCL-2 as well as the activated cleaved form of caspase-3 and the fragment of cytokeratin-18 (CK-18) cleaved at Asp396 (neoepitope M30). Phosphorylation of the Ser 46 residue on p53, the expression of P53AIP1, Mdm2, p21, as well as caspase-3 and CK-18 were significantly increased in explants at 40 °C and 42 °C. Conversely, these effects were significantly attenuated by leptin 10 nM at both 40 °C and 42 °C. The BCL2/BAX ratio was also significantly decreased in explants at 40 °C and 42 °C compared with explants incubated at 37 °C, which was prevented by leptin stimulation. These data illustrate the potential role of leptin for reducing apoptosis in trophoblast explants, including trophoblastic cells, triggered by high temperature, by preventing the activation of p53 signaling. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cytotoxic sesquiterpene lactones from the leaves of Vernonia guineensis Benth. (Asteraceae)

PubMed Central

Toyang, Ngeh J.; Wabo, Hippolyte K.; Ateh, Eugene N.; Davis, Harry; Tane, Pierre; Sondengam, Luc B.; Bryant, Joseph; Verpoorte, Rob

2015-01-01

Ethnopharmacological relevance Vernonia guineensis Benth. (Asteraceae) preparations are used in folk medicine in Cameroon to treat a number of ailments, including prostate cancer and malaria, and is used as an anthelmintic, adaptogen and antidote. The aim of this study was to continue the validation of the activity of Vernonia guineensis Benth. extracts and isolated molecules against cancer cell lines following the previous isolation of an anti-prostate cancer sugar ester from the root extract. Materials and methods Acetone extracts of Vernonia guineensis Benth. leaves were tested for activity against 10 cancer cell lines (Breast—MDA-MB-231, Breast—MCF-7, Colon—HCT-116, Leukemia—HL-60, Lung—A549, Melanoma—A375, Ovarian—OVCAR3, Pancreas—Mia-paca, Prostate—PC-3 and Prostate—DU-145). The acetone extract was subjected to bioactivity guided fractionation. Anti-proliferation and clonogenic activity of the isolated compounds were tested. The WST-1 assay was used for the anti-proliferation activity, while the standard clonogenic test was used to determine the clonogenic activity. Results The acetone extract of Vernonia guineensis Benth. demonstrated in vitro activity ranging from IC50 4–26 mg/mL against the 10 cell lines. Activity guided fractionation of this extract yielded two sesquiterpene lactones, isolated for the first time from the genus Vernonia. The compounds were characterized using spectroscopic experiments, including a combination of 1D and 2D NMR data. Vernopicrin (1) and Vernomelitensin (2) demonstrated in vitro activity against human cancer cell lines with IC50 ranging from 0.35–2.04 μM (P < 0.05) and 0.13–1.5 μM (P < 0.05), respectively, between the most and least sensitive cell lines for each compound. Vernopicrin was most active against the human melanoma (A375) cell line and least active against the lung cancer (A549) cell line, while Vernomelitensin was also most active against the human melanoma (A375) cell line and least active against the breast cancer (MCF-7) cell line. Both compounds also demonstrated anticlonogenic activity. Conclusion The cytotoxicity demonstrated by the crude extract and isolated sesquiterpenes against cancer cell lines highlights the medicinal potential of V. guineensis. The selective anti-proliferation and dose dependent anticlonogenic activities suggest that the identified sesquiterpenes could be potential antitumor agents.. PMID:23376285

The novel human influenza A(H7N9) virus is naturally adapted to efficient growth in human lung tissue.

PubMed

Knepper, Jessica; Schierhorn, Kristina L; Becher, Anne; Budt, Matthias; Tönnies, Mario; Bauer, Torsten T; Schneider, Paul; Neudecker, Jens; Rückert, Jens C; Gruber, Achim D; Suttorp, Norbert; Schweiger, Brunhilde; Hippenstiel, Stefan; Hocke, Andreas C; Wolff, Thorsten

2013-10-08

A novel influenza A virus (IAV) of the H7N9 subtype has been isolated from severely diseased patients with pneumonia and acute respiratory distress syndrome and, apparently, from healthy poultry in March 2013 in Eastern China. We evaluated replication, tropism, and cytokine induction of the A/Anhui/1/2013 (H7N9) virus isolated from a fatal human infection and two low-pathogenic avian H7 subtype viruses in a human lung organ culture system mimicking infection of the lower respiratory tract. The A(H7N9) patient isolate replicated similarly well as a seasonal IAV in explanted human lung tissue, whereas avian H7 subtype viruses propagated poorly. Interestingly, the avian H7 strains provoked a strong antiviral type I interferon (IFN-I) response, whereas the A(H7N9) virus induced only low IFN levels. Nevertheless, all viruses analyzed were detected predominantly in type II pneumocytes, indicating that the A(H7N9) virus does not differ in its cellular tropism from other avian or human influenza viruses. Tissue culture-based studies suggested that the low induction of the IFN-β promoter correlated with an efficient suppression by the viral NS1 protein. These findings demonstrate that the zoonotic A(H7N9) virus is unusually well adapted to efficient propagation in human alveolar tissue, which most likely contributes to the severity of lower respiratory tract disease seen in many patients. Humans are usually not infected by avian influenza A viruses (IAV), but this large group of viruses contributes to the emergence of human pandemic strains. Transmission of virulent avian IAV to humans is therefore an alarming event that requires assessment of the biology as well as pathogenic and pandemic potentials of the viruses in clinically relevant models. Here, we demonstrate that an early virus isolate from the recent A(H7N9) outbreak in Eastern China replicated as efficiently as human-adapted IAV in explanted human lung tissue, whereas avian H7 subtype viruses were unable to propagate. Robust replication of the H7N9 strain correlated with a low induction of antiviral beta interferon (IFN-β), and cell-based studies indicated that this is due to efficient suppression of the IFN response by the viral NS1 protein. Thus, explanted human lung tissue appears to be a useful experimental model to explore the determinants facilitating cross-species transmission of the H7N9 virus to humans.

Lubricin is expressed in chondrocytes derived from osteoarthritic cartilage encapsulated in poly (ethylene glycol) diacrylate scaffold

PubMed Central

Musumeci, G.; Loreto, C.; Carnazza, M.L.; Coppolino, F.; Cardile, V.; Leonardi, R.

2011-01-01

Osteoarthritis (OA) is characterized by degenerative changes within joints that involved quantitative and/or qualitative alterations of cartilage and synovial fluid lubricin, a mucinous glycoprotein secreted by synovial fibroblasts and chondrocytes. Modern therapeutic methods, including tissue-engineering techniques, have been used to treat mechanical damage of the articular cartilage but to date there is no specific and effective treatment. This study aimed at investigating lubricin immunohistochemical expression in cartilage explant from normal and OA patients and in cartilage constructions formed by Poly (ethylene glycol) (PEG) based hydrogels (PEG-DA) encapsulated OA chondrocytes. The expression levels of lubricin were studied by immunohistochemistry: i) in tissue explanted from OA and normal human cartilage; ii) in chondrocytes encapsulated in hydrogel PEGDA from OA and normal human cartilage. Moreover, immunocytochemical and western blot analysis were performed in monolayer cells from OA and normal cartilage. The results showed an increased expression of lubricin in explanted tissue and in monolayer cells from normal cartilage, and a decreased expression of lubricin in OA cartilage. The chondrocytes from OA cartilage after 5 weeks of culture in hydrogels (PEGDA) showed an increased expression of lubricin compared with the control cartilage. The present study demonstrated that OA chondrocytes encapsulated in PEGDA, grown in the scaffold and were able to restore lubricin biosynthesis. Thus our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repairing cartilage lesions in patients with OA to reduce at least the progression of the disease. PMID:22073377

Extracellular matrix and α5β1 integrin signaling control the maintenance of bone formation capacity by human adipose-derived stromal cells

PubMed Central

Di Maggio, Nunzia; Martella, Elisa; Frismantiene, Agne; Resink, Therese J.; Schreiner, Simone; Lucarelli, Enrico; Jaquiery, Claude; Schaefer, Dirk J.; Martin, Ivan; Scherberich, Arnaud

2017-01-01

Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, adipose-derived stromal/stem cells (ASC), even after minimal monolayer expansion, display poor osteogenic capacity in vivo. We investigated whether ASC bone-forming capacity may be maintained by culture within a self-produced extracellular matrix (ECM) that recapitulates the native environment. SVF cells expanded without passaging up to 28 days (Unpass-ASC) deposited a fibronectin-rich extracellular matrix and displayed greater clonogenicity and differentiation potential in vitro compared to ASC expanded only for 6 days (P0-ASC) or for 28 days with regular passaging (Pass-ASC). When implanted subcutaneously, Unpass-ASC produced bone tissue similarly to SVF cells, in contrast to P0- and Pass-ASC, which mainly formed fibrous tissue. Interestingly, clonogenic progenitors from native SVF and Unpass-ASC expressed low levels of the fibronectin receptor α5 integrin (CD49e), which was instead upregulated in P0- and Pass-ASC. Mechanistically, induced activation of α5β1 integrin in Unpass-ASC led to a significant loss of bone formation in vivo. This study shows that ECM and regulation of α5β1-integrin signaling preserve ASC progenitor properties, including bone tissue-forming capacity, during in vitro expansion. PMID:28290502

Density-dependent induction of apoptosis by transforming growth factor-beta 1 in a human ovarian carcinoma cell line.

PubMed

Mathieu, C; Jozan, S; Mazars, P; Côme, M G; Moisand, A; Valette, A

1995-01-01

Transforming growth factor-beta 1 inhibited proliferation of a human ovarian carcinoma cell line (NIH-OVCAR-3). The inhibition of NIH-OVCAR-3 cell proliferation was accompanied by a decrease in clonogenic potential, evidenced by the reduced ability of TGF-beta 1-treated NIH-OVCAR-3 cells to form colonies on a plastic substratum. This rapid decrease of clonogenic potential, which was detected 6 h after addition of TGF-beta 1 was dose-dependent (IC50 = 4 pM). Fluorescence microscopy of DAPI-stained cells supported by electron-microscopic examination showed that TGF-beta 1 induced chromatin condensation and nuclear fragmentation. In addition, oligonucleosomal-sized fragments were detected in the TGF-beta 1-treated cells. These features indicated that TGF-beta 1 induced NIH-OVCAR-3 cell death by an apoptosis-like mechanism. This TGF-beta 1 apoptotic effect was subject to modulation by cell density. It was observed that an increase in cell density (up to 20 x 10(3) cells/cm2) protected NIH-OVCAR-3 cells against apoptosis induced by TGF-beta 1. Conditioned medium from high-density cultures of NIH-OVCAR-3 cells did not inhibit apoptosis induced by TGF-beta 1 on NIH-OVCAR-3 cells cultured at low density, suggesting that the protective effect of cell density was not related to the cell secretion of a soluble survival factor.

Antitumor activity of curcumin is involved in down-regulation of YAP/TAZ expression in pancreatic cancer cells.

PubMed

Zhou, Xiuxia; Su, Jingna; Feng, Shaoyan; Wang, Lixia; Yin, Xuyuan; Yan, Jingzhe; Wang, Zhiwei

2016-11-29

Pancreatic cancer (PC) is one of the most aggressive human malignancies worldwide and is the fourth leading cause of cancer-related deaths. Curcumin (diferuloylmethane) is a polyphenol derived from the Curcuma longa plant. Certain studies have demonstrated that curcumin exerts its anti-tumor function in a variety of human cancers including PC, via targeting multiple therapeutically important cancer signaling pathways. However, the detailed molecular mechanisms are not fully understood. Two transcriptional co-activators, YAP (Yes-associated protein) and its close paralog TAZ (transcriptional coactivator with PDZ-binding motif) exert oncogenic activities in various cancers. Therefore, in this study we aimed to determine the molecular basis of curcumin-induced cell proliferation inhibition in PC cells. First, we detected the anti-tumor effects of curcumin on PC cell lines using CTG assay, Flow cytometry, clonogenic assay, wound healing assay and Transwell invasion assay. We found that curcumin significantly suppressed cell growth, weakened clonogenic potential, inhibited migration and invasion, and induced apoptosis and cell cycle arrest in PC cells. We further measured that overexpression of YAP enhanced cell proliferation and abrogated the cytotoxic effects of curcumin on PC cells. Moreover, we found that curcumin markedly down-regulated YAP and TAZ expression and subsequently suppressed Notch-1 expression. Collectively, these findings suggest that pharmacological inhibition of YAP and TAZ activity may be a promising anticancer strategy for the treatment of PC patients.

Antitumor activity of curcumin is involved in down-regulation of YAP/TAZ expression in pancreatic cancer cells

PubMed Central

Wang, Lixia; Yin, Xuyuan; Yan, Jingzhe; Wang, Zhiwei

2016-01-01

Pancreatic cancer (PC) is one of the most aggressive human malignancies worldwide and is the fourth leading cause of cancer-related deaths. Curcumin (diferuloylmethane) is a polyphenol derived from the Curcuma longa plant. Certain studies have demonstrated that curcumin exerts its anti-tumor function in a variety of human cancers including PC, via targeting multiple therapeutically important cancer signaling pathways. However, the detailed molecular mechanisms are not fully understood. Two transcriptional co-activators, YAP (Yes-associated protein) and its close paralog TAZ (transcriptional coactivator with PDZ-binding motif) exert oncogenic activities in various cancers. Therefore, in this study we aimed to determine the molecular basis of curcumin-induced cell proliferation inhibition in PC cells. First, we detected the anti-tumor effects of curcumin on PC cell lines using CTG assay, Flow cytometry, clonogenic assay, wound healing assay and Transwell invasion assay. We found that curcumin significantly suppressed cell growth, weakened clonogenic potential, inhibited migration and invasion, and induced apoptosis and cell cycle arrest in PC cells. We further measured that overexpression of YAP enhanced cell proliferation and abrogated the cytotoxic effects of curcumin on PC cells. Moreover, we found that curcumin markedly down-regulated YAP and TAZ expression and subsequently suppressed Notch-1 expression. Collectively, these findings suggest that pharmacological inhibition of YAP and TAZ activity may be a promising anticancer strategy for the treatment of PC patients. PMID:27738325

Effect of radiation and other cytotoxic agents on the growth of cells cultured from normal and tumor tissues from the female genital tract

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

Mothersill, C.; Seymour, C.B.; Bonnar, J.

1990-05-01

A technique is presented which allows the response of human gynecological tissue to radiation and cytotoxic drugs to be assessed using a tissue culture explant system. The technique is simple to use and gives results in line with those obtained for human tissues by more complex culture methods. Data are presented showing how the explant technique developed by the group for other tissues can be adapted to yield acceptable results for normal tissue response to radiation. The potential of the technique for use in predictive testing of individual tumor response is then assessed in five cases of gynecological malignancy. Itmore » is clear that variations in sensitivity to different radio- and chemotherapy agents and combinations can be detected. The results obtained require clinical validation and it is hoped that this will come over the next few years from evaluation of patient response to treatment using individually optimized, rather than empirical therapy.« less

In vitro progesterone modulation on bacterial endotoxin-induced production of IL-1β, TNFα, IL-6, IL-8, IL-10, MIP-1α, and MMP-9 in pre-labor human term placenta.

PubMed

Garcia-Ruíz, G; Flores-Espinosa, P; Preciado-Martínez, E; Bermejo-Martínez, L; Espejel-Nuñez, A; Estrada-Gutierrez, G; Maida-Claros, R; Flores-Pliego, A; Zaga-Clavellina, Veronica

2015-10-07

During human pregnancy, infection/inflammation represents an important factor that increases the risk of developing preterm labor. The purpose of this study was to determine if pre-treatment with progesterone has an immunomodulatory effect on human placenta production of endotoxin-induced inflammation and degradation of extracellular matrix markers. Placentas were obtained under sterile conditions from pregnancies delivered at term before the onset of labor by cesarean section. Explants from central cotyledons of 10 human placentas were pre-treated with different concentrations of progesterone (0.01, 01, 1.0 μM) and then stimulated with 1000 ng/mL of LPS of Escherichia coli. Cytokines TNFα, IL-1β, IL-6, IL-8, MIP-1α, IL-10 concentrations in the culture medium were then measured by specific ELISA. Secretion profile of MMP-9 was evaluated by ELISA and zymogram. Statistical differences were determined by one-way ANOVA followed by the appropriate ad hoc test; P < 0.05 was considered statistically significant. In comparison to the explants incubated with vehicle, the LPS treatment led to a significant increase in the level of all cytokines. In comparison to the explants treated only with LPS, pre-treatment with 0.01-1.0 μM progesterone significantly blunted (73, 56, 56, 75, 25, 48 %) the secretion of TNF-α, IL-1β, IL-6, IL-8, MIP-1α, IL-10, respectively. The MMP-9 induced by LPS treatment was inhibited only with the highest concentration of progesterone. Mifepristone (RU486) blocked the immunosuppressive effect of progesterone. The present results support the concept that progesterone could be part of the compensatory mechanism that limits the inflammation-induced cytotoxic effects associated with an infection process during gestation.

Effects of osteopontin inhibition on radiosensitivity of MDA-MB-231 breast cancer cells.

PubMed

Hahnel, Antje; Wichmann, Henri; Kappler, Matthias; Kotzsch, Matthias; Vordermark, Dirk; Taubert, Helge; Bache, Matthias

2010-09-17

Osteopontin (OPN) is a secreted glycophosphoprotein that is overexpressed in various tumors, and high levels of OPN have been associated with poor prognosis of cancer patients. In patients with head and neck cancer, high OPN plasma levels have been associated with poor prognosis following radiotherapy. Since little is known about the relationship between OPN expression and radiosensitivity, we investigated the cellular and radiation induced effects of OPN siRNA in human MDA-MB-231 breast cancer cells. MDA-MB-231 cells were transfected with OPN-specific siRNAs and irradiated after 24 h. To verify the OPN knockdown, we measured the OPN mRNA and protein levels using qRT-PCR and Western blot analysis. Furthermore, the functional effects of OPN siRNAs were studied by assays to assess clonogenic survival, migration and induction of apoptosis. Treatment of MDA-MB-231 cells with OPN siRNAs resulted in an 80% decrease in the OPN mRNA level and in a decrease in extracellular OPN protein level. Transfection reduced clonogenic survival to 42% (p = 0.008), decreased the migration rate to 60% (p = 0.15) and increased apoptosis from 0.3% to 1.7% (p = 0.04). Combination of OPN siRNA and irradiation at 2 Gy resulted in a further reduction of clonogenic survival to 27% (p < 0.001), decreased the migration rate to 40% (p = 0.03) and increased apoptosis to 4% (p < 0.005). Furthermore, OPN knockdown caused a weak radiosensitization with an enhancement factor of 1.5 at 6 Gy (p = 0.09) and a dose modifying factor (DMF10) of 1.1. Our results suggest that an OPN knockdown improves radiobiological effects in MDA-MB-231 cells. Therefore, OPN seems to be an attractive target to improve the effectiveness of radiotherapy.

The importance of serum serotonin levels in the measurement of radiation-induced bystander cell death in HaCaT cells.

PubMed

Lyng, Fiona M; Desplanques, Maxime; Jella, Kishore Kumar; Garcia, Amaya; McClean, Brendan

2012-10-01

The aim of this study was to investigate the importance of serum serotonin levels in the measurement of bystander cell death. The study was undertaken as part of an intercomparison exercise involving seven European laboratories funded under the European Union Sixth Framework Programme (FP6) Non-Targeted Effects (NOTE) integrated project. Three batches of foetal bovine serum were tested; serum with high and low serotonin content from the intercomparison exercise as well as serum from the home laboratory. Three sets of human keratinocytes (HaCaT cell line) were cultured in DMEM:F12 medium supplemented with serum with high or low serotonin content or serum from the home laboratory and both donor and recipient HaCaT cells were plated. The donor HaCaT cells were irradiated (0.5 Gy) using a cobalt 60 teletherapy unit, the medium was harvested 1 hour post irradiation and transferred to the recipient HaCaT cells. Bystander induced cell death was measured by the clonogenic survival assay and the Alamar blue viability assay. A significant reduction in cell survival, as measured by the clonogenic assay, and in cell viability, as measured by the Alamar blue assay, was observed in the recipient HaCaT cells treated with medium from irradiated cells compared to the cells treated with medium from unirradiated cells. No significant difference was found between the three batches of serum. The data suggest that in our cell system and with our endpoints (clonogenic assay and Alamar blue assay), serum serotonin levels do not play a role in bystander-induced cell death.

Transient elevation of glycolysis confers radio-resistance by facilitating DNA repair in cells.

PubMed

Bhatt, Anant Narayan; Chauhan, Ankit; Khanna, Suchit; Rai, Yogesh; Singh, Saurabh; Soni, Ravi; Kalra, Namita; Dwarakanath, Bilikere S

2015-05-01

Cancer cells exhibit increased glycolysis for ATP production (the Warburg effect) and macromolecular biosynthesis; it is also linked with therapeutic resistance that is generally associated with compromised respiratory metabolism. Molecular mechanisms underlying radio-resistance linked to elevated glycolysis remain incompletely understood. We stimulated glycolysis using mitochondrial respiratory modifiers (MRMs viz. di-nitro phenol, DNP; Photosan-3, PS3; Methylene blue, MB) in established human cell lines (HEK293, BMG-1 and OCT-1). Glucose utilization and lactate production, levels of glucose transporters and glycolytic enzymes were investigated as indices of glycolysis. Clonogenic survival, DNA repair and cytogenetic damage were studied as parameters of radiation response. MRMs induced the glycolysis by enhancing the levels of two important regulators of glucose metabolism GLUT-1 and HK-II and resulted in 2 fold increase in glucose consumption and lactate production. This increase in glycolysis resulted in resistance against radiation-induced cell death (clonogenic survival) in different cell lines at an absorbed dose of 5 Gy. Inhibition of glucose uptake and glycolysis (using fasentin, 2-deoxy-D-glucose and 3-bromopyruvate) in DNP treated cells failed to increase the clonogenic survival of irradiated cells, suggesting that radio-resistance linked to inhibition of mitochondrial respiration is glycolysis dependent. Elevated glycolysis also facilitated rejoining of radiation-induced DNA strand breaks by activating both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways of DNA double strand break repair leading to a reduction in radiation-induced cytogenetic damage (micronuclei formation) in these cells. These findings suggest that enhanced glycolysis generally observed in cancer cells may be responsible for the radio-resistance, partly by enhancing the repair of DNA damage.

The vascular disrupting agent ZD6126 shows increased antitumor efficacy and enhanced radiation response in large, advanced tumors

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

Siemann, Dietmar W.; Rojiani, Amyn M.

2005-07-01

Purpose: ZD6126 is a vascular-targeting agent that induces selective effects on the morphology of proliferating and immature endothelial cells by disrupting the tubulin cytoskeleton. The efficacy of ZD6126 was investigated in large vs. small tumors in a variety of animal models. Methods and Materials: Three rodent tumor models (KHT, SCCVII, RIF-1) and three human tumor xenografts (Caki-1, KSY-1, SKBR3) were used. Mice bearing leg tumors ranging in size from 0.1-2.0 g were injected intraperitoneally with a single 150 mg/kg dose of ZD6126. The response was assessed by morphologic and morphometric means as well as an in vivo to in vitromore » clonogenic cell survival assay. To examine the impact of tumor size on the extent of enhancement of radiation efficacy by ZD6126, KHT sarcomas of three different sizes were irradiated locally with a range of radiation doses, and cell survival was determined. Results: All rodent tumors and human tumor xenografts evaluated showed a strong correlation between increasing tumor size and treatment effect as determined by clonogenic cell survival. Detailed evaluation of KHT sarcomas treated with ZD6126 showed a reduction in patent tumor blood vessels that was {approx}20% in small (<0.3 g) vs. >90% in large (>1.0 g) tumors. Histologic assessment revealed that the extent of tumor necrosis after ZD6126 treatment, although minimal in small KHT sarcomas, became more extensive with increasing tumor size. Clonogenic cell survival after ZD6126 exposure showed a decrease in tumor surviving fraction from approximately 3 x 10{sup -1} to 1 x 10{sup -4} with increasing tumor size. When combined with radiotherapy, ZD6126 treatment resulted in little enhancement of the antitumor effect of radiation in small (<0.3 g) tumors but marked increases in cell kill in tumors larger than 1.0 g. Conclusions: Because bulky neoplastic disease is typically the most difficult to manage, the present findings provide further support for the continued development of vascular disrupting agents such as ZD6126 as a vascular-targeted approach to cancer therapy.« less

Effect of environmental and cultural conditions on medium pH and explant growth performance of Douglas-fir ( Pseudotsuga menziesii) shoot cultures

PubMed Central

Chen, Chien-Chih; Bates, Rick; Carlson, John

2015-01-01

The medium pH level of plant tissue cultures has been shown to be essential to many aspects of explant development and growth. Sensitivity or tolerance of medium pH change in vitro varies according to specific requirements of individual species. The objectives of this study are to 1) determine medium pH change over time in storage conditions and with presence of explants, 2) evaluate the effects of medium pH change on explant growth performance and 3) assess the effects of adding a pH stabilizer, 2-(N-morpholino)ethanesulfonic acid (MES) that is commonly used in Douglas-fir micropropagation medium. Vegetative buds were collected in the spring before breaking dormancy from juvenile and mature donor trees for conducting these evaluations. Medium, with or without MES, was pre-adjusted to five pH levels before adding MES, agar and autoclaving. Medium pH changes and explant growth parameters were measured at eight different incubation times. Overall, MES provided a more stable medium pH, relative to starting pH values, under both light and dark storage conditions as well as with presence of explants. A general trend of decreasing medium pH over time was found comparing explants from juvenile and mature donor genotypes. Explant height and weight growth increased over time, but differ among explants from juvenile and mature donor genotypes. Our findings suggest that a 21-day subculture practice may best sustain medium freshness, medium pH level and desirable explant growth. PMID:26535110

Radiologic Differences between Bone Marrow Stromal and Hematopoietic Progenitor Cell Lines from Fanconi Anemia (Fancd2−/−) Mice

PubMed Central

Berhane, Hebist; Epperly, Michael W.; Goff, Julie; Kalash, Ronny; Cao, Shaonan; Franicola, Darcy; Zhang, Xichen; Shields, Donna; Houghton, Frank; Wang, Hong; Wipf, Peter; Parmar, Kalindi; Greenberger, Joel S.

2014-01-01

FancD2 plays a central role in the human Fanconi anemia DNA damage response (DDR) pathway. Fancd2−/− mice exhibit many features of human Fanconi anemia including cellular DNA repair defects. Whether the DNA repair defect in Fancd2−/− mice results in radiologic changes in all cell lineages is unknown. We measured stress of hematopoiesis in long-term marrow cultures and radiosensitivity in clonogenic survival curves, as well as comet tail intensity, total antioxidant stores and radiation-induced gene expression in hematopoietic progenitor compared to bone marrow stromal cell lines. We further evaluated radioprotection by a mitochondrial-targeted antioxidant GS-nitroxide, JP4-039. Hematopoiesis longevity in Fancd2−/− mouse long-term marrow cultures was diminished and bone marrow stromal cell lines were radiosensitive compared to Fancd2+/+ stromal cells (Fancd2−/− D0 = 1.4 ± 0.1 Gy, ñ = 5.0 ± 0.6 vs. Fancd2+/+ D0 = 1.6 ± 0.1 Gy, ñ = 6.7 ± 1.6), P = 0.0124 for D0 and P = 0.0023 for ñ, respectively). In contrast, Fancd2−/− IL-3-dependent hematopoietic progenitor cells were radioresistant (D0 = 1.71 ± 0.04 Gy and ñ = 5.07 ± 0.52) compared to Fancd2+/+ (D0 = 1.39 ± 0.09 Gy and ñ = 2.31 ± 0.85, P = 0.001 for D0). CFU-GM from freshly explanted Fancd2−/− marrow was also radioresistant. Consistent with radiosensitivity, irradiated Fancd2−/− stromal cells had higher DNA damage by comet tail intensity assay compared to Fancd2+/+ cells (P < 0.0001), slower DNA damage recovery, lower baseline total antioxidant capacity, enhanced radiation-induced depletion of antioxidants, and increased CDKN1A-p21 gene transcripts and protein. Consistent with radioresistance, Fancd2−/− IL-3-dependent hematopoietic cells had higher baseline and post irradiation total antioxidant capacity. While, there was no detectable alteration of radiation-induced cell cycle arrest with Fancd2−/− stromal cells, hematopoietic progenitor cells showed reduced G2/M cell cycle arrest. The absence of the mouse Fancd2 gene product confers radiosensitivity to bone marrow stromal but not hematopoietic progenitor cells. PMID:24397476

Optic nerve compression as a late complication of a hydrogel explant with silicone encircling band.

PubMed

Crama, Niels; Kluijtmans, Leo; Klevering, B Jeroen

2018-06-01

To present a complication of compressive optic neuropathy caused by a swollen hydrogel explant and posteriorly displaced silicone encircling band. A 72-year-old female patient presented with progressive visual loss and a tilted optic disc. Her medical history included a retinal detachment in 1993 that was treated with a hydrogel explant under a solid silicone encircling band. Visual acuity had decreased from 6/10 to 6/20 and perimetry showed a scotoma in the temporal superior quadrant. On Magnetic Resonance Imaging (MRI), compression of the optic nerve by a displaced silicone encircling band inferior nasally in combination with a swollen episcleral hydrogel explant was observed. Surgical removal of the hydrogel explant and silicone encircling band was uneventful and resulted in improvement of visual acuity and visual field loss. This is the first report on compressive optic neuropathy caused by swelling of a hydrogel explant resulting in a dislocated silicone encircling band. The loss of visual function resolved upon removal of the explant and encircling band.

Peritumoral adipose tissue as a source of inflammatory and angiogenic factors in colorectal cancer.

PubMed

Amor, S; Iglesias-de la Cruz, M C; Ferrero, E; García-Villar, O; Barrios, V; Fernandez, N; Monge, L; García-Villalón, A L; Granado, M

2016-02-01

Obesity is a risk factor for the development of human colorectal cancer (CC). The aim of this work is to report the inflammatory and angiogenic scenario in lean (BMI < 25 kg/m2) and obese (BMI > 30 kg/m2) patients with and without CC and to assess the role of peritumoral adipose tissue in CC-induced inflammation. Patients were divided in four experimental groups: obese patients with CC (OB-CC), lean patients with CC (LEAN-CC), obese patients without CC (OB), and lean patients without CC (LEAN). Plasma levels of pro-inflammatory cytokines (interleukin (IL)-6, IL-4, IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in OB-CC patients. Peritumoral adipose tissue (TF) explants and cultured mature adipocytes secreted higher amounts of nitrites and nitrates than did control and non-tumoral (NTF) adipose tissue both alone and in response to lipopolysaccharide (LPS). Nitrite and nitrate secretion was also increased in TF explants from OB-CC patients compared with that from LEAN-CC patients. Gene expression of adiponectin, tumor necrosis factor alpha (TNF-α), insulin-like growth factor type I (IGF-I), cyclooxygenase-2 (COX-2), and peroxisome proliferator-activated receptor γ (PPAR-γ) was increased in TF explants from CC patients. LPS increased the gene expression of IL-6, IL-10, TNF-α, vascular endothelial growth factor (VEGF), and COX-2 in OB and in TF explants from OB-CC patients. COX-2 and PPAR-γ inhibition further increased LPS-induced release of nitrites and nitrates in TF explants and adipocytes from OB-CC patients. In conclusion, OB-CC patients have increased plasma levels of pro-inflammatory and angiogenic factors. TF from OB-CC patients shows an increased secretion of inflammatory markers compared with both TF from LEAN-CC and non-tumoral adipose tissue (AT) through a COX-2- and PPAR-γ-independent mechanism.

Effect of passage number on electrophoretic mobility distributions of cultured human embryonic kidney cells

NASA Technical Reports Server (NTRS)

Kunze, M. E.

1985-01-01

A systematic investigation was undertaken to characterize population shifts that occur in cultured human embryonic kidney cells as a function of passage number in vitro after original explantation. This approach to cell population shift analysis follows the suggestion of Mehreshi, Klein and Revesz that perturbed cell populations can be characterized by electrophoretic mobility distributions if they contain subpopulations with different electrophoretic mobilities. It was shown that this is the case with early passage cultured human embryo cells.

The xenoestrogens, bisphenol A and para-nonylphenol, decrease the expression of the ABCG2 transporter protein in human term placental explant cultures.

PubMed

Sieppi, E; Vähäkangas, K; Rautio, A; Ietta, F; Paulesu, L; Myllynen, P

2016-07-05

Many endogenous and xenobiotic compounds are substrates and regulators of human placental ABC transporters. ABCG2 is protecting fetus against foreign chemicals. Environmental xenoestrogens, like bisphenol A (BPA) and p-nonylphenol (p-NP), mimic natural estrogens and can affect hormonal systems. Effects of BPA, p-NP, DES (diethylstilbestrol) and estradiol (E2), on ABCG2 expression were studied using human first trimester and term placental explants. Role of estrogen receptors (ER) in the effects of chemicals was studied by ER antagonist. Term placenta expressed less ABCG2 protein. In term placentas BPA (p < 0.05), p-NP (p < 0.01) and E2 (p < 0.05) decreased the ABCG2 protein expression after 48 h exposure while after 24 h exposure, only E2 decreased the expression (p < 0.05). The chemicals did not affect ABCG2 in first trimester placentas. The ER antagonist affected differently the responses of chemicals. In conclusion, environmental xenoestrogens downregulate placental ABCG2 protein expression depending on gestational age. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Role of microRNA in Aggressive Prostate Cancer

DTIC Science & Technology

2015-09-01

j.issn.2223-4683.2013.08.01 Scan to your mobile device or view this article at: http://www.amepc.org/tau/article/view/2550/3635 229Translational Andrology...of Ezh2, accompanied with diminished clonogenic ability and sphere formation in PCa cells (45). Another let-7 target gene is High- mobility group AT...2013;32:4139-47. 89. Motoyama K, Inoue H, Nakamura Y, et al. Clinical significance of high mobility group A2 in human gastric cancer and its

Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines.

PubMed

Singh, Mahipal; Sharma, Anil K

2011-02-01

Three different commercially available media, known to support human and porcine-specific fibroblast cultures, were tested for their growth potential on goat skin explants. Although outgrowth of fibroblasts was observed in all media tested, irrespective of breed, porcine-specific media exhibited higher rate of growth. Using this media, three fibroblast cell lines (GSF289, GSF737, and GSF2010) from ear skin explants of normal healthy dairy goats of Kiko and Saanen breed were successfully established in culture. Liquid nitrogen stocks of these frozen cells had a viability rate of 96.2% in in vitro cultures. These cells were morphologically indistinguishable from the cell stocks prior to freezing. Analysis of the growth of a fifth passage culture revealed an 'S' shaped growth curve with a population doubling time of 25 h. The cell lines were found negative for microbial, fungal, and mycoplasma contaminations. These goat skin fibroblast lines and the simple method of their isolation and freezing with high rate of viability will provide additional tools to study molecular mechanisms that regulate fibroblast function and for genetic manipulation of small ruminants.

Reverse Transcriptase Inhibitors as Potential Colorectal Microbicides▿ †

PubMed Central

Herrera, Carolina; Cranage, Martin; McGowan, Ian; Anton, Peter; Shattock, Robin J.

2009-01-01

We investigated whether reverse transcriptase (RT) inhibitors (RTI) can be combined to inhibit human immunodeficiency virus type 1 (HIV-1) infection of colorectal tissue ex vivo as part of a strategy to develop an effective rectal microbicide. The nucleotide RTI (NRTI) PMPA (tenofovir) and two nonnucleoside RTI (NNRTI), UC-781 and TMC120 (dapivirine), were evaluated. Each compound inhibited the replication of the HIV isolates tested in TZM-bl cells, peripheral blood mononuclear cells, and colorectal explants. Dual combinations of the three compounds, either NRTI-NNRTI or NNRTI-NNRTI combinations, were more active than any of the individual compounds in both cellular and tissue models. Combinations were key to inhibiting infection by NRTI- and NNRTI-resistant isolates in all models tested. Moreover, we found that the replication capacities of HIV-1 isolates in colorectal explants were affected by single point mutations in RT that confer resistance to RTI. These data demonstrate that colorectal explants can be used to screen compounds for potential efficacy as part of a combination microbicide and to determine the mucosal fitness of RTI-resistant isolates. These findings may have important implications for the rational design of effective rectal microbicides. PMID:19258271

Reverse transcriptase inhibitors as potential colorectal microbicides.

PubMed

Herrera, Carolina; Cranage, Martin; McGowan, Ian; Anton, Peter; Shattock, Robin J

2009-05-01

We investigated whether reverse transcriptase (RT) inhibitors (RTI) can be combined to inhibit human immunodeficiency virus type 1 (HIV-1) infection of colorectal tissue ex vivo as part of a strategy to develop an effective rectal microbicide. The nucleotide RTI (NRTI) PMPA (tenofovir) and two nonnucleoside RTI (NNRTI), UC-781 and TMC120 (dapivirine), were evaluated. Each compound inhibited the replication of the HIV isolates tested in TZM-bl cells, peripheral blood mononuclear cells, and colorectal explants. Dual combinations of the three compounds, either NRTI-NNRTI or NNRTI-NNRTI combinations, were more active than any of the individual compounds in both cellular and tissue models. Combinations were key to inhibiting infection by NRTI- and NNRTI-resistant isolates in all models tested. Moreover, we found that the replication capacities of HIV-1 isolates in colorectal explants were affected by single point mutations in RT that confer resistance to RTI. These data demonstrate that colorectal explants can be used to screen compounds for potential efficacy as part of a combination microbicide and to determine the mucosal fitness of RTI-resistant isolates. These findings may have important implications for the rational design of effective rectal microbicides.

Genome-wide transcriptome and expression profile analysis of Phalaenopsis during explant browning.

PubMed

Xu, Chuanjun; Zeng, Biyu; Huang, Junmei; Huang, Wen; Liu, Yumei

2015-01-01

Explant browning presents a major problem for in vitro culture, and can lead to the death of the explant and failure of regeneration. Considerable work has examined the physiological mechanisms underlying Phalaenopsis leaf explant browning, but the molecular mechanisms of browning remain elusive. In this study, we used whole genome RNA sequencing to examine Phalaenopsis leaf explant browning at genome-wide level. We first used Illumina high-throughput technology to sequence the transcriptome of Phalaenopsis and then performed de novo transcriptome assembly. We assembled 79,434,350 clean reads into 31,708 isogenes and generated 26,565 annotated unigenes. We assigned Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, and potential Pfam domains to each transcript. Using the transcriptome data as a reference, we next analyzed the differential gene expression of explants cultured for 0, 3, and 6 d, respectively. We then identified differentially expressed genes (DEGs) before and after Phalaenopsis explant browning. We also performed GO, KEGG functional enrichment and Pfam analysis of all DEGs. Finally, we selected 11 genes for quantitative real-time PCR (qPCR) analysis to confirm the expression profile analysis. Here, we report the first comprehensive analysis of transcriptome and expression profiles during Phalaenopsis explant browning. Our results suggest that Phalaenopsis explant browning may be due in part to gene expression changes that affect the secondary metabolism, such as: phenylpropanoid pathway and flavonoid biosynthesis. Genes involved in photosynthesis and ATPase activity have been found to be changed at transcription level; these changes may perturb energy metabolism and thus lead to the decay of plant cells and tissues. This study provides comprehensive gene expression data for Phalaenopsis browning. Our data constitute an important resource for further functional studies to prevent explant browning.

Uneven drying of zygotic embryos and embryonic axes of recalcitrant seeds: challenges and considerations for cryopreservation.

PubMed

Ballesteros, Daniel; Sershen; Varghese, Boby; Berjak, Patricia; Pammenter, Norman W

2014-08-01

Cryopreservation is the most promising option for the long-term germplasm conservation of recalcitrant-seeded species. However, the variable post-cryo success achieved with the excised zygotic explants traditionally used for cryopreservation has been a concern for some time. Differential drying rates amongst explants of different species, uneven drying amongst explants within a batch of seeds and uneven drying across tissues within individual embryos could be contributory factors to this variable success and these phenomena form the foci of the present study. Using zygotic explants from a range of recalcitrant-seeded species, which included sub-tropical dicotyledonous trees and sub-tropical monocotyledonous geophytes, the study showed that embryo morphology and anatomy are critical determinants of the drying characteristics of the different tissues composing the explant and hence, post-cryo survival. The results suggest that the rates of drying of explants to water contents (WCs) in the theoretically optimal range for successful cryopreservation are species-specific, and that more rapid drying rates may promote post-cryo survival. However, the large variation in WC amongst individual explants in bulk samples challenges the selection of the theoretically optimum WC for cryopreservation. As a consequence of differential drying rates across the different tissues composing explants, either lethal ice crystal damage or desiccation damage may sometimes be likely in tissues responsible for the onwards development of the embryo. Drying times for cryopreservation of such explants should, therefore, be selected on the basis of WC of segments containing root or shoot meristem, rather than embryo bulk WC. Drying intensity and duration also interact with explant morphology and embryo/axis size and anatomy to bring about - or preclude - post-cryo survival. Copyright © 2014 Elsevier Inc. All rights reserved.

Fibrin glue inhibits migration of ocular surface epithelial cells

PubMed Central

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

2016-01-01

Purpose Fibrin 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. Methods Corneoscleral 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. Results Explants 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. Conclusions Fibrin 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. PMID:27367746

Genome-Wide Transcriptome and Expression Profile Analysis of Phalaenopsis during Explant Browning

PubMed Central

Xu, Chuanjun; Zeng, Biyu; Huang, Junmei; Huang, Wen; Liu, Yumei

2015-01-01

Background Explant browning presents a major problem for in vitro culture, and can lead to the death of the explant and failure of regeneration. Considerable work has examined the physiological mechanisms underlying Phalaenopsis leaf explant browning, but the molecular mechanisms of browning remain elusive. In this study, we used whole genome RNA sequencing to examine Phalaenopsis leaf explant browning at genome-wide level. Methodology/Principal Findings We first used Illumina high-throughput technology to sequence the transcriptome of Phalaenopsis and then performed de novo transcriptome assembly. We assembled 79,434,350 clean reads into 31,708 isogenes and generated 26,565 annotated unigenes. We assigned Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, and potential Pfam domains to each transcript. Using the transcriptome data as a reference, we next analyzed the differential gene expression of explants cultured for 0, 3, and 6 d, respectively. We then identified differentially expressed genes (DEGs) before and after Phalaenopsis explant browning. We also performed GO, KEGG functional enrichment and Pfam analysis of all DEGs. Finally, we selected 11 genes for quantitative real-time PCR (qPCR) analysis to confirm the expression profile analysis. Conclusions/Significance Here, we report the first comprehensive analysis of transcriptome and expression profiles during Phalaenopsis explant browning. Our results suggest that Phalaenopsis explant browning may be due in part to gene expression changes that affect the secondary metabolism, such as: phenylpropanoid pathway and flavonoid biosynthesis. Genes involved in photosynthesis and ATPase activity have been found to be changed at transcription level; these changes may perturb energy metabolism and thus lead to the decay of plant cells and tissues. This study provides comprehensive gene expression data for Phalaenopsis browning. Our data constitute an important resource for further functional studies to prevent explant browning. PMID:25874455

Induction of bulb organogenesis in in vitro cultures of tarda tulip (Tulipa tarda Stapf.) from seed-derived explants.

PubMed

Maślanka, Małgorzata; Bach, Anna

2014-01-01

A protocol for obtaining bulbs via in vitro organogenesis was developed for tarda tulip ( Tulipa tarda Stapf). Scale explants were obtained from bulbs formed at the base of seedlings or from adventitious bulbs that developed from callus tissue forming on stolons or on germinating seeds. Some explants were subjected to chilling at 5°C for 12 wk. The culture media contained 3 or 6% sucrose and was supplemented with either no growth regulators, either 0.5 μM 6-benzyl-aminopurine (BAP) or 18.9 or 94.6 μM abscisic acid (ABA). Cultures were maintained in the dark at 20°C. Callus tissue developed mainly on media without growth regulators or with BAP. Callus was formed from up to 96% of explants derived from non-chilled adventitious bulbs that were treated with 3% sucrose and 0.5 μM BAP. Less callus was formed from chilled explants compared with non-chilled explants. Newly formed adventitious bulbs appeared on the explants via direct and indirect organogenesis. The media with BAP promoted the formation of adventitious bulbs at a rate of 56-92% from non-chilled explants, whereas a maximum rate of 36% was observed from chilled explants. ABA inhibited the induction of adventitious bulbs and callus. The adventitious bulbs obtained in these experiments contained a meristem, which was evidence that they had developed properly.

Load-bearing capacity and biological allowable limit of biodegradable metal based on degradation rate in vivo.

PubMed

Cho, Sung Youn; Chae, Soo-Won; Choi, Kui Won; Seok, Hyun Kwang; Han, Hyung Seop; Yang, Seok Jo; Kim, Young Yul; Kim, Jong Tac; Jung, Jae Young; Assad, Michel

2012-08-01

In this study, a newly developed Mg-Ca-Zn alloy for low degradation rate and surface erosion properties was evaluated. The compressive, tensile, and fatigue strength were measured before implantation. The degradation behavior was evaluated by analyzing the microstructure and local hardness of the explanted specimen. Mean and maximum degradation rates were measured using micro CT equipment from 4-, 8-, and 16- week explants, and the alloy was shown to display surface erosion properties. Based on these characteristics, the average and minimum load bearing capacities in tension, compression, and bending modes were calculated. According to the degradation rate and references of recommended dietary intakes (RDI), the Mg-Ca-Zn alloy appears to be safe for human use. Copyright © 2012 Wiley Periodicals, Inc.

Does the Implantation Technique for Totally Implantable Venous Access Ports (TIVAPs) Influence Long-Term Outcome?

PubMed

Biacchi, Daniele; Sammartino, Paolo; Sibio, Simone; Accarpio, Fabio; Cardi, Maurizio; Sapienza, Paolo; De Cesare, Alessandro; Atta, Joseph Maher Fouad; Impagnatiello, Alessio; Di Giorgio, Angelo

2016-02-01

Totally implantable venous access ports (TIVAP) are eventually explanted for various reasons, related or unrelated to the implantation technique used. Having more information on long-term explantation would help improve placement techniques. From a series of 1572 cancer patients who had TIVAPs implanted in our center with the cutdown technique or Seldinger technique, we studied the 542 patients who returned to us to have their TIVAP explanted after 70 days or more. As outcome measures we distinguished between TIVAPs explanted for long-term complications (infection, catheter-, reservoir-, and patient-related complications) and TIVAPs no longer needed. Univariate and multivariate analyses were run to investigate the reasons for explantation and their possible correlation with implantation techniques. The most common reason for explantation was infection (47.6 %), followed by catheter-related (20.8 %), patient-related (14.7 %), and reservoir-related complications (4.7 %). In the remaining 12.2 % of cases, the TIVAP was explanted complication free after the planned treatments ended. Infection correlated closely with longer TIVAP use. Univariate and multivariate analyses identified the Seldinger technique as a major risk factor for venous thrombosis and catheter dislocation. The need for long-term TIVAP explantation in about one-third of cancer patients is related to the implantation techniques used.

Sweet Potato [Ipomoea batatas (L.) Lam].

PubMed

Song, Guo-qing; Yamaguchi, Ken-ichi

2006-01-01

Among the available transformation methods reported on sweet potato, Agrobacterium tumefaciens-mediated transformation is more successful and desirable. Stem explants have shown to be ideal for the transformation of sweet potato because of their ready availability as explants, the simple transformation process, and high-frequency-regeneration via somatic embryogenesis. Under the two-step kanamycin-hygromycin selection method and using the appropriate explants type (stem explants), the efficiency of transformation can be considerably improved in cv. Beniazuma. The high efficiency in the transformation of stem explants suggests that the transformation protocol described in this chapter warrants testing for routine stable transformation of diverse varieties of sweet potato.

Effects of laser irradiation on immature olfactory neuroepithelial explants from the rat

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

Mester, A.F.; Snow, J.B. Jr.

1988-07-01

The photobiological effect of low-output laser irradiation on the maturation and regeneration of immature olfactory bipolar receptor cells of the rat was studied. The maturation and regeneration of the receptor cells of rat fetuses were quantified in neuroepithelial explants with morphometric analysis. The number of explants with outgrowth and the number and length of neuritic outgrowths were determined on a regular basis for 12 days. Explants in the experimental group were irradiated with a helium-neon laser using different incident energy densities (IED). Explants in the fluorescent light control group were exposed to fluorescent light for the same periods of timemore » as those in the experimental group were exposed to laser irradiation. Explants in another control group were not exposed to laser or fluorescent light irradiation. The IED of 0.5 J/cm2 laser irradiation has been found to increase significantly the number of explants with outgrowth and the number and length of the outgrowths. Other laser IEDs or fluorescent light irradiation did not influence maturation or regeneration.« less

Collaborative Model for Acceleration of Individualized Therapy of Colon Cancer

DTIC Science & Technology

2012-10-01

human CRC explants will be assessed (in our CLIA-certified UCCC Pathology Core) using the DxS Scorpion method (DxS, Manchester, UK) according to the...for those treatments. Unfortunately the lack of such strategies is what led to thousands of CRC patients with KRAS mutations being treated with...KRAS mutant colorectal cancer (CRC) using a comprehensive bioinformatics approach and novel preclinical models of human CRC. This proposal has the

Brugia malayi infective larvae fail to activate Langerhans cells and dermal dendritic cells in human skin.

PubMed

Cotton, R N; McDonald-Fleming, R; Boyd, A; Spates, K; Nutman, T B; Tolouei Semnani, R

2015-02-01

Filarial infection in humans is initiated when a mosquito deposits third-stage parasite larvae (L3) in the skin. Langerhans cells (LCs) and dermal dendritic cells (DDCs) are the first cells that the parasite encounters, and L3s must evade these highly effective antigen-presenting cells to establish infection. To assess LC and DDC responses to L3 in human skin, we employed three models of increasing physiologic relevance: in vitro-generated LCs, epidermal blister explants and full-thickness human skin sections. In vitro-generated LCs expressed TLR1-10 and robustly produced IL-6 and TNF-α in response to PolyI:C, but pre-exposure to L3s did not alter inflammatory cytokine production or TLR expression. L3s did not modulate expression of LC markers CDH1, CD207, or CD1a, or the regulatory products TSLP or IDO in epidermal explants or in vitro-generated LC. LC, CD14+ DDC, CD1c+ DC and CD141+ DC from human skin sections were analysed by flow cytometry. While PolyI:C potently induced CCL22 production in LC, CD1c+ DC, and CD141+ DC, and IL-10 production in LC, L3s did not modulate the numbers of or cytokine production by any skin DC subset. L3s broadly failed to activate or modulate LCs or DDCs, suggesting filarial larvae expertly evade APC detection in human skin. © 2014 John Wiley & Sons Ltd.

Human Periodontal Ligament-Derived Stem Cells Promote Retinal Ganglion Cell Survival and Axon Regeneration After Optic Nerve Injury.

PubMed

Cen, Ling-Ping; Ng, Tsz Kin; Liang, Jia-Jian; Zhuang, Xi; Yao, Xiaowu; Yam, Gary Hin-Fai; Chen, Haoyu; Cheung, Herman S; Zhang, Mingzhi; Pang, Chi Pui

2018-06-01

Optic neuropathies are the leading cause of irreversible blindness and visual impairment in the developed countries, affecting more than 80 million people worldwide. While most optic neuropathies have no effective treatment, there is intensive research on retinal ganglion cell (RGC) protection and axon regeneration. We previously demonstrated potential of human periodontal ligament-derived stem cells (PDLSCs) for retinal cell replacement. Here, we report the neuroprotective effect of human PDLSCs to ameliorate RGC degeneration and promote axonal regeneration after optic nerve crush (ONC) injury. Human PDLSCs were intravitreally injected into the vitreous chamber of adult Fischer rats after ONC in vivo as well as cocultured with retinal explants in vitro. Human PDLSCs survived in the vitreous chamber and were maintained on the RGC layer even at 3 weeks after ONC. Immunofluorescence analysis of βIII-tubulin and Gap43 showed that the numbers of surviving RGCs and regenerating axons were significantly increased in the rats with human PDLSC transplantation. In vitro coculture experiments confirmed that PDLSCs enhanced RGC survival and neurite regeneration in retinal explants without inducing inflammatory responses. Direct cell-cell interaction and elevated brain-derived neurotrophic factor secretion, but not promoting endogenous progenitor cell regeneration, were the RGC protective mechanisms of human PDLSCs. In summary, our results revealed the neuroprotective role of human PDLSCs by strongly promoting RGC survival and axonal regeneration both in vivo and in vitro, indicating a therapeutic potential for RGC protection against optic neuropathies. Stem Cells 2018;36:844-855. © AlphaMed Press 2018.

Blood and clonogenic hemopoietic cells of newts after the space flight

NASA Astrophysics Data System (ADS)

Michurina, T. V.; Domaratskaya, E. I.; Nikonova, T. M.; Khrushchov, N. G.

Ribbed newts were used for studying the effect of space flight on board of the biosatellite (Cosmos-2229) on blood and clonogenic hemopoietic cells. In blood of newts of the flight group, the relative proportion of neutrophils increased, whereas that of lymphocytes and eosinophils decreased. Space flight did not result in loss of the ability of newt blood cells to incorporate H^3-thymidine. Analysis of clonogenic hemopoietic cells was performed using the method of hemopoietic colony formation on cellulose acetate membranes implanted into the peritoneal cavity of irradiated newts. To analyze reconstitution of hemopoiesis after irradiation donor hemopoietic cells from flight or control newts were transplanted into irradiated newts whose hemopoietic organs were investigated. The newt can be considered an adequate model for studying hemopoiesis under the conditions of the space flight. Previous studies on rats subjected to 5- to 19-day space flights revealed a decrease in the number of clonogenic cells in their hemopoietic organs accompanied by specific changes in the precursor cell compartment and in blood /1,2/. Hence, it was interesting to analyze blood and hemopoietic tissue of lower vertebrates after a space flight and to compare the response to it of animals belonging to different taxonomic groups. We analyzed blood and clonogenic hemopoietic cells of ribbed newts, Pleurodeles waltl (age one year, weight 20-28 g) subjected to a 12-day space flight on board of a Cosmos-2229 biosatellite. The same animals were used in studies on limb and lens regeneration. The results were compared with those obtained with control groups of newts: (1) basic control, operated newts sacrificed on the day of biosatellite launching (BC); (2) synchronous control, operated newts kept in the laboratory under simulated space flight conditions (SC); and (3) intact newts (IC).

Influence of environmental pH on G2-phase arrest caused by ionizing radiation.

PubMed

Park, Heon Joo; Lee, Sang Hwa; Chung, HyunSook; Rhee, Yun Hee; Lim, Byung Uk; Ha, Sung Whan; Griffin, Robert J; Lee, Hyung Sik; Song, Chang Won; Choi, Eun Kyung

2003-01-01

We investigated the effects of an acidic environment on the G2/M-phase arrest, apoptosis, clonogenic death, and changes in cyclin B1-CDC2 kinase activity caused by a 4-Gy irradiation in RKO.C human colorectal cancer cells in vitro. The time to reach peak G2/M-phase arrest after irradiation was delayed in pH 6.6 medium compared to that in pH 7.5 medium. Furthermore, the radiation-induced G2/M-phase arrest decayed more slowly in pH 6.6 medium than in pH 7.5 medium. Finally, there was less radiation-induced apoptosis and clonogenic cell death in pH 6.6 medium than in pH 7.5 medium. It appeared that the prolongation of G2-phase arrest after irradiation in the acidic environment allowed for greater repair of radiation-induced DNA damage, thereby decreasing the radiation-induced cell death. The prolongation of G2-phase arrest after irradiation in the acidic pH environment appeared to be related at least in part to a prolongation of the phosphorylation of CDC2, which inhibited cyclin B1-CDC2 kinase activity.

MiR-204 down-regulation elicited perturbation of a gene target signature common to human cholangiocarcinoma and gastric cancer.

PubMed

Canu, Valeria; Sacconi, Andrea; Lorenzon, Laura; Biagioni, Francesca; Lo Sardo, Federica; Diodoro, Maria Grazia; Muti, Paola; Garofalo, Alfredo; Strano, Sabrina; D'Errico, Antonietta; Grazi, Gian Luca; Cioce, Mario; Blandino, Giovanni

2017-05-02

There is high need of novel diagnostic and prognostic tools for tumors of the digestive system, such as gastric cancer and cholangiocarcinoma. We recently found that miR-204 was deeply downregulated in gastric cancer tissues. Here we investigated whether this was common to other tumors of the digestive system and whether this elicited a miR-204-dependent gene target signature, diagnostically and therapeutically relevant. Finally, we assessed the contribution of the identified target genes to the cell cycle progression and clonogenicity of gastric cancer and cholangiocarcinoma cell lines. We employed quantitative PCR and Affymetrix profiling for gene expression studies. In silico analysis aided us to identifying a miR-204 target signature in publicly available databases (TGCA). We employed transient transfection experiments, clonogenic assays and cell cycle profiling to evaluate the biological consequences of miR-204 perturbation. We identified a novel miR-204 gene target signature perturbed in gastric cancer and in cholangiocarcinoma specimens. We validated its prognostic relevance and mechanistically addressed its biological relevance in GC and CC cell lines. We suggest that restoring the physiological levels of miR-204 in some gastrointestinal cancers might be exploited therapeutically.

Survival and characteristics of murine leukaemic and normal stem cells after hyperthermia: a murine model for human bone marrow purging.

PubMed

Gidáli, J; Szamosvölgyi, S; Fehér, I; Kovács, P

1990-01-01

The effect of hyperthermia in vitro on the survival and leukaemogenic effectiveness of WEHI 3-B cells and on the survival and transplantation efficiency of bone marrow cells was compared in a murine model system. Normal murine clonogenic haemopoietic cells (day 9 CFU-S and CFU-GM) proved to be significantly less sensitive to 42.5 degrees C hyperthermia (Do values: 54.3 and 41.1 min, respectively) than leukaemic clonogenic cells (CFU-L) derived from suspension culture or from bone marrow of leukaemic mice (Do: 17.8 min). Exposure for 120 min to 42.5 degrees C reduced the surviving fraction of CFU-L to 0.002 and that of CFU-S to 0.2. If comparable graft sizes were transplanted from normal or heat exposed bone marrow, 60-day survival of supralethally irradiated mice was similar. Surviving WEHI 3-B cells were capable of inducing leukaemia in vivo. The two log difference in the surviving fraction of CFU-L and CFU-S after 120 min exposure to 42.5 degrees C suggests that hyperthermia ex vivo may be a suitable purging method for autologous bone marrow transplantation.

Nonablative skin rejuvenation devices and the role of heat shock protein 70: results of a human skin explant model

NASA Astrophysics Data System (ADS)

Helbig, Doris; Moebius, Anne; Simon, Jan C.; Paasch, Uwe

2010-05-01

Nonablative thermal laser therapy with a 1540-nm laser induces controlled, spatially determined thermal damage, allowing subsequent collagen remodeling while preserving the epidermis. A photorejuvenation effect using nonthermal nonablative stimulation of cells with low energy and narrow band light has been termed photomodulation. Light emitting diodes (LEDs) are narrow band emitters that lead to photomodulation via stimulation of mitochondrial cell organelles. In a previous study, we demonstrated in a human skin explant model that heat shock protein 70 (HSP70) plays a pivotal role in the initiation of skin remodeling after ablative fractional photothermolysis. To test its importance in nonablative laser therapy and photomodulation, the spatio-temporal expression of HSP70 is investigated in response to a 1540-nm laser treatment and six different LED therapies. An Er:glass laser is used with a 1-Hz repetition rate, 30-J/cm2 fluence, and a hand piece with a 2-mm spot size. Nonthermal nonablative treatment is performed using two LED (LEDA SCR red light: 635 nm, 40 to 120 W/cm2, 40 to 120 J/cm2 LEDA SCR yellow light: 585 nm, 16 to 35 W/cm2, 20 to 100 J/cm2 spot size 16×10 cm). Immediate responses as well as responses 1, 3, or 7 days postprocedure are studied; untreated skin explants serve as control. Immunohistochemical investigation (HSP70) is performed in all native, nontreated, and Er:glass laser- or LED-treated samples (n=175). Nonablative laser therapy leads to a clear time-dependent induction of epidermally expressed HSP70, peaking between one to three days post-treatment. In contrast, none of the various LED treatments up-regulated the HSP70 expression in our skin explant model. HSP70 is up-regulated by nonablative but thermal laser devices, but does not seem to play a significant role in the induction of skin remodeling induced by photomodulation. The maximum of HSP70 expression is reached later after Er:glass laser intervention compared to ablative fractional (AFP) treatment.

Explanted Diseased Livers – A Possible Source of Metabolic Competent Primary Human Hepatocytes

PubMed Central

Krech, Till; DeTemple, Daphne; Jäger, Mark D.; Lehner, Frank; Manns, Michael P.; Klempnauer, Jürgen; Borlak, Jürgen; Bektas, Hueseyin; Vondran, Florian W. R.

2014-01-01

Being an integral part of basic, translational and clinical research, the demand for primary human hepatocytes (PHH) is continuously growing while the availability of tissue resection material for the isolation of metabolically competent PHH remains limited. To overcome current shortcomings, this study evaluated the use of explanted diseased organs from liver transplantation patients as a potential source of PHH. Therefore, PHH were isolated from resected surgical specimens (Rx-group; n = 60) and explanted diseased livers obtained from graft recipients with low labMELD-score (Ex-group; n = 5). Using established protocols PHH were subsequently cultured for a period of 7 days. The viability and metabolic competence of cultured PHH was assessed by the following parameters: morphology and cell count (CyQuant assay), albumin synthesis, urea production, AST-leakage, and phase I and II metabolism. Both groups were compared in terms of cell yield and metabolic function, and results were correlated with clinical parameters of tissue donors. Notably, cellular yields and viabilities were comparable between the Rx- and Ex-group and were 5.3±0.5 and 2.9±0.7×106 cells/g liver tissue with 84.3±1.3 and 76.0±8.6% viability, respectively. Moreover, PHH isolated from the Rx- or Ex-group did not differ in regards to loss of cell number in culture, albumin synthesis, urea production, AST-leakage, and phase I and II metabolism (measured by the 7-ethoxycoumarin-O-deethylase and uracil-5′-diphosphate-glucuronyltransferase activity). Likewise, basal transcript expressions of the CYP monooxygenases 1A1, 2C8 and 3A4 were comparable as was their induction when treated with a cocktail that consisted of 3-methylcholantren, rifampicin and phenobarbital, with increased expression of CYP 1A1 and 3A4 mRNA while transcript expression of CYP 2C8 was only marginally changed. In conclusion, the use of explanted diseased livers obtained from recipients with low labMELD-score might represent a valuable source of metabolically competent PHH which are comparable in viability and function to cells obtained from specimens following partial liver resection. PMID:24999631

Enhanced radiosensitization of p53 mutant cells by oleamide.

PubMed

Lee, Yoon-Jin; Chung, Da Yeon; Lee, Su-Jae; Ja Jhon, Gil; Lee, Yun-Sil

2006-04-01

Effect of oleamide, an endogenous fatty-acid primary amide, on tumor cells exposed to ionizing radiation (IR) has never before been explored. NCI H460, human lung cancer cells, and human astrocytoma cell lines, U87 and U251, were used. The cytotoxicity of oleamide alone or in combination with IR was determined by clonogenic survival assay, and induction of apoptosis was estimated by FACS analysis. Protein expressions were confirmed by Western blotting, and immunofluorescence analysis of Bax by use of confocal microscopy was also performed. The combined effect of IR and oleamide to suppress tumor growth was studied by use of xenografts in the thighs of nude mice. Oleamide in combination with IR had a synergistic effect that decreased clonogenic survival of lung-carcinoma cell lines and also sensitized xenografts in nude mice. Enhanced induction of apoptosis of the cells by the combined treatment was mediated by loss of mitochondrial membrane potential, which resulted in the activation of caspase-8, caspase-9, and caspase-3 accompanied by cytochrome c release and Bid cleavage. The synergistic effects of the combined treatment were more enhanced in p53 mutant cells than in p53 wild-type cells. In p53 wild-type cells, both oleamide and radiation induced Bax translocation to mitochondria. On the other hand, in p53 mutant cells, radiation alone slightly induced Bax translocation to mitochondria, whereas oleamide induced a larger translocation. Oleamide may exhibit synergistic radiosensitization in p53 mutant cells through p53-independent Bax translocation to mitochondria.

Relative biological effectiveness (RBE) of alpha radiation in cultured porcine aortic endothelial cells.

PubMed

Thomas, Patricia; Tracy, Bliss; Ping, Tilly; Baweja, Anar; Wickstrom, Mark; Sidhu, Narinder; Hiebert, Linda

2007-03-01

Northern peoples can receive elevated radiation doses (1- 10 mSv/y) from transfer of polonium-210 (210Po) through the lichen-caribou-human food chain. Ingested 210Po is primarily blood-borne and thus many of its short range alpha particles irradiate the endothelial cells lining the blood vessels. The relative biological effectiveness (RBE) of alpha particles vs. x-rays was examined in porcine aortic endothelial cells as a surrogate for understanding what might happen to human endothelial cells in northern populations consuming traditional foods. Cultured porcine aortic endothelial cells were exposed to x-ray and 210Po alpha particle radiation. Alpha irradiation was applied to the cell cultures internally via the culture medium and externally, using thin-bottomed culture dishes. The results given here are based on the external irradiation method, which was found to be more reliable. Dose-response curves were compared for four lethal endpoints (cell viability, live cell fraction, release of lactate dehydrogenase [LDH] and clonogenic survival) to determine the relative biological effectiveness (RBE) of alpha radiation. The alpha RBE for porcine cells varied from 1.6-21, depending on the endpoint: 21.2+/-4.5 for cell viability, 12.9+/-2.7 for decrease in live cell number, 5.3+/-0.4 for LDH release to the medium but only 1.6 +/-0.1 for clonogenic survival. The low RBE of 1.6 was due to x-ray hypersensitivity of endothelial cells at low doses.

Upregulation of NAD(P)H:Quinone Oxidoreductase By Radiation Potentiates the Effect of Bioreductive β-Lapachone on Cancer Cells1

PubMed Central

Choi, Eun K; Terai, Kaoru; Ji, In-Mi; Kook, Yeon H; Park, Kyung H; Oh, Eun T; Griffin, Robert J; Lim, Byung U; Kim, Jin-Seok; Lee, Doo S; Boothman, David A; Loren, Melissa; Song, Chang W; Park, Heon Joo

2007-01-01

We found that β-lapachone (β-lap), a novel bioreductive drug, caused rapid apoptosis and clonogenic cell death in A549 human lung epithelial cancer cells in vitro in a dose-dependent manner. The clonogenic cell death caused by β-lap could be significantly inhibited by dicoumarol, an inhibitor of NAD(P)H:quinone oxido-reductase (NQO1), and also by siRNA for NQO1, demonstrating that NQO1-induced bioreduction of β-lap is an essential step in β-lap-induced cell death. Irradiation of A549 cells with 4 Gy caused a long-lasting upregulation of NQO1, thereby increasing NQO1-mediated β-lap-induced cell deaths. Although the direct cause of β-lap-induced apoptosis is not yet clear, β-lap treatment reduced the expression of p53 and NF-κB, whereas it increased cytochrome C release, caspase-3 activity, and γH2AX foci formation. Importantly, β-lap treatment immediately after irradiation enhanced radiation-induced cell death, indicating that β-lap sensitizes cancer cells to radiation, in addition to directly killing some of the cells. The growth of A549 tumors induced in immunocompromised mice could be markedly suppressed by local radiation therapy when followed by β-lap treatment. This is the first study to demonstrate that combined radiotherapy and β-lap treatment can have a significant effect on human tumor xenografts. PMID:17786182

Correlation of Particle Traversals with Clonogenic Survival Using Cell-Fluorescent Ion Track Hybrid Detector.

PubMed

Dokic, Ivana; Niklas, Martin; Zimmermann, Ferdinand; Mairani, Andrea; Seidel, Philipp; Krunic, Damir; Jäkel, Oliver; Debus, Jürgen; Greilich, Steffen; Abdollahi, Amir

2015-01-01

Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD.

Characterization and Multilineage Potential of Cells Derived from Isolated Microvascular Fragments

DTIC Science & Technology

2014-05-24

three in vitro human ’angiogenesis’ assays with capillaries formed in vivo. Angiogenesis 2001;4:113. [18] Gimble JM, Katz AJ , Bunnell BA. Adipose derived...Cell Cycle 2005;4:1338. [31] Rosenblatt JD, Lunt AI, Parry DJ, et al. Culturing satellite cells from living single muscle fiber explants. In Vitro Cell

Risk factors for explantation due to infection after sacral neuromodulation: a multicenter retrospective case-control study.

PubMed

Myer, Emily N B; Petrikovets, Andrey; Slocum, Paul D; Lee, Toy Gee; Carter-Brooks, Charelle M; Noor, Nabila; Carlos, Daniela M; Wu, Emily; Van Eck, Kathryn; Fashokun, Tola B; Yurteri-Kaplan, Ladin; Chen, Chi Chiung Grace

2018-04-07

Sacral neuromodulation is an effective therapy for overactive bladder, urinary retention, and fecal incontinence. Infection after sacral neurostimulation is costly and burdensome. Determining optimal perioperative management strategies to reduce the risk of infection is important to reduce this burden. We sought to identify risk factors associated with sacral neurostimulator infection requiring explantation, to estimate the incidence of infection requiring explantation, and identify associated microbial pathogens. This is a multicenter retrospective case-control study of sacral neuromodulation procedures completed from Jan. 1, 2004, through Dec. 31, 2014. We identified all sacral neuromodulation implantable pulse generator implants as well as explants due to infection at 8 participating institutions. Cases were patients who required implantable pulse generator explantation for infection during the review period. Cases were included if age ≥18 years old, follow-up data were available ≥30 days after implantable pulse generator implant, and the implant was performed at the institution performing the explant. Two controls were matched to each case. These controls were the patients who had an implantable pulse generator implanted by the same surgeon immediately preceding and immediately following the identified case who met inclusion criteria. Controls were included if age ≥18 years old, no infection after implantable pulse generator implant, follow-up data were available ≥180 days after implant, and no explant for any reason <180 days from implant. Controls may have had an explant for reasons other than infection at >180 days after implant. Fisher exact test (for categorical variables) and Student t test (for continuous variables) were used to test the strength of the association between infection and patient and surgery characteristics. Significant variables were then considered in a multivariable logistic regression model to determine risk factors independently associated with infection. Over a 10-year period at 8 academic institutions, 1930 sacral neuromodulator implants were performed by 17 surgeons. In all, 38 cases requiring device explant for infection and 72 corresponding controls were identified. The incidence of infection requiring explant was 1.97%. Hematoma formation (13% cases, 0% controls; P = .004) and pocket depth of ≥3 cm (21% cases, 0% controls; P = .031) were independently associated with an increased risk of infection requiring explant. On multivariable regression analysis controlling for significant variables, both hematoma formation (P = .006) and pocket depth ≥3 cm (P = .020, odds ratio 3.26; 95% confidence interval, 1.20-8.89) remained significantly associated with infection requiring explant. Of the 38 cases requiring explant, 32 had cultures collected and 24 had positive cultures. All 5 cases with a hematoma had a positive culture (100%). Of the 4 cases with a pocket depth ≥3 cm, 2 had positive cultures, 1 had negative cultures, and 1 had a missing culture result. The most common organism identified was methicillin-resistant Staphylococcus aureus (38%). Infection after sacral neuromodulation requiring device explant is low. The most common infectious pathogen identified was methicillin-resistant S aureus. Demographic and health characteristics did not predict risk of explant due to infection, however, having a postoperative hematoma or a deep pocket ≥3 cm significantly increased the risk of explant due to infection. These findings highlight the importance of meticulous hemostasis as well as ensuring the pocket depth is <3 cm at the time of device implant. Copyright © 2018 Elsevier Inc. All rights reserved.

Effect of various factors on shoot regeneration from citrus epicotyl explants

USDA-ARS?s Scientific Manuscript database

The effect of various treatments on shoot organogenesis from seedling epicotyl explants from various scion and rootstock polyembryonic citrus types was determined. Treatments included water source, gelling agent, explant insertion, seed size, light intensity, malachite green, nonionic surfactants, a...

Laparoscopic Adjustable Gastric Band Explantation and Implantation at Academic Centers.

PubMed

Koh, Christina Y; Inaba, Colette S; Sujatha-Bhaskar, Sarath; Hohmann, Samuel; Ponce, Jaime; Nguyen, Ninh T

2017-10-01

The laparoscopic adjustable gastric band (LAGB) was approved for use in the US in 2001 and has been found to be a safe and effective surgical treatment for morbid obesity. However, there is a recent trend toward reduced use of LAGB nationwide. The objective of this study was to examine the prevalence and outcomes of primary LAGB implantation compared with revision and explantation at academic centers. Data were obtained from the Vizient database from 2007 through 2015. The ICD-9-Clinical Modification and ICD-10-Clinical Modification were used to select patients with a primary diagnosis of obesity who had undergone LAGB implantation, revision, or explantation. Prevalence and outcomes of primary LAGB implantation compared with revision or explantation were analyzed. Outcomes measures included length of stay, ICU admission, morbidity, mortality, and cost. From 2007 through 2015, a total of 28,202 patients underwent LAGB implantation for surgical weight loss. The annual number of LAGB implantation procedures decreased steadily after 2010. In the same time period, 12,157 patients underwent LAGB explantation. In 2013, the number of LAGB explantation procedures exceeded that of implantation. Laparoscopic adjustable gastric band revision rates remained stable throughout the study period. Mean length of stay, serious morbidity, and proportion of patients requiring ICU admission were higher for gastric band revision and explantation cases compared with primary LAGB implantation cases. There was no statistically significant difference in mortality or mean cost between the 2 groups. Since 2013, the number of gastric band explantation procedures has exceeded that of implantation procedures at academic centers. Laparoscopic adjustable gastric band revision or explantation is associated with longer length of stay, higher rate of postoperative ICU admissions, and higher overall morbidity compared with LAGB implantation. Copyright © 2017 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

The Regulation of Vascular Endothelial Growth Factor by Hypoxia and Prostaglandin F2α during Human Endometrial Repair

PubMed Central

Maybin, Jacqueline A.; Hirani, Nikhil; Brown, Pamela; Jabbour, Henry N.

2011-01-01

Context: The human endometrium has an exceptional capacity for repeated repair after menses, but its regulation remains undefined. Premenstrually, progesterone levels fall and prostaglandin (PG) F2α synthesis increases, causing spiral arteriole constriction. We hypothesized that progesterone withdrawal, PGF2α, and hypoxia increase vascular endothelial growth factor (VEGF), an endometrial repair factor. Design and Results: Endometrial biopsies were collected (n = 47) with ethical approval and consent. VEGF mRNA, quantified by quantitative RT-PCR, was increased during menstruation (P < 0.01).VEGF protein was maximally secreted from proliferative endometrial explants. Treatment of an endometrial epithelial cell line and primary human endometrial stromal cells with 100 nm PGF2α or hypoxia (0.5% O2) resulted in significant increases in VEGF mRNA and protein. VEGF was maximal when cells were cotreated with PGF2α and hypoxia simultaneously (P < 0.05–0.001). Secretory-phase endometrial explants also showed an increase in VEGF with cotreatment (P < 0.05). However, proliferative-phase explants showed no increase in VEGF on treatment with PGF2α and/or hypoxia. Proliferative tissue was induced to increase VEGF mRNA expression when exposed to progesterone and its withdrawal in vitro but only in the presence of hypoxia and PG. Hypoxia-inducible factor-1α (HIF-1α) silencing with RNA interference suppressed hypoxia-induced VEGF expression in endometrial cells but did not alter PGF2α-induced VEGF expression. Conclusions: Endometrial VEGF is increased at the time of endometrial repair. Progesterone withdrawal, PGF2α, and hypoxia are necessary for this perimenstrual VEGF expression. Hypoxia acts via HIF-1α to increase VEGF, whereas PGF2α acts in a HIF-1α-independent manner. Hence, two pathways regulate the expression of VEGF during endometrial repair. PMID:21677035

Bisphenol A alters β-hCG and MIF release by human placenta: an in vitro study to understand the role of endometrial cells.

PubMed

Mannelli, C; Ietta, F; Carotenuto, C; Romagnoli, R; Szostek, A Z; Wasniewski, T; Skarzynski, D J; Paulesu, Luana

2014-01-01

A proper fetomaternal immune-endocrine cross-talk in pregnancy is fundamental for reproductive success. This might be unbalanced by exposure to environmental chemicals, such as bisphenol A (BPA). As fetoplacental contamination with BPA originates from the maternal compartment, this study investigated the role of the endometrium in BPA effects on the placenta. To this end, in vitro decidualized stromal cells were exposed to BPA 1 nM, and their conditioned medium (diluted 1 : 2) was used on chorionic villous explants from human placenta. Parallel cultures of placental explants were directly exposed to 0.5 nM BPA while, control cultures were exposed to the vehicle (EtOH 0.1%). After 24-48 h, culture medium from BPA-treated and control cultures was assayed for concentration of hormone human Chorionic Gonadotropin ( β -hCG) and cytokine Macrophage Migration Inhibitory Factor (MIF). The results showed that direct exposure to BPA stimulated the release of both MIF and β -hCG. These effects were abolished/diminished in placental cultures exposed to endometrial cell-conditioned medium. GM-MS analysis revealed that endometrial cells retain BPA, thus reducing the availability of this chemical for the placenta. The data obtained highlight the importance of in vitro models including the maternal component in reproducing the effects of environmental chemicals on human fetus/placenta.

Bisphenol A Alters β-hCG and MIF Release by Human Placenta: An In Vitro Study to Understand the Role of Endometrial Cells

PubMed Central

Mannelli, C.; Ietta, F.; Carotenuto, C.; Romagnoli, R.; Szostek, A. Z.; Wasniewski, T.; Skarzynski, D. J.

2014-01-01

A proper fetomaternal immune-endocrine cross-talk in pregnancy is fundamental for reproductive success. This might be unbalanced by exposure to environmental chemicals, such as bisphenol A (BPA). As fetoplacental contamination with BPA originates from the maternal compartment, this study investigated the role of the endometrium in BPA effects on the placenta. To this end, in vitro decidualized stromal cells were exposed to BPA 1 nM, and their conditioned medium (diluted 1 : 2) was used on chorionic villous explants from human placenta. Parallel cultures of placental explants were directly exposed to 0.5 nM BPA while, control cultures were exposed to the vehicle (EtOH 0.1%). After 24–48 h, culture medium from BPA-treated and control cultures was assayed for concentration of hormone human Chorionic Gonadotropin (β-hCG) and cytokine Macrophage Migration Inhibitory Factor (MIF). The results showed that direct exposure to BPA stimulated the release of both MIF and β-hCG. These effects were abolished/diminished in placental cultures exposed to endometrial cell-conditioned medium. GM-MS analysis revealed that endometrial cells retain BPA, thus reducing the availability of this chemical for the placenta. The data obtained highlight the importance of in vitro models including the maternal component in reproducing the effects of environmental chemicals on human fetus/placenta. PMID:24737926

A novel method for coral explant culture and micropropagation.

PubMed

Vizel, Maya; Loya, Yossi; Downs, Craig A; Kramarsky-Winter, Esti

2011-06-01

We describe here a method for the micropropagation of coral that creates progeny from tissue explants derived from a single polyp or colonial corals. Coral tissue explants of various sizes (0.5-2.5 mm in diameter) were manually microdissected from the solitary coral Fungia granulosa. Explants could be maintained in an undeveloped state or induced to develop into polyps by manipulating environmental parameters such as light and temperature regimes, as well as substrate type. Fully developed polyps were able to be maintained for a long-term in a closed sea water system. Further, we demonstrate that mature explants are also amenable to this technique with the micropropagation of second-generation explants and their development into mature polyps. We thereby experimentally have established coral clonal lines that maintain their ability to differentiate without the need for chemical induction or genetic manipulation. The versatility of this method is also demonstrated through its application to two other coral species, the colonial corals Oculina patigonica and Favia favus.

Dissecting and Culturing Animal Cap Explants.

PubMed

Dingwell, Kevin S; Smith, James C

2018-05-16

The animal cap explant is a simple but adaptable tool available to developmental biologists. The use of animal cap explants in demonstrating the presence of mesoderm-inducting activity in the Xenopus embryo vegetal pole is one of many elegant examples of their worth. Animal caps respond to a range of growth factors (e.g., Wnts, FGF, TGF-β), making them especially useful for studying signal transduction pathways and gene regulatory networks. Explants are also suitable for examining cell behavior and have provided key insights into the molecular mechanisms controlling vertebrate morphogenesis. In this protocol, we outline two methods to isolate animal cap explants from Xenopus laevis , both of which can be applied easily to Xenopus tropicalis The first method is a standard manual method that can be used in any laboratory equipped with a standard dissecting microscope. For labs planning on dissecting large numbers of explants on a regular basis, a second, high throughput method is described that uses a specialized microcautery surgical instrument. © 2018 Cold Spring Harbor Laboratory Press.

Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo - Implications for Tissue Engineering and Clinical Applications.

PubMed

Szabó, Dóra Júlia; Noer, Agate; Nagymihály, Richárd; Josifovska, Natasha; Andjelic, Sofija; Veréb, Zoltán; Facskó, Andrea; Moe, Morten C; Petrovski, Goran

2015-01-01

Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.

Mathematics as a Conduit for Translational Research in Post-Traumatic Osteoarthritis

PubMed Central

Ayati, Bruce P.; Kapitanov, Georgi I.; Coleman, Mitchell C.; Anderson, Donald D.; Martin, James A.

2016-01-01

Biomathematical models offer a powerful method of clarifying complex temporal interactions and the relationships among multiple variables in a system. We present a coupled in silico biomathematical model of articular cartilage degeneration in response to impact and/or aberrant loading such as would be associated with injury to an articular joint. The model incorporates fundamental biological and mechanical information obtained from explant and small animal studies to predict post-traumatic osteoarthritis (PTOA) progression, with an eye toward eventual application in human patients. In this sense, we refer to the mathematics as a “conduit of translation”. The new in silico framework presented in this paper involves a biomathematical model for the cellular and biochemical response to strains computed using finite element analysis. The model predicts qualitative responses presently, utilizing system parameter values largely taken from the literature. To contribute to accurate predictions, models need to be accurately parameterized with values that are based on solid science. We discuss a parameter identification protocol that will enable us to make increasingly accurate predictions of PTOA progression using additional data from smaller scale explant and small animal assays as they become available. By distilling the data from the explant and animal assays into parameters for biomathematical models, mathematics can translate experimental data to clinically relevant knowledge. PMID:27653021

T cells fail to develop in the human skin-cell explants system; an inconvenient truth.

PubMed

Meek, Bob; Van Elssen, Catharina H M J; Huijskens, Mirelle J A J; van der Stegen, Sjoukje J C; Tonnaer, Siebe; Lumeij, Stijn B J; Vanderlocht, Joris; Kirkland, Mark A; Hesselink, Reinout; Germeraad, Wilfred T V; Bos, Gerard M J

2011-02-18

Haplo-identical hematopoietic stem cell (HSC) transplantation is very successful in eradicating haematological tumours, but the long post-transplant T-lymphopenic phase is responsible for high morbidity and mortality rates. Clark et al. have described a skin-explant system capable of producing host-tolerant donor-HSC derived T-cells. Because this T-cell production platform has the potential to replenish the T-cell levels following transplantation, we set out to validate the skin-explant system. Following the published procedures, while using the same commercial components, it was impossible to reproduce the skin-explant conditions required for HSC differentiation towards mature T-cells. The keratinocyte maturation procedure resulted in fragile cells with minimum expression of delta-like ligand (DLL). In most experiments the generated cells failed to adhere to carriers or were quickly outcompeted by fibroblasts. Consequently it was not possible to reproduce cell-culture conditions required for HSC differentiation into functional T-cells. Using cell-lines over-expressing DLL, we showed that the antibodies used by Clark et al. were unable to detect native DLL, but instead stained 7AAD+ cells. Therefore, it is unlikely that the observed T-lineage commitment from HSC is mediated by DLL expressed on keratinocytes. In addition, we did confirm expression of the Notch-ligand Jagged-1 by keratinocytes. Currently, and unfortunately, it remains difficult to explain the development or growth of T-cells described by Clark et al., but for the fate of patients suffering from lymphopenia it is essential to both reproduce and understand how these co-cultures really "work". Fortunately, alternative procedures to speed-up T-cell reconstitution are being established and validated and may become available for patients in the near future.

Modulation of Female Genital Tract-Derived Dendritic Cell Migration and Activation in Response to Inflammatory Cytokines and Toll-Like Receptor Agonists.

PubMed

Shey, Muki S; Maharaj, Niren; Archary, Derseree; Ngcapu, Sinaye; Garrett, Nigel; Abdool Karim, Salim; Passmore, Jo-Ann S

2016-01-01

HIV transmission across the genital mucosa is a major mode of new HIV infections in women. The probability of infection may be influenced by several factors including recruitment and activation of HIV target cells, such as dendritic cells (DCs) and cytokine production, associated with genital inflammation. We evaluated the role of inflammatory cytokines and TLR signaling in migration and activation of genital tract DCs in the human cervical explant model. Hysterectomy tissues from 10 HIV-negative and 7 HIV-positive donor women were separated into ecto- and endocervical explants, and incubated with inflammatory cytokines (TNF-α, IL-1β, IL-8, MIP-1β) or agonists for TLR4 (LPS), TLR2/1 (PAM3) and TLR7/8 (R848). Migration (frequency) and activation (HLA-DR expression) of myeloid and plasmacytoid DCs and Langerhans cells were measured by flow cytometry. We observed that cytokines, LPS and PAM3 induced activation of migrating myeloid and plasmacytoid DCs. LPS induced a 3.6 fold lower levels of migration of plasmacytoid DCs from HIV-infected women compared with HIV-uninfected women (median activation indices of 2.932 vs 0.833). There was however a 4.5 fold increase in migration of Langerhans cells in HIV-infected compared with HIV-uninfected women in response to cytokines (median activation indices of 3.539 vs 0.77). Only TLR agonists induced migration and activation of DCs from endocervical explants. Hormonal contraception use was associated with an increase in activation of DC subsets in the endo and ectocervical explants. We conclude that inflammatory signals in the female genital tract induced DC migration and activation, with possible important implications for HIV susceptibility of cervical tissues.

Frequent Infection of Human Cancer Xenografts with Murine Endogenous Retroviruses in Vivo

PubMed Central

Naseer, Asif; Terry, Anne; Gilroy, Kathryn; Kilbey, Anna; Watts, Ciorsdaidh; Mackay, Nancy; Bell, Margaret; Mason, Susan; Blyth, Karen; Cameron, Ewan; Neil, James C.

2015-01-01

Infection of human cancer xenografts in mice with murine leukemia viruses (MLVs) is a long-standing observation, but the likelihood of infection in vivo and its biological consequences are poorly understood. We therefore conducted a prospective study in commonly used xenograft recipient strains. From BALB/c nude mice engrafted with MCF7 human mammary carcinoma cells, we isolated a virus that was virtually identical to Bxv1, a locus encoding replication-competent xenotropic MLV (XMLV). XMLV was detected in 9/17 (53%) independently isolated explants. XMLV was not found in primary leukemias or in THP1 leukemia cells grown in Bxv1-negative NSG (NOD/SCID/γCnull) mice, although MCF7 explants harbored replication-defective MLV proviruses. To assess the significance of infection for xenograft behavior in vivo, we examined changes in growth and global transcription in MCF7 and the highly susceptible Raji Burkitt lymphoma cell line chronically infected with XMLV. Raji cells showed a stronger transcriptional response that included up-regulation of chemokines and effectors of innate antiviral immunity. In conclusion, the risk of de novo XMLV infection of xenografts is high in Bxv1 positive mice, while infection can have positive or negative effects on xenograft growth potential with significant consequences for interpretation of many xenograft studies. PMID:25912714

Single-Center Experience With HeartMate II Left Ventricular Assist Device Explantation.

PubMed

Tchantchaleishvili, Vakhtang; Cheyne, Christina; Sherazi, Saadia; Melvin, Amber L; Hallinan, William; Chen, Leway; Todd Massey, Howard

2016-12-01

In patients with continuous flow left ventricular assist devices (CF-LVADs) myocardial recovery is uncommon. Given the heterogeneity of the population implanted and low incidence of recovery, the discovery of native left ventricular (LV) recovery and criteria for explantation of CF-LVAD system is not clearly determined. We sought to analyze the characteristics of the patients who underwent CF-LVAD explantation at our institution. Prospectively collected data on patients supported with CF-LVADs were reviewed retrospectively. Patients who underwent CF-LVAD explants were identified and their characteristics were analyzed with a focus on patient presentation and determinants of explantability. From November 2006 to June 2014, 223 patients (181 male, 42 female) underwent implantation of HeartMate II LVAD. Seven female (16.7%) and one male (0.6%) patients were explanted (P < 0.001). Mean age was 43 ± 9 years and etiology for cardiomyopathy was ischemic in three (37.5%) patients, nonischemic in four (50%) patients, and mixed in the one (12.5%) male patient of the cohort. Five (62.5%) patients presented acutely with significant hemolysis, and were found to have LV improvement as well as reduced, absent, or reversed diastolic flow velocities on echocardiography. Overall, mean lactate dehydrogenase level before explantation was 1709 ± 1168 U/L compared to the mean baseline level of 601 ± 316 U/L (P = 0.048). Mean LV ejection fraction (LVEF) improved from 17 ± 7% preimplant to 56 ± 11% pre-explantation (P < 0.001). Median number of days on CF-LVAD support was 870 (interquartile range, 209-975) while mean duration of follow-up after the CF-LVAD explantation was 276 ± 240 days. Mean LVEF dropped from 46 ± 19% postexplantation to 34 ± 10% during the most recent follow-up (P = 0.015). At our institution, patients who underwent LVAD explants were predominantly women with nonischemic cardiomyopathy. Clinical evidence of hemolysis and echocardiographic evidence of reduced or absent diastolic flow velocities were common findings in these patients. Over time, patient's native LV function declined in the absence of LVAD (after LVAD explantation). Significant challenges remain in predicting LV recovery and identifying those individuals who have recovered myocardial function significant enough to be explanted. Copyright © 2016 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Identification of a small molecule that facilitates the differentiation of human iPSCs/ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells.

PubMed

Kondo, Yasushi; Toyoda, Taro; Ito, Ryo; Funato, Michinori; Hosokawa, Yoshiya; Matsui, Satoshi; Sudo, Tomomi; Nakamura, Masahiro; Okada, Chihiro; Zhuang, Xiaotong; Watanabe, Akira; Ohta, Akira; Inagaki, Nobuya; Osafune, Kenji

2017-08-01

Pancreatic beta-like cells generated from human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs) offer an appealing donor tissue source. However, differentiation protocols that mainly use growth factors are costly. Therefore, in this study, we aimed to establish efficient differentiation protocols to change hiPSCs/hESCs to insulin (INS) + cells using novel small-molecule inducers. We screened small molecules that increased the induction rate of INS + cells from hESC-derived pancreatic and duodenal homeobox 1 (PDX1) + pancreatic progenitor cells. The differentiation protocol to generate INS + cells from hiPSCs/hESCs was optimised using hit compounds, and INS + cells induced with the compounds were characterised for their in vitro and in vivo functions. The inducing activity of the hit compounds was also examined using mouse embryonic pancreatic tissues in an explant culture system. Finally, RNA sequencing analyses were performed on the INS + cells to elucidate the mechanisms of action by which the hit compounds induced pancreatic endocrine differentiation. One hit compound, sodium cromoglicate (SCG), was identified out of approximately 1250 small molecules screened. When SCG was combined with a previously described protocol, the induction rate of INS + cells increased from a mean ± SD of 5.9 ± 1.5% (n = 3) to 16.5 ± 2.1% (n = 3). SCG induced neurogenin 3-positive cells at a mean ± SD of 32.6 ± 4.6% (n = 3) compared with 14.2 ± 3.6% (n = 3) for control treatment without SCG, resulting in an increased generation of endocrine cells including insulin-producing cells. Similar induction by SCG was confirmed using mouse embryonic pancreatic explants. We also confirmed that the mechanisms of action by which SCG induced pancreatic endocrine differentiation included the inhibition of bone morphogenetic protein 4 signalling. SCG improves the generation of pancreatic endocrine cells from multiple hiPSC/hESC lines and mouse embryonic pancreatic explants by facilitating the differentiation of endocrine precursors. This discovery will contribute to elucidating the mechanisms of pancreatic endocrine development and facilitate cost-effective generation of INS + cells from hiPSCs/hESCs. The RNA sequencing data generated during the current study are available in the Gene Expression Omnibus ( www.ncbi.nlm.nih.gov/geo ) with series accession number GSE89973.

Proteomic Profile of Brucella abortus-Infected Bovine Chorioallantoic Membrane Explants

PubMed Central

Mol, Juliana P. S.; Pires, Simone F.; Chapeaurouge, Alexander D.; Perales, Jonas; Santos, Renato L.; Andrade, Hélida M.; Lage, Andrey P.

2016-01-01

Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation. PMID:27104343

Noninvasive assessment of articular cartilage surface damage using reflected polarized light microscopy

NASA Astrophysics Data System (ADS)

Huynh, Ruby N.; Nehmetallah, George; Raub, Christopher B.

2017-06-01

Articular surface damage occurs to cartilage during normal aging, osteoarthritis, and in trauma. A noninvasive assessment of cartilage microstructural alterations is useful for studies involving cartilage explants. This study evaluates polarized reflectance microscopy as a tool to assess surface damage to cartilage explants caused by mechanical scraping and enzymatic degradation. Adult bovine articular cartilage explants were scraped, incubated in collagenase, or underwent scrape and collagenase treatments. In an additional experiment, cartilage explants were subject to scrapes at graduated levels of severity. Polarized reflectance parameters were compared with India ink surface staining, features of histological sections, changes in explant wet weight and thickness, and chondrocyte viability. The polarized reflectance signal was sensitive to surface scrape damage and revealed individual scrape features consistent with India ink marks. Following surface treatments, the reflectance contrast parameter was elevated and correlated with image area fraction of India ink. After extensive scraping, polarized reflectance contrast and chondrocyte viability were lower than that from untreated explants. As part of this work, a mathematical model was developed and confirmed the trend in the reflectance signal due to changes in surface scattering and subsurface birefringence. These results demonstrate the effectiveness of polarized reflectance microscopy to sensitively assess surface microstructural alterations in articular cartilage explants.

Effects of pinealectomy and melatonin supplementation on endometrial explants in a rat model.

PubMed

Koc, Onder; Gunduz, Bülent; Topcuoglu, Ata; Bugdayci, Güler; Yilmaz, Fahri; Duran, Bülent

2010-11-01

To determine the effects of pinealectomy on endometrial explants in rats and evaluate the activity of superoxide dismutase (SOD) and catalase (CAT) and the levels of malondialdehyde (MDA) in the rat endometriosis model. Rats with experimentally induced endometriosis were randomly divided into three groups after second-look laparotomies. Group 1 (pinealectomy, n = 8) and Group 2 (pinealectomy+melatonin, n = 8) underwent pinealectomies after the second-look laparotomies. Group 3 was presented as control group (vehicle solution+without pinealectomy (n = 6)). Melatonin was administered intraperitoneally for 4 weeks in Group 2, whereas an equal volume of vehicle solution was given to Groups 1 and 3. Evaluation of the volume of the endometrial explants, histopathological examination and preservation of explant epitheliums according to the scoring system were undertaken. There was a statistically significant increase in spherical explant volumes of Group 1 compared to Groups 2 and 3. In Group 1, the level of MDA was significantly higher and SOD and CAT activity was significantly lower compared to Groups 2 and 3. A statistically significant increase in the epithelial lining scores of explants was noted in Group 1 compared to Groups 2 and 3. The effects of pinealectomy on the progression of endometriosis explants were reversed by melatonin. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Anti-Cancer Effects of Imperata cylindrica Leaf Extract on Human Oral Squamous Carcinoma Cell Line SCC-9 in Vitro.

PubMed

Keshava, Rohini; Muniyappa, Nagesh; Gope, Rajalakshmi; Ramaswamaiah, Ananthanarayana Saligrama

2016-01-01

Imperata cylindrica, a tall tufted grass which has multiple pharmacological applications is one of the key ingredients in various traditional medicinal formula used in India. Previous reports have shown that I. cylindrica plant extract inhibited cell proliferation and induced apoptosis in various cancer cell lines. To our knowledge, no studies have been published on the effect of I. cylindrica leaf extract on human oral cancers. The present study was undertaken in order to evaluate the anticancer properties of the leaf extract of I. cylindrica using an oral squamous cell carcinoma cell line SCC-9 as an in vitro model system. A methanol extract from dried leaves of I. cylindrica (ICL) was prepared by standard procedures. Effects of the ICL extract on the morphology of SCC-9 cells was visualized by microscopy. Cytotoxicity was determined by MTT assay. Effects of the ICL extract on colony forming ability of SCC-9 cells was evaluated using clonogenic assay. Cell cycle analysis was performed by flow cytometry and induction of apoptosis was determined by DNA fragmentation assay. The ICL extract treatment caused cytotoxicity and induced cell death in vitro in SCC-9 cells in a dose-dependent manner. This treatment also significantly reduced the clonogenic potential and inhibited cell proliferation by arresting the cell cycle in the G2/M phase. Furthermore, DNA fragmentation assays showed that the observed cell death was caused by apoptosis. This is the first report showing the anticancer activity of the methanol extracts from the leaves of I. cylindrica in human oral cancer cell line. Our data indicates that ICL extract could be considered as one of the lead compounds for the formulation of anticancer therapeutic agents to treat/manage human oral cancers. The natural abundance of I. cylindrica and its wide geographic distribution could render it one of the primary resource materials for preparation of anticancer therapeutic agents.

Investigation of biomaterials by human epithelial gingiva cells: an in vitro study

PubMed Central

2012-01-01

Introduction In modern medicine and dentistry the use of biomaterials is a fast developing field of increasing interest. Especially in dentistry the interaction between biomaterials like implant materials and the soft tissue in the oral cavity is in the focus of daily research. In this context the high importance of testing materials and their surfaces concerning their biocompatibility towards corresponding cells is very likely. For this purpose this study investigates cells derived from human gingival biopsies on different materials and surfaces. Methods Cells in this study were cultivated out of human biopsies by a grow out explant technique and were sub cultivated on titanium, zirconium dioxide and collagen membrane specimens. To characterise the cells on the material surfaces used in this study immunohistochemical and histological staining techniques as well as different methods of microscopy (light microscopy and SEM) were applied. Results With the aid of the explant technique and the chosen cell cultivation method it was possible to investigate the human gingiva derived cells on different materials. The data of the present study show that the human gingival cells attach and proliferate on all three tested materials by exhibiting characteristic gingival keratinocyte protein expression even after long periods of culture e.g. up to 70 days. Conclusions It could be shown that the three tested materials titanium, zirconium dioxide and collagen membrane (and their special surfaces) are good candidates for the application as materials in the dental gingival environment or, in the case of the collagen membrane as scaffold/cell-carrier for human gingival cells in tissue engineering. PMID:23241143

Preservation of Differentiation and Clonogenic Potential of Human Hematopoietic Stem and Progenitor Cells during Lyophilization and Ambient Storage

PubMed Central

Buchanan, Sandhya S.; Pyatt, David W.; Carpenter, John F.

2010-01-01

Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC) and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450±230 CFU-GM, 430±140 BFU-E, and 50±40 CFU-GEMM per 50 µL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25°C in the dark. Cells reconstituted immediately after lyophilization produced 580±90 CFU-GM (∼40%, relative to unprocessed controls p<0.0001), 170±70 BFU-E (∼40%, p<0.0001), and 41±22 CFU-GEMM (∼82%, p = 0.4171), and cells reconstituted after 28 days at room temperature produced 513±170 CFU-GM (∼35%, relative to unprocessed controls, p<0.0001), 112±68 BFU-E (∼26%, p<0.0001), and 36±17 CFU-GEMM (∼82%, p = 0.2164) These studies are the first to document high level retention of CFU-GEMM following lyophilization and storage for 4 weeks at 25°C. This type of flexible storage stability would potentially permit the ability to ship and store HPC without the need for refrigeration. PMID:20824143

Preservation of differentiation and clonogenic potential of human hematopoietic stem and progenitor cells during lyophilization and ambient storage.

PubMed

Buchanan, Sandhya S; Pyatt, David W; Carpenter, John F

2010-09-01

Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC) and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450+/-230 CFU-GM, 430+/-140 BFU-E, and 50+/-40 CFU-GEMM per 50 microL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25 degrees C in the dark. Cells reconstituted immediately after lyophilization produced 580+/-90 CFU-GM ( approximately 40%, relative to unprocessed controls p<0.0001), 170+/-70 BFU-E (approximately 40%, p<0.0001), and 41+/-22 CFU-GEMM (approximately 82%, p = 0.4171), and cells reconstituted after 28 days at room temperature produced 513+/-170 CFU-GM (approximately 35%, relative to unprocessed controls, p<0.0001), 112+/-68 BFU-E (approximately 26%, p<0.0001), and 36+/-17 CFU-GEMM ( approximately 82%, p = 0.2164) These studies are the first to document high level retention of CFU-GEMM following lyophilization and storage for 4 weeks at 25 degrees C. This type of flexible storage stability would potentially permit the ability to ship and store HPC without the need for refrigeration.

Identification of a role for the nuclear receptor EAR-2 in the maintenance of clonogenic status within the leukemia cell hierarchy

PubMed Central

Ichim, CV; Atkins, HL; Iscove, NN; Wells, RA

2016-01-01

Identification of genes that regulate clonogenicity of acute myelogenous leukemia (AML) cells is hindered by the difficulty of isolating pure populations of cells with defined proliferative abilities. By analyzing the growth of clonal siblings in low passage cultures of the cell line OCI/AML4 we resolved this heterogeneous population into strata of distinct clonogenic potential, permitting analysis of the transcriptional signature of single cells with defined proliferative abilities. By microarray analysis we showed that the expression of the orphan nuclear receptor EAR-2 (NR2F6) is greater in leukemia cells with extensive proliferative capacity than in those that have lost proliferative ability. EAR-2 is expressed highly in long-term hematopoietic stem cells, relative to short-term hematopoietic stem and progenitor cells, and is downregulated in AML cells after induction of differentiation. Exogenous expression of EAR-2 increased the growth of U937 cells and prevented the proliferative arrest associated with terminal differentiation, and blocked differentiation of U937 and 32Dcl3 cells. Conversely, silencing of EAR-2 by short-hairpin RNA initiated terminal differentiation of these cell lines. These data identify EAR-2 as an important factor in the regulation of clonogenicity and differentiation, and establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy. PMID:21637284

Sulfur Mustard (SM) Lesions in Organ-Cultured Human Skin: Markers of Injury and Inflammatory Mediators

DTIC Science & Technology

1990-04-16

18. SUB3ECT TERMS (oont’d) epidermal injury organ culture •ranuaear vacuoles C-leucine incorpora’tion by full-thickness human akin explants hi stamine ...mast- cell degranulation prostaglandin E2 lysobomal enzymes: acid phosphatase, B-glucuronidase, 0-galactcsidase, lysozyme and lactic dehydrogenase...that histamline (from local mast cells ), and PA and POgk (probably from mast cells and epidermal cells ) are s3e of the early mediators of the inflmma

"Not all breast implants are equal: a 13-year review of implant longevity and reasons for explantation".

PubMed

Van Slyke, Aaron C; Carr, Michael; Carr, Nicholas J

2018-06-04

Augmentation mammoplasty is the most common aesthetic procedure. Textured implants control implant position and have improved capsular contracture rates; however, the impact of texturing on longevity and clinical findings at explantation is unclear. All cases of explantation between January 2005 - April 2017 from an aesthetic practice were reviewed retrospectively. Patient demographics, implant characteristics, time-to-explantation, and clinical presentation and intraoperative findings at explantation were analyzed. 539 breast implants were explanted during the study period: 249 saline, 147 smooth gel, 123 Biocell, and 20 other nonaggressively textured breast implants. Average time from placement to explantation was 7.5 years, 5.6 years, 4.9 years, and 4.0 years for saline, other textured, smooth gel, and Biocell implants, respectively (p-value = 3.25e-08). The percentage of implants removed associated with implant performance failure was 50.3%, 57.5%, 75.0%, and 85.4% for smooth gel, saline, other textured, and Biocell implants, respectively (p-value = 7.25e-09). 21.1% of Biocell implants versus 1.5% of all other implants presented with pain (p-value = 2.71e-15). 45 Biocell implants had double capsules; this phenomenon was not observed with any other implant type (p-value = 5.85e-37). Seven Biocell implants had late seromas, compared to three late seromas with any other implant type (p-value = 0.0013). Here, we provide evidence that Biocell implants have the shortest time-to-explantation and the highest proportion of implants associated with implant performance failure. This information should complement the informed consent process when selecting an appropriate implant.

Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

PubMed

Lin, Yang; Gil, Chang-Hyun; Yoder, Mervin C

2017-11-01

The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony-forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols. © 2017 American Heart Association, Inc.

Cell motion predicts human epidermal stemness

PubMed Central

Toki, Fujio; Tate, Sota; Imai, Matome; Matsushita, Natsuki; Shiraishi, Ken; Sayama, Koji; Toki, Hiroshi; Higashiyama, Shigeki

2015-01-01

Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that α6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation. PMID:25897083

Oncogenic Ras: A double-edged sword for human epidermal stem and transient amplifying cells

PubMed Central

Dellambra, Elena

2016-01-01

ABSTRACT The human epidermal clonal evolution, i.e. the transition from stem cells (SCs) to transient amplifying (TA)-cells and post-mitotic cells, is a continuous and tightly regulated process that ensures physiologic tissue homeostasis. The Ras family of small GTPases has a key role in skin homeostasis and tumorigenesis. Indeed, activating mutations in Ras genes have been found in human cutaneous squamous cell carcinomas (cSCCs) and in experimentally-induced murine cSCCs. In mouse models, the Ras signaling might lead to hyperproliferative phenotypes, including the development of cSCCs, depending on the nature of the founding cells. Tumor-initiating cells or Cancer Stem Cells (CSCs) have been demonstrated in murine and human cSCCs even if the mechanism of their development from normal SCs or TA-cells is not completely elucidated. Here, the relation between the Ras expression outcome and the clonogenic potential of the target keratinocyte is discussed. PMID:27111451

Effects of NAA and BAP, double-layered media, and light distance on in vitro regeneration of Nelumbo nucifera Gaertn. (lotus), an aquatic edible plant.

PubMed

Mahmad, Noraini; Taha, Rosna Mat; Othman, Rashidi; Saleh, Azani; Hasbullah, Nor Azlina; Elias, Hashimah

2014-01-01

In vitro direct regeneration of Nelumbo nucifera Gaertn. was successfully achieved from immature explants (yellow plumule) cultured on a solid MS media supplemented with combinations of 0.5 mg/L BAP and 1.5 mg/L NAA which resulted in 16.00 ± 0.30 number of shoots per explant and exhibited a new characteristic of layered multiple shoots, while normal roots formed on the solid MS basal media. The double-layered media gave the highest number of shoots per explant with a ratio of 2 : 1 (liquid to solid) with a mean number of 16.67 ± 0.23 shoots per explant with the formation of primary and secondary roots from immature explants. In the study involving light distance, the tallest shoot (16.67 ± 0.23 mm) obtained from the immature explants was at a light distance of 200 mm from the source of inflorescent light (1000 lux). The plantlets were successfully acclimatized in clay loam soil after 8 months being maintained under in vitro conditions.

Mechanisms underlying the organizer formation in Bufo arenarum embryos.

PubMed

Manes, M E; Nieto, O L

1989-06-01

In the early gastrula of Bufo arenarum the prospective mesoderm was previously identified as a marginal belt of grey cells. To analyze their differentiation capacity explants of these cells were cultured within ectodermal vesicles, in isolation and in combination with vegetal components. When cultured in isolation, dorsal and ventral fragments from the deep marginal zone behaved differently. Whilst ventral explants produced blood cells, dorsal explants failed to differentiate, remaining as masses of yolk-laden cells. On the other hand, both cultures were drastically modified when associated with superficial cells from the blastoporal zone, which caused the following effects: a) Promotion of differentiation in dorsal marginal explants, able now to produce notochordal and somitic structures, in addition to mesenchymatic cells. b) Promotion of dorsalization in ventral marginal explants, which changed their expected destiny developing axial components, similar to those furnished by "activated" dorso marginal explants. On the contrary, combined cultures of animal and vegetal pieces were unable to generate mesodermal structures. These studies suggest that the axial mesoderm, identified as the "organizer", develops from a marginal substrate of genuine mesodermal cells through a dorsalizing inductive stimulus originated in superficial periblastoporal cells.

FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2

PubMed Central

Kim, Dong Geon; Cho, Hee Jin; Kim, Yeonghwan; Rheey, Jinguen; Shin, Kayoung; Seo, Yun Jee; Choi, Yeon-Sook; Lee, Jung-Il; Lee, Jeongwu; Joo, Kyeung Min; Nam, Do-Hyun

2015-01-01

Glioblastoma (GBM) is the most aggressive and most lethal brain tumor. As current standard therapy consisting of surgery and chemo-irradiation provides limited benefit for GBM patients, novel therapeutic options are urgently required. Forkhead box M1 (FoxM1) transcription factor is an oncogenic regulator that promotes the proliferation, survival, and treatment resistance of various human cancers. The roles of FoxM1 in GBM remain incompletely understood, due in part to pleotropic nature of the FoxM1 pathway. Here, we show the roles of FoxM1 in GBM stem cell maintenance and radioresistance. ShRNA-mediated FoxM1 inhibition significantly impeded clonogenic growth and survival of patient-derived primary GBM cells with marked downregulation of Sox2, a master regulator of stem cell phenotype. Ectopic expression of Sox2 partially rescued FoxM1 inhibition-mediated effects. Conversely, FoxM1 overexpression upregulated Sox2 expression and promoted clonogenic growth of GBM cells. These data, with a direct binding of FoxM1 in the Sox2 promoter region in GBM cells, suggest that FoxM1 regulates stemness of primary GBM cells via Sox2. We also found significant increases in FoxM1 and Sox2 expression in GBM cells after irradiation both in vitro and in vivo orthotopic tumor models. Notably, genetic or a small-molecule FoxM1 inhibitor-mediated FoxM1 targeting significantly sensitized GBM cells to irradiation, accompanying with Sox2 downregulation. Finally, FoxM1 inhibition combined with irradiation in a patient GBM-derived orthotopic model significantly impeded tumor growth and prolonged the survival of tumor bearing mice. Taken together, these results indicate that the FoxM1-Sox2 signaling axis promotes clonogenic growth and radiation resistance of GBM, and suggest that FoxM1 targeting combined with irradiation is a potentially effective therapeutic approach for GBM. PMID:26444992

FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2.

PubMed

Lee, Yeri; Kim, Kang Ho; Kim, Dong Geon; Cho, Hee Jin; Kim, Yeonghwan; Rheey, Jinguen; Shin, Kayoung; Seo, Yun Jee; Choi, Yeon-Sook; Lee, Jung-Il; Lee, Jeongwu; Joo, Kyeung Min; Nam, Do-Hyun

2015-01-01

Glioblastoma (GBM) is the most aggressive and most lethal brain tumor. As current standard therapy consisting of surgery and chemo-irradiation provides limited benefit for GBM patients, novel therapeutic options are urgently required. Forkhead box M1 (FoxM1) transcription factor is an oncogenic regulator that promotes the proliferation, survival, and treatment resistance of various human cancers. The roles of FoxM1 in GBM remain incompletely understood, due in part to pleotropic nature of the FoxM1 pathway. Here, we show the roles of FoxM1 in GBM stem cell maintenance and radioresistance. ShRNA-mediated FoxM1 inhibition significantly impeded clonogenic growth and survival of patient-derived primary GBM cells with marked downregulation of Sox2, a master regulator of stem cell phenotype. Ectopic expression of Sox2 partially rescued FoxM1 inhibition-mediated effects. Conversely, FoxM1 overexpression upregulated Sox2 expression and promoted clonogenic growth of GBM cells. These data, with a direct binding of FoxM1 in the Sox2 promoter region in GBM cells, suggest that FoxM1 regulates stemness of primary GBM cells via Sox2. We also found significant increases in FoxM1 and Sox2 expression in GBM cells after irradiation both in vitro and in vivo orthotopic tumor models. Notably, genetic or a small-molecule FoxM1 inhibitor-mediated FoxM1 targeting significantly sensitized GBM cells to irradiation, accompanying with Sox2 downregulation. Finally, FoxM1 inhibition combined with irradiation in a patient GBM-derived orthotopic model significantly impeded tumor growth and prolonged the survival of tumor bearing mice. Taken together, these results indicate that the FoxM1-Sox2 signaling axis promotes clonogenic growth and radiation resistance of GBM, and suggest that FoxM1 targeting combined with irradiation is a potentially effective therapeutic approach for GBM.

WE-G-BRE-08: Radiosensitization by Olaparib Eluting Nanospheres

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

Tangutoori, S; Kumar, R; Sridhar, S

2014-06-15

Purpose: Permanent prostate brachytherapy often uses inert bio-absorbable spacers to achieve the desired geometric distribution of sources within the prostate. Transforming these spacers into implantable nanoplatforms for chemo-radiation therapy (INCeRT) provides a means of providing sustained in-situ release of radiosensitizers in the prostate to enhance the therapeutic ratio of the procedure. Olaparib, a PARP inhibitor, suppresses DNA repair processes present during low dose rate continuous irradiation. This work investigates the radiosensitizing/DNA damage repair inhibition by NanoOlaparib eluting nanospheres. Methods: Human cell line PC3 (from ATCC), was maintained in F12-k medium supplemented with fetal bovine serum. Clonogenic assay kit (from Fischermore » Scientific) was used to fix and stain the cells to determine the long term effects of irradiation. Nanoparticle size and zeta potential of nanospheres were determined using a Zeta particle size analyzer. The incorporation of Olaparib in nanospheres was evaluated by HPLC. Irradiation was performed in a small animal irradiator operating at 220 KeV.The long term effects of radio-sensitization with olaparib and nanoolaparib was determined using the clonogenic assay at 2 Gy and 4 Gy doses. The cells were allowed to grow for around 10 doubling cycles, The colonies were fixed and stained using clonogenic assay kit. The excess stain was washed off using DI water and the images were taken using a digital camera. Results: Radiosensitization studies were carried out in prostate cancer cell line, PC3 radiation at 0, 2 and 4Gy doses. Strongest dose response was observed with nanoolaparib treated cells compared to untreated cells. Conclusion: A two stage drug release of drug eluting nanospheres from a biodegradable spacer has been suggested for sustained in-situ release of Olaparib to suppress DNA repair processes during prostate brachytherapy. The Olaparib eluting nanospheres had the same in-vitro radiosensitizing effect as free olaparib. DOD 1R21CA16977501, A. David Mazzone Awards Program 2012PD164.« less

miR-219-5p inhibits proliferation and clonogenicity in chordoma cells and is associated with tumor recurrence

PubMed Central

Wei, Wei; Zhang, Qiuhang; Wang, Zhenlin; Yan, Bo; Feng, Yanjun; Li, Pu

2016-01-01

Chordoma is a rare malignant bone tumor that is usually localized to the skull base, vertebral column and sacrum. The transcription factor brachyury, which is encoded by the T gene, has a critical role in the development and progression of chordoma, although the mechanisms underlying brachyury regulation remain unclear. The aim of the current study was to identify and characterize microRNAs (miRs) that regulate brachyury expression in chordoma. MicroRNAs that target brachyury were predicted using miRanda and TargetScan. Using reverse transcription-quantitative polymerase chain reaction, miR-219-5p was shown to be significantly downregulated in chordoma tissues and the U-CH2 chordoma cell lines. A dual-luciferase reporter assay was used to validate the inhibitory effect of miR-219-5p on brachyury mRNA expression. The expression level of brachyury was downregulated in U-CH2 cells following transfection with miR-219-5p mimics and upregulated following transfection with the miR-219-5p inhibitor. The effects of miR-219-5p on the proliferation and clonogenicity of chordoma cells were assessed using cell counting kit-8, EdU and clone formation assays. These in vitro results indicated that miR-219-5p may have an important role in regulating the cell proliferation and clonogenicity of human chordoma cells, potentially by targeting brachyury. Furthermore, the associations between the expression levels of miR-219-5p and various clinicopathological factors were analyzed, and miR-219-5p expression was shown to correlate with tumor extent and recurrence. These results suggested that miR-219-5p functions as a tumor suppressor in chordoma and, therefore, that miR-219-50 may be a potential target for therapeutic intervention. PMID:28105164

A Caspase-Resistant Form of Bcl-XL, but Not Wild Type Bcl-XL, Promotes Clonogenic Survival After Ionizing Radiation

PubMed Central

Rehemtulla, Alnawaz; Hamilton, A Christin; Taneja, Neelam; Fridman, Jordan; Juan, Todd SC; Maybaum, Jonathan; Chinnaiyan, Arul

1999-01-01

Abstract Bcl-2 and Bcl-XL belong to a family of proteins overexpressed in a variety of human cancers which inhibit apoptosis in response to a number of stimuli including chemotherapeutic agents and ionizing radiation. To better understand the role of these polypeptides in modulating the response of cancer cells to ionizing radiation we used cell lines that were engineered to overexpress the two polypeptides. Although Bcl-2 and Bcl-XL overexpression resulted in inhibition of radiation-induced apoptosis, it did not result in enhanced clonogenic survival. Consistent with this was the observation that Bcl-2 and Bcl-XL protected cells from DNA fragmentation, loss of mitochondrial membrane potential, and caspase activation for up to 72 hours after irradiation. Beyond 72 hours, there was a rapid loss in the ability of Bcl-2 and Bcl-XL to inhibit these markers of apoptosis. When Bcl-XL was analyzed at 72 hours after irradiation and beyond, a rapid accumulation of a 16-kDa form of Bcl-XL was observed. To test the hypothesis that cleavage of the 29-kDa form of Bcl-XL by caspases to a 16-kDa polypeptide results in its inability to inhibit apoptosis beyond 72 hours, we constructed a cell line that overexpressed a caspase-resistant form of Bcl-XL Bcl-XLΔloop. Cells overexpressing Bcl-XL-Δloop were resistant to apoptosis beyond 72 hours after irradiation and did not contain the 16-kDa form at these time points. In addition, Bcl-XL-Δloop overexpression resulted in enhanced clonogenic survival compared with control or Bcl-XL overexpressing cells. These results provide a molecular basis for the observation that expression of Bcl-2 or Bcl-XL is not a prognostic marker of tumor response to cancer therapy. PMID:10935471

Combinatorial cytotoxic effects of Curcuma longa and Zingiber officinale on the PC-3M prostate cancer cell line

PubMed Central

Kurapati, Kesava Rao V.; Samikkannu, Thangavel; Kadiyala, Dakshayani B.; Zainulabedin, Saiyed M.; Gandhi, Nimisha; Sathaye, Sadhana S.; Indap, Manohar A.; Boukli, Nawal; Rodriguez, Jose W.; Nair, Madhavan P.N.

2015-01-01

Background Many plant-derived products exhibit potent chemopreventive activity against animal tumor models as well as rodent and human cancer cell lines. They have low side effects and toxicity and presumably modulate the factors that are critical for cell proliferation, differentiation, senescence and apoptosis. The present study investigates the effects of some medicinal plant extracts from generally recognized as safe plants that may be useful in the prevention and treatment of cancer. Methods Clonogenic assays using logarithmically-growing cells were performed to test the effect. The cytotoxic effects of Curcuma longa and Zingiber officinale were studied using sulforhodamine B assay, tetrazolium dye assay, colony morphology and microscopic analysis. Results Out of the 13 lyophilized plant-derived extracts evaluated for growth-inhibitory effects on the PC-3M prostate cancer cell line, two extracts derived from C. longa and Z. officinale showed significant inhibitory effects on colony-forming ability. The individual and augmentative effects of these two extracts were tested for their narrow range effective lower concentration on PC-3M in clonogenic assays. At relatively lower concentrations, C. longa showed significant inhibition of colony formation in clonogenic assays; whereas at same concentrations Z. officinale showed only moderate inhibitory effects. However, when both the agents were tested together at the same concentrations, the combined effects were much more significant than their individual ones. On normal prostate epithelial cells both C. longa and Z. officinale had similar effects but at a lower magnitude. These observations were confirmed by several cytotoxicity assays involving the morphological appearance of the colonies, microscopic observations, per cent inhibition in comparison to control by sulforhodamine B and tetrazolium dye assay. Conclusions From these observations, it was concluded that the combined effects of C. longa and Z. officinale are much greater than their individual effects, suggesting the role of multiple components and their synergistic mode of actions to elicit stronger beneficial effects. PMID:23072849

Multiple shoot production from seedling explants of slash pine (Pinus elliottii, Engelm.).

PubMed

Burns, J A; Schwarz, O J; Schlarbaum, S E

1991-11-01

Hypocotylary explants obtained from 30- to 40-day-old slash pine (Pinus elliottii, Engelm.) seedlings treated with 6-benzylaminopurine produced multiple buds that eventually elongated into axillary shoots. The explants were pulse treated (45-s dip) with 6-benzylaminopurine (22.2, 111, 222 μM) plus a control and cultured on three different basal media containing activated charcoal (0.5% w/v). Hormonal concentration and basal medium were compared for the number and size of axillary shoots induced after 12 and 29 days. The greatest number of axillary shoots was produced by explants that were pulse treated with 111 μM 6-benzylaminopurine and cultured on Gresshoff and Doy medium. The axillary shoots were fewer in number per explant than shoots previously reported resulting from hormonally induced advantitious buds of slash pine, but the axillary shoots developed more rapidly.

Organogenesis from transformed tomato explants.

PubMed

Frary, Anne; Van Eck, Joyce

2005-01-01

Tomato was one of the first crops for which a genetic transformation system was reported involving regeneration by organogenesis from Agrobacterium-transformed explants. Since the initial reports, various factors have been studied that affect the efficiency of tomato transformation and the technique has been useful for the isolation and identification of many genes involved in plant disease resistance, morphology and development. In this method, cotyledon explants from in vitro-grown seedlings are precultured overnight on a tobacco suspension feeder layer. The explants are then inoculated with Agrobacterium and returned to the feeder layer for a 2-d period of cocultivation. After cocultivation, the explants are transferred to an MS-based selective regeneration medium containing zeatin. Regenerated shoots are then rooted on a separate selective medium. This protocol has been used with several tomato cultivars and routinely yields transformation efficiencies of 10-15%.

1,25D3 potentiates cisplatin antitumor activity by p73 induction in a squamous cell carcinoma model

PubMed Central

Ma, Yingyu; Yu, Wei-Dong; Hershberger, Pamela A.; Flynn, Geraldine; Kong, Rui-Xian; Trump, Donald L.; Johnson, Candace S.

2008-01-01

1,25D3 exhibits anti-tumor activity in a variety of cancers including squamous cell carcinoma (SCC). Intrinsic resistance of SCC cells to cisplatin was observed and led to the investigation into whether 1,25D3 sensitizes SCC cells to cisplatin. Pretreatment with 1,25D3 followed by cisplatin enhanced growth inhibition in SCC cells compared with 1,25D3 alone, as assessed by cytotoxicity and in vitro clonogenic assays. In addition, 1,25D3 sensitized SCC cells to cisplatin-mediated apoptosis. Treatment of tumor-bearing C3H mice with 1,25D3 prior to cisplatin reduced clonogenic survival using in vivo excision clonogenic assay. These results were not observed in a 1,25D3-resistant SCC variant, indicating the critical role of 1,25D3 in sensitizing SCC cells to cisplatin. Further, a marked decrease in fractional tumor volume was observed when SCC tumor-bearing mice were treated with 1,25D3 prior to cisplatin as compared to either agent administered alone. Cisplatin has been shown to modulate p73 protein level in certain cancer cells. Our data showed that p73 level was not affected by cisplatin, but increased by 1,25D3 in SCC cells. Knocking down p73 by siRNA protected SCC cells against 1,25D3 and cisplatin-mediated clonogenic cell kill and apoptosis. Increasing p73 protein level by knocking down UFD2a, which mediates p73 degradation, promoted 1,25D3 and cisplatin-mediated clonogenic cell kill. These results suggest that 1,25D3 potentiates cisplatin anti-tumor activity in vitro and in vivo in a SCC model system, possibly through p73 induction and apoptosis. The combination treatment may provide a more effective therapeutic regimen in cancer treatment. PMID:18790784

An early look at the Organ Procurement and Transplantation Network explant pathology form data.

PubMed

Harper, Ann M; Edwards, Erick; Washburn, W Kenneth; Heimbach, Julie

2016-06-01

In April 2012, the Organ Procurement and Transplantation Network (OPTN) implemented an online explant pathology form for recipients of liver transplantation who received additional wait-list priority for their diagnosis of hepatocellular carcinoma (HCC). The purpose of the form was to standardize the data being reported to the OPTN, which had been required since 2002 but were submitted to the OPTN in a variety of formats via facsimile. From April 2012 to December 2014, over 4500 explant forms were submitted, allowing for detailed analysis of the characteristics of the explanted livers. Data from the explant pathology forms were used to assess agreement with pretransplant imaging. Explant data were also used to assess the risk of recurrence. Of those with T2 priority, 55.7% were found to be stage T2 on explant. Extrahepatic spread (odds ratio [OR] = 6.8; P < 0.01), poor tumor differentiation (OR = 2.8; P < 0.01), microvascular invasion (OR = 2.6; P < 0.01), macrovascular invasion (OR = 3.2; P < 0.01), and whether the Milan stage based on the number and size of tumors on the explant form was T4 (OR = 2.4; P < 0.01) were the strongest predictors of recurrence. In conclusion, this analysis confirms earlier findings that showed an incomplete agreement between pretransplant imaging and posttransplant pathology in terms of HCC staging, though the number of patients with both no pretransplant treatment and no tumor in the explant was reduced from 20% to <1%. In addition, several factors were identified (eg, tumor burden, age, sex, region, ablative therapy, alpha-fetoprotein, Milan stage, vascular invasion, satellite lesions, etc.) that were predictive of HCC recurrence, allowing for more targeted surveillance of high-risk recipients. Continued evaluation of these data will help shape future guidelines or policy recommendations. Liver Transplantation 22 757-764 2016 AASLD. © 2016 American Association for the Study of Liver Diseases.

Interleukin-1beta-induced extracellular matrix degradation and glycosaminoglycan release is inhibited by curcumin in an explant model of cartilage inflammation.

PubMed

Clutterbuck, Abigail L; Mobasheri, Ali; Shakibaei, Mehdi; Allaway, David; Harris, Pat

2009-08-01

Osteoarthritis (OA) is a degenerative and inflammatory disease of synovial joints that is characterized by the loss of articular cartilage, for which there is increasing interest in natural remedies. Curcumin (diferuloylmethane) is the main polyphenol in the spice turmeric, derived from rhizomes of the plant Curcuma longa. Curcumin has potent chemopreventive properties and has been shown to inhibit nuclear factor kappaB-mediated inflammatory signaling in many cell types, including chondrocytes. In this study, normal articular cartilage was harvested from metacarpophalangeal and metatarsophalangeal joints of eight horses, euthanized for reasons other than research purposes, to establish an explant model mimicking the inflammatory events that occur in OA. Initially, cartilage explants (N= 8) were stimulated with increasing concentrations of the proinflammatory cytokine IL-1beta to select effective doses for inducing cartilage degeneration in the explant model. Separate cartilage explants were then cotreated with IL-1beta at either 10 ng/mL (n= 3) or 25 ng/mL (n= 3) and curcumin (0.1 micromol/L, 0.5 micromol/L, 1 micromol/L, 10 micromol/L, and 100 micromol/L). After 5 days, the percentage of glycosaminoglycan (GAG) release from the explants was assessed using a dimethylmethylene blue colorimetric assay. Curcumin (100 micromol/L) significantly reduced IL-1beta-stimulated GAG release in the explants by an average of 20% at 10 ng/mL and 27% at 25 ng/mL back to unstimulated control levels (P < 0.001). Our results suggest that this explant model effectively simulates the proinflammatory cytokine-mediated release of articular cartilage components seen in OA. Furthermore, the evidence suggests that the inflammatory cartilage explant model is useful for studying the effects of curcumin on inflammatory pathways and gene expression in IL-1beta-stimulated chondrocytes.

Hypothalamic interactions between neuropeptide Y, agouti-related protein, cocaine- and amphetamine-regulated transcript and alpha-melanocyte-stimulating hormone in vitro in male rats.

PubMed

Dhillo, W S; Small, C J; Stanley, S A; Jethwa, P H; Seal, L J; Murphy, K G; Ghatei, M A; Bloom, S R

2002-09-01

A number of neuropeptides implicated in the hypothalamic regulation of appetite are synthesized in the arcuate nucleus (Arc). Neuropeptide Y (NPY) and agouti-related protein (Agrp) are orexigenic. The pro-opiomelanocortin (POMC) product alpha-melanocyte-stimulating hormone (alpha-MSH) is anorectic. Intracerebroventricular administration of cocaine- and amphetamine-regulated transcript (CART) decreases food intake. However, recent results show that CART is orexigenic when injected into discrete hypothalamic nuclei. There is almost complete coexpression of NPY and Agrp mRNA in Arc neurones, and the majority of CART-containing neurones in the Arc also contain POMC mRNA. We investigated possible interactions between these neuropeptides in vitro using a rat hypothalamic explant system. Administration of 1, 10 and 100 nm of NPY to hypothalamic explants significantly increased release of Agrp(83-132)-immunoreactivity (IR). NPY (10 and 100 nm) significantly increased the release of CART(55-102)-IR and alpha-MSH-IR from hypothalamic explants. Agrp(83-132) (10 nm) administered to hypothalamic explants significantly increased the release of NPY-IR. Agrp(83-132) (10 and 100 nm) significantly decreased the release of CART(55-102)-IR from hypothalamic explants. Administration of 1, 10 and 100 nm CART(55-102) to hypothalamic explants resulted in a significant increase in NPY-IR release. Administration of 10 nm CART(55-102) to hypothalamic explants significantly increased the release of Agrp(83-132)-IR. NDP-MSH (10 nm) administered to hypothalamic explants significantly increased the release of NPY-IR. NDP-MSH (10 and 100 nm) significantly increased the release of Agrp(83-132)-IR from hypothalamic explants. These data suggest that orexigenic neuropeptides in the arcuate nucleus stimulate the release of each other, perhaps reinforcing orexigenic behaviour via a positive-feedback loop. Our results are also in keeping with the possibility that the melanocortin-3 receptor in the arcuate nucleus may influence the release of arcuate neuropeptides.

High frequency organogenesis in hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

PubMed

Kumar, Pankaj; Srivastava, D K

2015-04-01

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.

In Vitro and Cryopreservation Techniques for Conservation of Snow Mountain Garlic.

PubMed

Mahajan, Ritu

2016-01-01

Garlic is an important medicinal herb of culinary value by imparting its flavors and odors to the food. Allicin, a notable flavonoid in garlic, is a powerful antibiotic and antifungal compound. Due to poor bioavailability, garlic is of limited use for oral human consumption. Being sexually sterile, propagation of garlic is done by individual cloves from a bulb which increases the chances of transfer of viral diseases. In this chapter, an efficient and improved regeneration protocol for explant establishment and shoot multiplication under in vitro conditions is described. A high rate of shoot multiplication is obtained on MS medium supplemented with 0.5 mg/l BAP, 1.0 mg/l KN, and 2.0 mg/l GA3. Addition of 1.0 mg/l NAA to MS medium resulted in rooting at the shoot bases. A detailed method for encapsulation of explant in sodium alginate beads and their cryopreservation using encapsulation-dehydration is also described.

Effects of Selective Checkpoint Kinase 1 Inhibition on Cytarabine Cytotoxicity in Acute Myelogenous Leukemia Cells in Vitro

PubMed Central

Schenk, Erin L.; Koh, Brian D.; Flatten, Karen S.; Peterson, Kevin L.; Parry, David; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Karnitz, Larry M.; Kaufmann, Scott H.

2012-01-01

Purpose Previous studies have demonstrated that the replication checkpoint, which involves the kinases ATR and Chk1, contributes to cytarabine resistance in cell lines. In the present study, we examined whether this checkpoint is activated in clinical AML during cytarabine infusion in vivo and then assessed the impact of combining cytarabine with the recently described Chk1 inhibitor SCH 900776 in vitro. Experimental design AML marrow aspirates harvested before and during cytarabine infusion were examined by immunoblotting. Human AML lines treated with cytarabine in the absence or presence of SCH 900776 were assayed for checkpoint activation by immunoblotting, nucleotide incorporation into DNA and flow cytometry. Long-term effects in AML lines, clinical AML isolates, and normal myeloid progenitors were assayed using clonogenic assays. Results Immunoblotting demonstrated increased Chk1 phosphorylation, a marker of checkpoint activation, in over half of Chk1-containing AMLs after 48 h of cytarabine infusion. In human AML lines, SCH 900776 not only disrupted cytarabine-induced Chk1 activation and S phase arrest, but also markedly increased cytarabine-induced apoptosis. Clonogenic assays demonstrated that SCH 900776 enhanced the anti-proliferative effects of cytarabine in AML cell lines and clinical AML samples at concentrations that had negligible impact on normal myeloid progenitors. Conclusions These results not only provide evidence for cytarabine-induced S phase checkpoint activation in AML in the clinical setting, but also show that a selective Chk1 inhibitor can overcome the S phase checkpoint and enhance the cytotoxicity of cytarabine. Accordingly, further investigation of the cytarabine/SCH 900776 combination in AML appears warranted. PMID:22869869

The effects of the covalent attachment of 3-(4-hydroxy-3,5-di-tert-butylphenyl) propyl amine to glutaraldehyde pretreated bovine pericardium on structural degeneration, oxidative modification, and calcification of rat subdermal implants.

PubMed

Christian, Abigail J; Alferiev, Ivan S; Connolly, Jeanne M; Ischiropoulos, Harry; Levy, Robert J

2015-07-01

Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated heterograft materials, porcine aortic valves or bovine pericardium (BP), are widely used in cardiac surgery. BHV progressively fail in clinical use due to structural degeneration. Previously we reported that dityrosine, an oxidized amino acid, was present in failed clinical BP-BHV explants; unimplanted BP had no detectable dityrosine. In the same studies BP were demonstrated in vitro to be susceptible to oxidative damage, that could be mitigated with BP covalently modified with the antioxidant, 3-(4-hydroxy-3,5-di-tert-butylphenyl)propyl amine (DBP). The present studies compared in rat subdermal implants glutaraldehyde pretreated BP to BP modified with either DBP or the chemical reactions used to link DBP. All BP explants regardless of DBP demonstrated reduced hydroxyproline and increased digestibility by collagenase. However, the DBP-BP explants showed significant inhibition of reduced explant shrink temperatures (an index of crosslinking) as compared with control BP. Significant mitigation of calcification was observed in both the BP-DBP and chemically modified explants as compared with BP. Dityrosine was not detectable in the 90 day explants. It is concluded that rat subdermal BP implants undergo both calcific and noncalcific structural degeneration, but without the formation of dityrosine, unlike clinical BP explants. © 2014 Wiley Periodicals, Inc.

The effects of the covalent attachment of 3-(4-hydroxy-3,5-di-tert-butylphenyl)propyl amine to glutaraldehyde pre-treated bovine pericardium on structural degeneration, oxidative modification and calcification of rat subdermal implants

PubMed Central

Christian, Abigail J.; Alferiev, Ivan S.; Connolly, Jeanne M.; Ischiropoulos, Harry; Levy, Robert J.

2014-01-01

Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated heterograft materials, porcine aortic valves or bovine pericardium (BP), are widely used in cardiac surgery. BHV progressively fail in clinical use due to structural degeneration. Previously we reported that dityrosine, an oxidized amino acid, was present in failed clinical BP-BHV explants; unimplanted BP had no detectable dityrosine. In the same studies BP were demonstrated in vitro to be susceptible to oxidative damage, that could be mitigated with BP covalently modified with the antioxidant, 3-(4-hydroxy-3,5-di-tert-butylphenyl)propyl amine (DBP). The present studies compared in rat subdermal implants glutaraldehyde pretreated BP to BP modified with either DBP or the chemical reactions used to link DBP. All BP explants regardless of DBP demonstrated reduced hydroxyproline and increased digestibility by collagenase. However, the DBP-BP explants showed significant inhibition of reduced explant shrink temperatures (an index of crosslinking) compared to control BP. Significant mitigation of calcification was observed in both the BP-DBP and chemically modified explants compared to BP. Dityrosine was not detectable in the 90 day explants. It is concluded that rat subdermal BP implants undergo both calcific and non-calcific structural degeneration, but without the formation of dityrosine, unlike clinical BP explants. PMID:25546235

Highly Tumorigenic Diffuse Large B Cell Lymphoma Cells Are Produced by Coculture with Stromal Cells.

PubMed

Lin, Zhiguang; Chen, Bobin; Wu, Ting; Xu, Xiaoping

2018-05-23

Diffuse large B cell lymphoma (DLBCL) is heterogeneous. We aimed to explore how tumor microenvironment promotes lymphoma cell aggressiveness and heterogeneity. We created a coculture system using human DLBCL cells and mouse bone marrow stromal cells. Proliferative capacity, drug resistance, clonogenicity, and tumorigenicity were compared in lymphoma cells from the coculture system and lymphoma cells cultured alone. Expression of Notch signaling associated genes was evaluated using real-time reverse transcriptase PCR and Western blot. Lymphoma cells in the coculture system differentiated into a suspended cell group and an adherent cell group. They acquired a stronger proliferative capacity and drug resistance than lymphoma cells cultured alone, and differences existed between the adherent cell and suspended cell groups. The suspended cell group acquired the most powerful clonogenic and tumorigenic potential. However, Notch3 was exclusively expressed in the adherent lymphoma cell group and the use of N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, an inhibitor of Notch pathway, could abolish the emergence of highly aggressive lymphoma cells. Highly tumorigenic lymphoma cells could be generated by coculture with stromal cells, and it was dependent on Notch3 expression in the adjacent lymphoma cells through interaction with stromal cells. © 2018 S. Karger AG, Basel.

Synergistic Integration of Mesenchymal Stem Cells and Hydrostatic Pressure in the Expansion and Maintenance of Human Hematopoietic/Progenitor Cells

PubMed Central

2018-01-01

Ex vivo expansion of hematopoietic stem/progenitor cell (HSPC) has been investigated to improve the clinical outcome of HSPC transplantation. However, ex vivo expansion of HSPCs still faces a major obstacle in that HPSCs tend to differentiate when proliferating. Here, we cocultured HSPCs with mesenchymal stem cells (MSCs) and divided the HSPCs into two fractions according to whether they came into adherent to MSCs or not. Additionally, we used hydrostatic pressure (HP) to mimic the physical conditions in vivo. Even nonadherent cells expanded to yield a significantly larger number of total nucleated cells (TNCs), adherent cells maintained the HSPC phenotype (CD34+, CD34+CD38−, and CD133+CD38−) to a greater extent than nonadherent cells and had superior clonogenic potential. Moreover, applying HP significantly increased the number of TNCs, the frequency of the immature HSPC phenotype, and the clonogenic potential. Furthermore, the genetic markers for the HSPC niche were significantly increased under HP. Our data suggest that the nonadherent fraction is the predominant site of HSPC expansion, whereas the adherent fraction seems to mimic the HSPC niche for immature cells. Moreover, HP has a synergistic effect on expansion and functional maintenance. This first study utilizing HP has a potential of designing clinically applicable expansion systems. PMID:29681947

Identification of multipotent stem cells from adult dog periodontal ligament.

PubMed

Wang, Wen-Jun; Zhao, Yu-Ming; Lin, Bi-Chen; Yang, Jie; Ge, Li-Hong

2012-08-01

Periodontal diseases, which are characterized by destruction of the connective tissues responsible for restraining the teeth within the jaw, are the main cause of tooth loss. Periodontal regeneration mediated by human periodontal ligament stem cells (hPDLSCs) may offer an alternative strategy for the treatment of periodontal disease. Dogs are a widely used large-animal model for the study of periodontal-disease progression, tissue regeneration, and dental implants, but little attention has been paid to the identification of the cells involved in this species. This study aimed to characterize stem cells isolated from canine periodontal ligament (cPDLSCs). The cPDLSCs, like hPDLSCs, showed clonogenic capability and expressed the mesenchymal stem cell markers STRO-1, CD146, and CD105, but not CD34. After induction of osteogenesis, cPDLSCs showed calcium accumulation in vitro. Moreover, cPDLSCs also showed both adipogenic and chondrogenic potential. Compared with cell-free controls, more cementum/periodontal ligament-like structures were observed in CB-17/SCID mice into which cPDLSCs had been transplanted. These results suggest that cPDLSCs are clonogenic, highly proliferative, and have multidifferentiation potential, and that they could be used as a new cellular therapeutic approach to facilitate successful and more predictable regeneration of periodontal tissue using a canine model of periodontal disease. © 2012 Eur J Oral Sci.

Zeatin and Thidiazuron Induced Embryogenic Calli From In Vitro Leaf and Stem of Jojoba (Simmondsia chinensis).

PubMed

El-Ashry, Amal Abd El-Latif; Gabr, Ahmed Mohamed Magdy; Bekheet, Shawky Abd El-Hamid

2017-01-01

Jojoba is a promising industrial plant, which recommended with pharmaceutical benefits. The present study was conducted to stimulate embryogenic calli formation from jojoba using zeatin and thidiazuron (TDZ), as well as determination of the antioxidant activity of proliferated calli. For callus induction, leaf and stem explants derived from in vitro grown shootlets, were cultured on Murashige and Skoog (MS) medium with different combinations of 0.5 mg L-1 benzyl adenine (BA) or kinetin with 2,4-Dichlorophenoxyacetic acid (2,4-D), Naphthalene acetic acid (NAA) and picloram at 0.5 or 1mg L-1. To stimulate embryogenic calli, friable callus were transferred to woody plant medium (WPM) supplemented with different concentrations of zeatin or TDZ. Antioxidant activity of different treatments was determined using hexane or petroleum ether extraction. Data was analyzed as mean±standard deviation (SD). The MS medium supplemented with 0.5 mg L-1 BA+0.5 or 1 mg L-1 picloram was the best treatment to obtain friable calli from both explants types. WPM medium supplemented with 2 mg L-1 zeatin gave the highest percentage of embryogenic calli derived from leaf explants. While the highest percentage of embryogenic calli derived from stem explants was registered using 1 or 4 mg L-1 TDZ containing medium. Embryogenic calli originated from leaves explants on 1.5 mg L-1 zeatin showed promising activity of antioxidant with hexane extraction. However, embryogenic calli originated from stem explants on 1 mg L-1 TDZ showed the highest antioxidant activity with petroleum ether extraction. TDZ has promising effect on embryogenic callus induction from stem explants. While, zeatin has promising effect on embryogenic callus induction from leaf explants.

Induction of hairy roots by various strains of Agrobacterium rhizogenes in different types of Capsicum species explants.

PubMed

Md Setamam, Nursuria; Jaafar Sidik, Norrizah; Abdul Rahman, Zainon; Che Mohd Zain, Che Radziah

2014-06-30

Capsicum annuum and Capsicum frutescens, also known as "chilies", belong to the Solanaceae family and have tremendous beneficial properties. The application of hairy root culture may become an alternative method for future development of these species by adding value, such as by increasing secondary metabolites and improving genetic and biochemical stability compared with normal Capsicum plants. Therefore, in this research, different types of explants of both species were infected with various Agrobacterium rhizogenes strains to provide more information about the morphology and induction efficiency of hairy roots. After 2 weeks of in vitro seed germination, young seedling explants were cut into three segments; the cotyledon, hypocotyl, and radical. Then, the explants were co-cultured with four isolated A. rhizogenes strains in Murashige & Skoog culture media (MS) containing decreasing carbenicillin disodium concentrations for one month. In this experiment, thick and short hairy roots were induced at all induction sites of C. annuum while thin, elongated hairy roots appeared mostly at wound sites of C. frutescens. Overall, the hairy root induction percentages of C. frutescens were higher compared with C. annuum. Hairy root initiation was observed earliest using radicles (1st week), followed by cotyledons (2nd week), and hypocotyls (3rd week). Cotyledon explants of both species had the highest induction frequency with all strains compared with the other explants types. Strains ATCC 13333 and ATCC 15834 were the most favourable for C. frutescens while ATCC 43056 and ATCC 43057 were the most favourable for C. annuum. The interactions between the different explants and strains showed significant differences with p-values < 0.0001 in both Capsicum species. Both Capsicum species were amenable to A. rhizogenes infection and hairy root induction is recommended for use as an alternative explants in future plant-based studies.

Tissue-tissue interaction-triggered calcium elevation is required for cell polarization during Xenopus gastrulation.

PubMed

Shindo, Asako; Hara, Yusuke; Yamamoto, Takamasa S; Ohkura, Masamichi; Nakai, Junichi; Ueno, Naoto

2010-02-02

The establishment of cell polarity is crucial for embryonic cells to acquire their proper morphologies and functions, because cell alignment and intracellular events are coordinated in tissues during embryogenesis according to the cell polarity. Although much is known about the molecules involved in cell polarization, the direct trigger of the process remains largely obscure. We previously demonstrated that the tissue boundary between the chordamesoderm and lateral mesoderm of Xenopus laevis is important for chordamesodermal cell polarity. Here, we examined the intracellular calcium dynamics during boundary formation between two different tissues. In a combination culture of nodal-induced chordamesodermal explants and a heterogeneous tissue, such as ectoderm or lateral mesoderm, the chordamesodermal cells near the boundary frequently displayed intracellular calcium elevation; this frequency was significantly less when homogeneous explants were used. Inhibition of the intracellular calcium elevation blocked cell polarization in the chordamesodermal explants. We also observed frequent calcium waves near the boundary of the dorsal marginal zone (DMZ) dissected from an early gastrula-stage embryo. Optical sectioning revealed that where heterogeneous explants touched, the chordamesodermal surface formed a wedge with the narrow end tucked under the heterogeneous explant. No such configuration was seen between homogeneous explants. When physical force was exerted against a chordamesodermal explant with a glass needle at an angle similar to that created in the explant, or migrating chordamesodermal cells crawled beneath a silicone block, intracellular calcium elevation was frequent and cell polarization was induced. Finally, we demonstrated that a purinergic receptor, which is implicated in mechano-sensing, is required for such frequent calcium elevation in chordamesoderm and for cell polarization. This study raises the possibility that tissue-tissue interaction generates mechanical forces through cell-cell contact that initiates coordinated cell polarization through a transient increase in intracellular calcium.

Ventricular Recovery and Pump Explantation in Patients Supported by Left Ventricular Assist Devices: A Systematic Review.

PubMed

Phan, Kevin; Huo, Ya Ruth; Zhao, Dong Fang; Yan, Tristan D; Tchantchaleishvili, Vakhtang

2016-01-01

Several studies have reported that a portion of patients who exhibit cardiac recovery during left ventricular assist device (LVAD) support can have their device explanted with reasonable long-term survival. The aim of this systematic review is to assess the survival and cardiac function in patients with explanted LVADs from the current literature. Electronic search was performed to identify all studies in English literature assessing LVAD explantation. All identified articles were systematically assessed using the inclusion and exclusion criteria. Selected studies were subjected to quantitative assessment. From 5 electronic databases, 11 studies (213 patients) were included. Pooled mean perioperative mortality rate of those explanted was 9.2% (95% CI, 5.0-14.5%; I = 0). Pooled mean late mortality rate was 15% (95% CI, 9.0-22.1%; I = 31%). The pooled 1, 5, and 10 year survival postexplant was 91, 76, and 65.7%, respectively. Pooled postweaning freedom from heart failure (HF) recurrence reached 81.3%. Subset analysis demonstrated that patients explanted from a continuous-flow LVAD versus pulsatile LVAD had a lower rate of HF recurrence (6.6 vs. 28.3%, p = 0.03) and LVAD reimplantation (7.5 vs. 37%, p = 0.001). Before LVAD explantation, overall mean left ventricular ejection fraction (LVEF) was 49%. Weighted pooled early and late postexplantation mean LVEF was 47.3 and 41.2%, respectively. Late postexplantation LVEF was significantly higher in the continuous-flow versus pulsatile LVAD subgroup (41.5 vs. 24%, p = 0.001). This review shows encouraging safety and 10 year survival outcomes after explantation of LVADs in carefully selected patients, with rates better than expected after a heart transplant. Recovery of the native heart is the most desirable clinical outcome in patients supported with LVADs and should be actively sought.

Refining the application of direct embryogenesis in sugarcane: Effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency.

PubMed

Snyman, S J; Meyer, G M; Richards, J M; Haricharan, N; Ramgareeb, S; Huckett, B I

2006-10-01

A rapid in vitro protocol using direct somatic embryogenesis and microprojectile bombardment was investigated to establish the developmental phases most suitable for efficient sugarcane transformation. Immature leaf roll disc explants with and without pre-emergent inflorescence tissue were compared. It was shown that for effective transformation to occur, explants should be cultured for several days to allow initiation of embryo development prior to bombardment. Leaf roll discs with pre-emergent inflorescences showed a higher degree of embryogenic competence than non-flowering explants, and transformation efficiency was higher when explants containing floral initials were bombarded. Despite the occurrence of high numbers of phenotypically negative plants, combining the use of inflorescent leaf roll discs with direct embryogenic regeneration has the potential to improve the speed and efficiency of transgenesis in sugarcane.

Regulation of EGF and Prostaglandin Expression during Neonatal Gastrointestinal Injury in a Non-Human Primate Explant Model

DTIC Science & Technology

2017-05-05

an inflammatory agent (TNF 10 nglmI), or anti-inflammatory agent (indomethacin 50 mM). Secreted PGE2 in culture media was quantified by ELISA . mRNA...measured by ELISA for EGFR, p-EGFR, and AKT. RESULTS: PGE2 secretion was similar between DOand 014 controls. PGE2 secretion varied across intestinal

Anti-glycation activities of phenolic constituents from Silybum marianum (Milk Thistle) flower in vitro and on human explants.

PubMed

Shin, Seoungwoo; Lee, Jung-A; Kim, Minkyung; Kum, Hyunwoo; Jung, Eunsun; Park, Deokhoon

2015-02-19

Glycation is an ageing reaction of naturally occurring sugars with dermal proteins, with clinical signs appearing in vivo around age 30, and increasing steadily/regularly with age. The suppleness of the dermis is affected by the formation of bridges between proteins and sugars (Maillard's reaction). The accumulation of advanced glycation end products (AGEs) in skin plays a very important role in skin ageing. Therefore, natural compounds or extracts that possess antiglycation activities may have great anti-ageing potential. In the present study, Silybum marianum flower extract (SMFE) was demonstrated to possess antiglycation activity. We found that SMFE inhibits glycation reaction between BSA and glucose. In addition, antiglycation activity of SMFE was confirmed in a human skin explants model. SMFE reduced Nε-(carboxymethyl) lysine (CML) expression, whereas SMFE stimulated fibrillin-1 expression compared to treatment with methyglyoxal. An active ingredient contributing to the observed activities was identified as silibinin. The antiglycation activity of silibinin was dose-dependent. The beneficial effects of silibinin may be applied to prevention or management of AGE-mediated pathologies, targeting in a pleiotropic and complementary way the biochemical and cellular bases of skin aging.

Visible effects of rapamycin (sirolimus) on human skin explants in vitro.

PubMed

Peramo, Antonio; Marcelo, Cynthia L

2013-03-01

In this manuscript, we report observations of the effects of rapamycin in an organotypic culture of human skin explants. The tissues were cultured for 5 days at the air-liquid interface or in submersed conditions with media with and without rapamycin at 2 nM concentration. Histological analysis of tissue sections indicated that rapamycin-treated samples maintained a better epidermal structure in the upper layers of the tissue than untreated samples, mostly evident when skin was cultured in submersed conditions. A significant decrease in the number of positive proliferative cells using the Ki67 antigen was observed when specimens were treated with rapamycin, in both air-liquid and submersed conditions but apoptosis differences between treated and untreated specimens, as seen by cleaved caspase-3 positive cells, were only observed in submersed specimens. Finally, a decrease and variability in the location in the expression of the differentiation marker involucrin and in E-cadherin were also evident in submersed samples. These results suggest that the development of topical applications containing rapamycin, instead of systemic delivery, may be a useful tool in the treatment of skin diseases that require reduction of proliferation and modulation or control of keratinocyte differentiation.

/sup 125/I interstitial implants in the RIF-1 murine flank tumor: an animal model for brachytherapy

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

Bernstein, M.; Gutin, P.H.; Weaver, D.A.

1982-09-01

The development of a model for interstitial brachytherapy that uses high-activity, removable /sup 125/I sources in the RIF-1 murine flank tumor is reported. Experimental end points are clonogenic cell and tumor regrowth delay assays. For the clonogenic cell assay, interestitial radiation is delivered at total doses of 500-10,000 rad at dose rates of 0.9-2.7 rad/min to cells in annuli of tissue in the tumor. Dose-survival curves are characterized by an initial shoulder followed by a straight (exponential) portion, with D/sub 0/ similar to that of the curve obtained by external irradiation of the RIF-1 tumor in a self-contained cesium irradiatormore » at similar dose rates. Tumor regrowth curves have been obtained for minimum tumor doses of 500-5000 rad; marked tumor regression has been observed with minimum tumor doses as low as 2000 rad, but results are not as reproducible as the results obtained with the clonogenic cell assay.« less

Surface crystalline phases and nanoindentation hardness of explanted zirconia femoral heads.

PubMed

Catledge, Shane A; Cook, Monique; Vohra, Yogesh K; Santos, Erick M; McClenny, Michelle D; David Moore, K

2003-10-01

One new and nine explanted zirconia femoral heads were studied using glancing angle X-ray diffraction, scanning electron microscopy, and nanoindentation hardness techniques. All starting zirconia implants consisted only of tetragonal zirconia polycrystals (TZP). For comparison, one explanted alumina femoral head was also studied. Evidence for a surface tetragonal-to-monoclinic zirconia phase transformation was observed in some implants, the extent of which was varied for different in-service conditions. A strong correlation was found between increasing transformation to the monoclinic phase and decreasing surface hardness. Microscopic investigations of some of the explanted femoral heads revealed ultra high molecular weight polyethylene and metallic transfer wear debris.

Carnation (Dianthus caryophylus L.).

PubMed

Nontaswatsri, Chalermsri; Fukai, Seiichi

2006-01-01

Carnation is a valuable crop for the cut flower industry and demand for new and improved varieties is growing. However, genetic transformation of carnations is currently limited because of a lack of efficient routine technique. In this chapter, we present an easy and effective protocol for gene transfer to carnation node explants and subsequent adventitious shoot regeneration. For high-adventitious shoot regeneration, node explants from first to third node of 5- to 8-cm long shoots were cultured on Murashige and Skoog (MS) medium, containing 1.0 mg/Lthidiazuron (TDZ), 0.1 mg/L alpha-napthalenoacetic acid (NAA), 20 g/L sucrose, and 2 g/L Gellan gum for 10 d. Then the explants were cut into 8 radial segments and subcultured onto MS medium, containing 1.0 mg/L BA, 0.1 mg/L NAA, 20 g/L sucrose and 2 g/L Gellan Gum. For effective genetic transformation, 3- to 5-d precultured node explants were submerged in an Agrobacerium suspension for 10 min, then cocultivated on filter paper soaked with water and 50 microM acetosyringone (AS). After cocultivation, the explants were cut into eight radial segments and subcultured onto selection medium until transformed shoots regenerated from the explants.

Progress of tissue culture and genetic transformation research in pigeon pea [Cajanus cajan (L.) Millsp.].

PubMed

Krishna, Gaurav; Reddy, P Sairam; Ramteke, P W; Bhattacharya, P S

2010-10-01

Pigeon pea [Cajanus cajan (L.) Millsp.] (Family: Fabaceae) is an important legume crop cultivated across 50 countries in Asia, Africa, and the Americas; and ranks fifth in area among pulses after soybean, common bean, peanut, and chickpea. It is consumed as a major source of protein (21%) to the human population in many developing countries. In India, it is the second important food legume contributing to 80% of the global production. Several biotic and abiotic stresses are posing a big threat to its production and productivity. Attempts to address these problems through conventional breeding methods have met with partial success. This paper reviews the chronological progress made in tissue culture through organogenesis and somatic embryogenesis, including the influence of factors such as genotypes, explant sources, and culture media including the supplementation of plant growth regulators. Comprehensive lists of morphogenetic pathways involved in in vitro regeneration through organogenesis and somatic embryogenesis using different explant tissues of diverse pigeon pea genotypes are presented. Similarly, the establishment of protocols for the production of transgenics via particle bombardment and Agrobacterium-mediated transformation using different explant tissues, Agrobacterium strains, Ti plasmids, and plant selectable markers, as well as their interactions on transformation efficiency have been discussed. Future research thrusts on the use of different promoters and stacking of genes for various biotic and abiotic stresses in pigeon pea are suggested.

Novel Roles for Hypoxia and Prostaglandin E2 in the Regulation of IL-8 During Endometrial Repair

PubMed Central

Maybin, Jacqueline A.; Hirani, Nikhil; Jabbour, Henry N.; Critchley, Hilary O.D.

2011-01-01

The endometrium has a remarkable capacity for efficient repair; however, factors involved remain undefined. Premenstrual progesterone withdrawal leads to increased prostaglandin (PG) production and local hypoxia. Here we determined human endometrial expression of interleukin-8 (IL-8) and the roles of PGE2 and hypoxia in its regulation. Endometrial biopsy specimens (n = 51) were collected. Endometrial cells and explants were exposed to 100 nmol/L of PGE2 or 0.5% O2. The endometrial IL-8 concentration peaked during menstruation (P < 0.001) and had a significant proangiogenic effect. IL-8 was increased by PGE2 and hypoxia in secretory but not proliferative explants, which suggests that exposure to progesterone is essential. In vitro progesterone withdrawal induced significant IL-8 up-regulation in proliferative explants primed with progestins, but only in the presence of hypoxia. Epithelial cells treated simultaneously with PGE2 and hypoxia demonstrated synergistic increases in IL-8. Inhibition of HIF-1 by short hairpin RNA abolished hypoxic IL-8 induction, and inhibition of NF-κB by an adenoviral dominant negative inhibitor decreased PGE2-induced IL-8 expression (P > 0.05). Increased menstrual IL-8 is consistent with a role in repair. Progesterone withdrawal, hypoxia, and PGE2 regulate endometrial IL-8 by acting via HIF-1 and NF-κB. Hence, progesterone withdrawal may activate two distinct pathways to initiate endometrial repair. PMID:21356375

Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

PubMed

Saury, Charlotte; Lardenois, Aurélie; Schleder, Cindy; Leroux, Isabelle; Lieubeau, Blandine; David, Laurent; Charrier, Marine; Guével, Laëtitia; Viau, Sabrina; Delorme, Bruno; Rouger, Karl

2018-05-02

Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL). hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3'-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL. HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives. Our findings indicate that HS and hPL are efficient and viable alternatives to FBS for the preparation of hMuStem cell batches in compliance with GMPs.

Amniotic fluid derived mesenchymal stromal cells augment fetal lung growth in a nitrofen explant model.

PubMed

Di Bernardo, Julie; Maiden, Michael M; Hershenson, Marc B; Kunisaki, Shaun M

2014-06-01

Recent experimental work suggests the therapeutic role of mesenchymal stromal cells (MSCs) during lung morphogenesis. The purpose of this study was to investigate the potential paracrine effects of amniotic fluid-derived MSCs (AF-MSCs) on fetal lung growth in a nitrofen explant model. Pregnant Sprague-Dawley dams were gavage fed nitrofen on gestational day 9.5 (E9.5). E14.5 lung explants were subsequently harvested and cultured ex vivo for three days on filter membranes in conditioned media from rat AF-MSCs isolated from control (AF-Ctr) or nitrofen-exposed (AF-Nitro) dams. The lungs were analyzed morphometrically and by quantitative gene expression. Although there were no significant differences in total lung surface area among hypoplastic lungs, there were significant increases in terminal budding among E14.5+3 nitrofen explants exposed to AF-Ctr compared to explants exposed to medium alone (58.8±8.4 vs. 39.0±10.0 terminal buds, respectively; p<0.05). In contrast, lungs cultured in AF-Nitro medium failed to augment terminal budding. Nitrofen explants exposed to AF-Ctr showed significant upregulation of surfactant protein C to levels observed in normal fetal lungs. AF-MSCs can augment branching morphogenesis and lung epithelial maturation in a fetal explant model of pulmonary hypoplasia. Cell therapy using donor-derived AF-MSCs may represent a novel strategy for the treatment of fetal congenital diaphragmatic hernia. Copyright © 2014 Elsevier Inc. All rights reserved.

Risk Factors for Erosion of Artificial Urinary Sphincters: A Multicenter Prospective Study

PubMed Central

Brant, William O.; Erickson, Bradley A.; Elliott, Sean P.; Powell, Christopher; Alsikafi, Nejd; McClung, Christopher; Myers, Jeremy B.; Voelzke, Bryan B.; Smith, Thomas G.; Broghammer, Joshua A.

2015-01-01

OBJECTIVE To evaluate the short- to medium-term outcomes after artificial urinary sphincter (AUS) placement from a large, multi-institutional, prospective, follow-up study. We hypothesize that along with radiation, patients with any history of a direct surgery to the urethra will have higher rates of eventual AUS explantation for erosion and/or infection. MATERIALS AND METHODS A prospective outcome analysis was performed on 386 patients treated with AUS placement from April 2009 to December 2012 at 8 institutions with at least 3 months of follow-up. Charts were analyzed for preoperative risk factors and postoperative complications requiring explantation. RESULTS Approximately 50% of patients were considered high risk. High risk was defined as patients having undergone radiation therapy, urethroplasty, multiple treatments for bladder neck contracture or urethral stricture, urethral stent placement, or a history of erosion or infection in a previous AUS. A total of 31 explantations (8.03%) were performed during the follow-up period. Overall explantation rates were higher in those with prior radiation and prior UroLume. Men with prior AUS infection or erosion also had a trend for higher rates of subsequent explantation. Men receiving 3.5-cm cuffs had significantly higher explantation rates than those receiving larger cuffs. CONCLUSION This outcomes study confirms that urethral risk factors, including radiation history, prior AUS erosion, and a history of urethral stent placement, increase the risk of AUS explantation in short-term follow-up. PMID:25109562

Chondrogenic properties of collagen type XI, a component of cartilage extracellular matrix.

PubMed

Li, Ang; Wei, Yiyong; Hung, Clark; Vunjak-Novakovic, Gordana

2018-08-01

Cartilage extracellular matrix (ECM) has been used for promoting tissue engineering. However, the exact effects of ECM on chondrogenesis and the acting mechanisms are not well understood. In this study, we investigated the chondrogenic effects of cartilage ECM on human mesenchymal stem cells (MSCs) and identified the contributing molecular components. To this end, a preparation of articular cartilage ECM was supplemented to pellets of chondrogenically differentiating MSCs, pellets of human chondrocytes, and bovine articular cartilage explants to evaluate the effects on cell proliferation and the production of cartilaginous matrix. Selective enzymatic digestion and screening of ECM components were conducted to identify matrix molecules with chondrogenic properties. Cartilage ECM promoted MSC proliferation, production of cartilaginous matrix, and maturity of chondrogenic differentiation, and inhibited the hypertrophic differentiation of MSC-derived chondrocytes. Selective digestion of ECM components revealed a contributory role of collagens in promoting chondrogenesis. The screening of various collagen subtypes revealed strong chondrogenic effect of collagen type XI. Finally, collagen XI was found to promote production and inhibit degradation of cartilage matrix in human articular chondrocyte pellets and bovine articular cartilage explants. Our results indicate that cartilage ECM promotes chondrogenesis and inhibits hypertrophic differentiation in MSCs. Collagen type XI is the ECM component that has the strongest effects on enhancing the production and inhibiting the degradation of cartilage matrix. Copyright © 2018 Elsevier Ltd. All rights reserved.

Tumor necrosis factor-alpha converting enzyme in the human placenta throughout gestation.

PubMed

Hung, Tai-Ho; Chen, Szu-Fu; Hsieh, Ching-Chang; Hsu, Jenn-Jeih; Li, Meng-Jen; Yeh, Yi-Lin; Hsieh, T'sang-T'ang

2008-02-01

Ectodomain shedding of epidermal growth factor receptor ligands such as transforming growth factor- alpha (TGF-alpha), heparin-binding epidermal growth factor-like growth factor (HBEGF), and amphiregulin (AREG) is considered to be important during implantation. Tumor necrosis factor-alpha converting enzyme (TACE) has been suggested as the major sheddase for these molecules. The objectives of this study are (1) to characterize the expression of TACE in the human placenta throughout gestation; (2) to determine the association between the expression of TACE with TGF-alpha, HBEGF, and AREG; (3) to ascertain whether TACE mediates TGF-alpha, HBEGF, and AREG shedding; and (4) to examine the effect of hypoxia on the expression of TACE. By analyzing a total of 55 villous samples representing different gestational ages, the authors found that TACE was continuously expressed in the placentas throughout gestation and that the levels of TACE were positively correlated with the levels of TGF-alpha, HBEGF, and AREG. Preadministration of a TACE inhibitor in villous explant cultures or transfection of cytotrophoblastic cells with TACE-specific small interference RNA decreased the shedding of HBEGF and AREG. Moreover, hypoxia (2% O(2)) caused an increase in the levels of TACE mRNA and protein in villous explants and primary cytotrophoblastic cells in vitro. These results indicate that oxygen regulates the expression of TACE and that TACE may be important for placental development during human pregnancy.

Metformin increases the novel adipokine adipolin/CTRP12: role of the AMPK pathway.

PubMed

Tan, Bee K; Chen, Jing; Adya, Raghu; Ramanjaneya, Manjunath; Patel, Vanlata; Randeva, Harpal S

2013-11-01

Adipolin is a novel adipokine with anti-inflammatory and glucose-lowering properties. Lower levels of adipolin are found in obese and diabetic mice. Polycystic ovary syndrome (PCOS) is a pro-inflammatory state associated with obesity and diabetes. To date, there are no human studies on adipolin. Therefore, we measured serum (ELISA) and adipose tissue adipolin mRNA expression (RT-PCR) and protein concentrations (western blotting) in PCOS and control subjects. We also investigated the ex vivo effect of glucose and metformin on adipolin protein production in human subcutaneous adipose tissue explants. We report novel data that serum and subcutaneous adipose tissue adipolin mRNA expression and protein concentrations were significantly lower in women with PCOS compared with control subjects. Furthermore, Spearman's rank analysis showed that serum adipolin concentrations were significantly negatively correlated with BMI, waist-to-hip ratio, and glucose (P<0.05). However, when subjected to multiple regression analysis, none of these variables were predictive of serum adipolin concentrations (P>0.05). Also, subcutaneous adipose tissue adipolin mRNA expression and protein concentrations were only significantly negatively correlated with glucose (P<0.05). No significant correlations were found with omental adipose tissue adipolin mRNA expression and protein concentrations (P>0.05). Moreover, glucose profoundly reduced and metformin significantly increased adipolin protein production in human adipose tissue explants respectively. Importantly, metformin's effects appear to be via the AMP-activated protein kinase signaling pathway.

No Evidence for a Low Linear Energy Transfer Adaptive Response in Irradiated RKO Cells

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

Sowa, Marianne B.; Goetz, Wilfried; Baulch, Janet E.

2011-01-06

It has become increasingly evident from reports in the literature that there are many confounding factors that are capable of modulating radiation induced non-targeted responses such as the bystander effect and the adaptive response. In this paper we examine recent data that suggest that the observation of non-targeted responses may not be universally observable for differing radiation qualities. We have conducted a study of the adaptive response following low LET exposures for human colon carcinoma cells and failed to observe adaption for the endpoints of clonogenic survival or micronucleus formation.

Endocrine gland-derived endothelial growth factor (EG-VEGF) is a potential novel regulator of human parturition.

PubMed

Dunand, C; Hoffmann, P; Sapin, V; Blanchon, L; Salomon, A; Sergent, F; Benharouga, M; Sabra, S; Guibourdenche, J; Lye, S J; Feige, J J; Alfaidy, N

2014-09-01

EG-VEGF is an angiogenic factor that we identified as a new placental growth factor during human pregnancy. EG-VEGF is also expressed in the mouse fetal membrane (FM) by the end of gestation, suggesting a local role for this protein in the mechanism of parturition. However, injection of EG-VEGF to gravid mice did not induce labor, suggesting a different role for EG-VEGF in parturition. Here, we searched for its role in the FM in relation to human parturition. Human pregnant sera and total FM, chorion, and amnion were collected during the second and third trimesters from preterm no labor, term no labor, and term labor patients. Primary human chorion trophoblast and FM explants cultures were also used. We demonstrate that circulating EG-VEGF increased toward term and significantly decreased at the time of labor. EG-VEGF production was higher in the FM compared to placentas matched for gestational age. Within the FM, the chorion was the main source of EG-VEGF. EG-VEGF receptors, PROKR1 and PROKR2, were differentially expressed within the FM with increased expression toward term and an abrupt decrease with the onset of labor. In chorion trophoblast and FM explants collected from nonlaboring patients, EG-VEGF decreased metalloproteinase-2 and -9 activities and increased PGDH (prostaglandin-metabolizing enzyme) expression. Altogether these data demonstrate that EG-VEGF is a new cytokine that acts locally to ensure FM protection in late pregnancy. Its fine contribution to the initiation of human labor is exhibited by the abrupt decrease in its levels as well as a reduction in its receptors. © 2014 by the Society for the Study of Reproduction, Inc.

Pomegranate juice and punicalagin attenuate oxidative stress and apoptosis in human placenta and in human placental trophoblasts

PubMed Central

Tuuli, Methodius G.; Longtine, Mark S.; Shin, Joong Sik; Lawrence, Russell; Inder, Terrie; Michael Nelson, D.

2012-01-01

The human placenta is key to pregnancy outcome, and the elevated oxidative stress present in many complicated pregnancies contributes to placental dysfunction and suboptimal pregnancy outcomes. We tested the hypothesis that pomegranate juice, which is rich in polyphenolic antioxidants, limits placental trophoblast injury in vivo and in vitro. Pregnant women with singleton pregnancies were randomized at 35∼38 wk gestation to 8 oz/day of pomegranate juice or apple juice (placebo) until the time of delivery. Placental tissues from 12 patients (4 in the pomegranate group and 8 in the control group) were collected for analysis of oxidative stress. The preliminary in vivo results were extended to oxidative stress and cell death assays in vitro. Placental explants and cultured primary human trophoblasts were exposed to pomegranate juice or glucose (control) under defined oxygen tensions and chemical stimuli. We found decreased oxidative stress in term human placentas from women who labored after prenatal ingestion of pomegranate juice compared with apple juice as control. Moreover, pomegranate juice reduced in vitro oxidative stress, apoptosis, and global cell death in term villous explants and primary trophoblast cultures exposed to hypoxia, the hypoxia mimetic cobalt chloride, and the kinase inhibitor staurosporine. Punicalagin, but not ellagic acid, both prominent polyphenols in pomegranate juice, reduced oxidative stress and stimulus-induced apoptosis in cultured syncytiotrophoblasts. We conclude that pomegranate juice reduces placental oxidative stress in vivo and in vitro while limiting stimulus-induced death of human trophoblasts in culture. The polyphenol punicalagin mimics this protective effect. We speculate that antenatal intake of pomegranate may limit placental injury and thereby may confer protection to the exposed fetus. PMID:22374759

Enhancement of RNA Synthesis, Protein Synthesis, and Abscission by Ethylene

PubMed Central

Abeles, F. B.; Holm, R. E.

1966-01-01

Ethylene stimulated RNA and protein synthesis in bean (Phaseolus vulgaris L. var. Red Kidney) abscission zone explants prior to abscission. The effect of ethylene on RNA synthesis and abscission was blocked by actinomycin D. Carbon dioxide, which inhibits the effect of ethylene on abscission, also inhibited the influence of ethylene on protein synthesis. An aging period appears to be essential before bean explants respond to ethylene. Stimulation of protein synthesis by ethylene occurred only in receptive or senescent explants. Treatment of juvenile explants with ethylene, which has no effect on abscission also has no effect on protein synthesis. Evidence in favor of a hormonal role for ethylene during abscission is discussed. PMID:16656405

Ultrastructural evaluation of explanted opacified Hydroview (H60M) intraocular lenses

PubMed Central

Cartwright, Nathaniel E Knox; Mayer, Eric J; McDonald, Brendan M; Skinner, Andrew; Salter, Chris J; Tole, Derek M; Sparrow, John M; Dick, Andrew D; Group, The Bristol IOL Study; Ferguson, David J P

2007-01-01

Aim To describe the ultrastructural appearance of explanted opacified Hydroview H60M intraocular lenses. Methods 14 explanted lenses were examined by scanning electron microscopy, and their appearance compared with a non‐implanted H60M lens from the same time period. Wavelength‐dispersive x ray spectroscopy (WDX) was performed on two opacified lenses. Results Subsurface deposits were seen in all explanted opacified lenses. These deposits broke only onto the surface of more densely opacified lenses. WDX confirmed that the deposits contained both calcium and phosphorous, consistent with their being calcium apatite. Conclusion These findings challenge the widely accepted opinion that H60M intraocular lens opacification begins on the surface of the optic. PMID:16987894

Characterisation of coral explants: a model organism for cnidarian-dinoflagellate studies

NASA Astrophysics Data System (ADS)

Gardner, S. G.; Nielsen, D. A.; Petrou, K.; Larkum, A. W. D.; Ralph, P. J.

2015-03-01

Coral cell cultures made from reef-building scleractinian corals have the potential to aid in the pursuit of understanding of the cnidarian-dinoflagellate symbiosis. Various methods have previously been described for the production of cell cultures in vitro with a range of success and longevity. In this study, viable tissue spheroids containing host tissue and symbionts (coral explants) were grown from the tissues of Fungia granulosa. The cultured explants remained viable for over 2 months and showed morphological similarities in tissue structure and internal microenvironment to reef-building scleractinian corals. The photophysiology of the explants (1 week old) closely matched that of the parent coral F. granulosa. This study provides the first empirical basis for supporting the use of coral explants as laboratory models for studying coral symbioses. In particular, it highlights how these small, self-sustaining, skeleton-free models can be useful for a number of molecular, genetic and physiological analyses necessary for investigating host-symbiont interactions at the microscale.

A putative mesenchymal stem cells population isolated from adult human testes.

PubMed

Gonzalez, R; Griparic, L; Vargas, V; Burgee, K; Santacruz, P; Anderson, R; Schiewe, M; Silva, F; Patel, A

2009-08-07

Mesenchymal stem cells (MSCs) isolated from several adult human tissues are reported to be a promising tool for regenerative medicine. In order to broaden the array of tools for therapeutic application, we isolated a new population of cells from adult human testis termed gonadal stem cells (GSCs). GSCs express CD105, CD166, CD73, CD90, STRO-1 and lack hematopoietic markers CD34, CD45, and HLA-DR which are characteristic identifiers of MSCs. In addition, GSCs express pluripotent markers Oct4, Nanog, and SSEA-4. GSCs propagated for at least 64 population doublings and exhibited clonogenic capability. GSCs have a broad plasticity and the potential to differentiate into adipogenic, osteogenic, and chondrogenic cells. These studies demonstrate that GSCs are easily obtainable stem cells, have growth kinetics and marker expression similar to MSCs, and differentiate into mesodermal lineage cells. Therefore, GSCs may be a valuable tool for therapeutic applications.

Targeting SYK kinase-dependent anti-apoptotic resistance pathway in B-lineage acute lymphoblastic leukaemia (ALL) cells with a potent SYK inhibitory pentapeptide mimic.

PubMed

Uckun, Fatih M; Ek, Rauf O; Jan, Shyi-Tai; Chen, Chun-Lin; Qazi, Sanjive

2010-05-01

The present study found that the pentapeptide mimic C-61, targeting the substrate binding P-site of SYK tyrosine kinase acted as a potent inducer of apoptosis in chemotherapy-resistant SYK-expressing primary leukemic B-cell precursors taken directly from relapsed B-precursor leukaemia (BPL) patients (but not SYK-deficient infant pro-B leukaemia cells), exhibited favourable pharmacokinetics in mice and non-human primates, and eradicated in vivo clonogenic leukaemia cells in severe combined immunodeficient mouse xenograft models of chemotherapy-resistant human BPL at dose levels non-toxic to mice and non-human primates. These in vitro and in vivo findings provide proof of principle for effective treatment of chemotherapy-resistant BPL by targeting SYK-dependent anti-apoptotic blast cell survival machinery with a SYK P-Site inhibitor. Further development of C-61 may provide the foundation for therapeutic innovation against chemotherapy-resistant BPL.

NAA-Induced Direct Organogenesis from Female Immature Inflorescence Explants of Date Palm.

PubMed

Khierallah, Hussam S M; Bader, Saleh M; Al-Khafaji, Makki A

2017-01-01

Micropropagation has great potential for the multiplication of female and male date palms of commercially grown cultivars by using inflorescences. This approach is simple, convenient, and much faster than the conventional method of using shoot-tip explants. We describe here a stepwise micropropagation procedure using inflorescence explants of Iraqi date palm cultivar Maktoom. Cultured explants were derived from 0.5-cm-long spike segments excised from 8 to 10-cm-long spathes. About 70% formed adventitious buds on Murashige and Skoog (MS) medium supplemented with 2 mg/L naphthalene acetic acid (NAA), 4 mg/L benzylaminopurine (BAP), and 40 g/L sucrose and maintained in the dark for 16 weeks before transferring to normal light conditions. The best multiplication rate was achieved with 3 mg/L 2ip and 2 mg/L; for shoot elongation, the best medium is MS containing 0.5 mg/L BAP, 0.5 mg/L 2ip, and 1 mg/L GA 3 . Well-developed shoots were cultured for rooting in half MS medium amended with 1 mg/L NAA and 45 g/L sucrose. Plantlets with well-developed roots were successfully hardened in the greenhouse. Inflorescence explants proved to be a promising alternative explant source for micropropagation of date palm cultivars.

Complications and Short-Term Explantation Rate Following Artificial Urinary Sphincter Implantation: Results from a Large Middle European Multi-Institutional Case Series.

PubMed

Kretschmer, Alexander; Hüsch, Tanja; Thomsen, Frauke; Kronlachner, Dominik; Obaje, Alice; Anding, Ralf; Pottek, Tobias; Rose, Achim; Olianas, Roberto; Friedl, Alexander; Hübner, Wilhelm; Homberg, Roland; Pfitzenmaier, Jesco; Grein, Ulrich; Queissert, Fabian; Naumann, Carsten Maik; Schweiger, Josef; Wotzka, Carola; Nyarangi-Dix, Joanne N; Hofmann, Torben; Seiler, Roland; Haferkamp, Axel; Bauer, Ricarda M

2016-01-01

Background/Aims/Objectives: To analyze perioperative complication and short-term explantation rates after perineal or penoscrotal single-cuff and double-cuff artificial urinary sphincter (AUS) implantation in a large middle European multi-institutional patient cohort. 467 male patients with stress urinary incontinence underwent implantation of a perineal single-cuff (n = 152), penoscrotal single-cuff (n = 99), or perineal double-cuff (n = 216) AUS between 2010 and 2012. Postoperative complications and 6-month explantation rates were assessed. For statistical analysis, Fisher's exact test and Kruskal-Wallis rank sum test, and a multiple logistic regression model were used (p < 0.05). Compared to perineal single-cuff AUS, penoscrotal single-cuff implantation led to significantly increased short-term explantation rates (8.6% (perineal) vs. 19.2% (penoscrotal), p = 0.019). The postoperative infection rate was significantly higher after double-cuff compared to single-cuff implantation (6.0% (single-cuff) vs. 13.9% (double-cuff), p = 0.019). The short-term explantation rate after primary double-cuff placement was 6.5% (p = 0.543 vs. perineal single-cuff). In multivariate analysis, the penoscrotal approach (p = 0.004), intraoperative complications (p = 0.005), postoperative bleeding (p = 0.011), and perioperative infection (p < 0.001) were independent risk factors for short-term explantation. Providing data from a large contemporary multi-institutional patient cohort from high-volume and low-volume institutions, our results reflect the current standard of care in middle Europe. We indicate that the penoscrotal approach is an independent risk factor for increased short-term explantation rates. © 2016 S. Karger AG, Basel.

An Efficient Method for Adventitious Root Induction from Stem Segments of Brassica Species

PubMed Central

Srikanth, Sandhya; Choong, Tsui Wei; Yan, An; He, Jie; Chen, Zhong

2016-01-01

Plant propagation via in vitro culture is a very laborious and time-consuming process. The growth cycle of some of the crop species is slow even in the field and the consistent commercial production is hard to maintain. Enhanced methods of reduced cost, materials and labor significantly impact the research and commercial production of field crops. In our studies, stem-segment explants of Brassica species were found to generate adventitious roots (AR) in aeroponic systems in less than a week. As such, the efficiency of rooting from stem explants of six cultivar varieties of Brassica spp was tested without using any plant hormones. New roots and shoots were developed from Brassica alboglabra (Kai Lan), B. oleracea var. acephala (purple kale), B. rapa L. ssp. chinensis L (Pai Tsai, Nai Bai C, and Nai Bai T) explants after 3 to 5 days of growing under 20 ± 2°C cool root zone temperature (C-RZT) and 4 to 7 days in 30 ± 2°C ambient root zone temperature (A-RZT). At the base of cut end, anticlinal and periclinal divisions of the cambial cells resulted in secondary xylem toward pith and secondary phloem toward cortex. The continuing mitotic activity of phloem parenchyma cells led to a ring of conspicuous white callus. Root initials formed from the callus which in turn developed into ARs. However, B. rapa var. nipposinica (Mizuna) explants were only able to root in C-RZT. All rooted explants were able to develop into whole plants, with higher biomass obtained from plants that grown in C-RZT. Moreover, explants from both RZTs produced higher biomass than plants grown from seeds (control plants). Rooting efficiency was affected by RZTs and explant cuttings of donor plants. Photosynthetic CO2 assimilation rate (Asat) and stomatal conductance (gssat) were significantly differentiated between plants derived from seeds and explants at both RZTs. All plants in A-RZT had highest transpiration rates. PMID:27446170

Induction of hairy roots by various strains of Agrobacterium rhizogenes in different types of Capsicum species explants

PubMed Central

2014-01-01

Background Capsicum annuum and Capsicum frutescens, also known as “chilies”, belong to the Solanaceae family and have tremendous beneficial properties. The application of hairy root culture may become an alternative method for future development of these species by adding value, such as by increasing secondary metabolites and improving genetic and biochemical stability compared with normal Capsicum plants. Therefore, in this research, different types of explants of both species were infected with various Agrobacterium rhizogenes strains to provide more information about the morphology and induction efficiency of hairy roots. After 2 weeks of in vitro seed germination, young seedling explants were cut into three segments; the cotyledon, hypocotyl, and radical. Then, the explants were co-cultured with four isolated A. rhizogenes strains in Murashige & Skoog culture media (MS) containing decreasing carbenicillin disodium concentrations for one month. Results In this experiment, thick and short hairy roots were induced at all induction sites of C. annuum while thin, elongated hairy roots appeared mostly at wound sites of C. frutescens. Overall, the hairy root induction percentages of C. frutescens were higher compared with C. annuum. Hairy root initiation was observed earliest using radicles (1st week), followed by cotyledons (2nd week), and hypocotyls (3rd week). Cotyledon explants of both species had the highest induction frequency with all strains compared with the other explants types. Strains ATCC 13333 and ATCC 15834 were the most favourable for C. frutescens while ATCC 43056 and ATCC 43057 were the most favourable for C. annuum. The interactions between the different explants and strains showed significant differences with p-values < 0.0001 in both Capsicum species. Conclusions Both Capsicum species were amenable to A. rhizogenes infection and hairy root induction is recommended for use as an alternative explants in future plant-based studies. PMID:24981787

Association between IL-6 production in synovial explants from rheumatoid arthritis patients and clinical and imaging response to biologic treatment: A pilot study.

PubMed

Andersen, Martin; Boesen, Mikael; Ellegaard, Karen; Söderström, Kalle; Søe, Niels H; Spee, Pieter; Mørch, Ulrik G W; Torp-Pedersen, Søren; Bartels, Else M; Danneskiold-Samsøe, Bente; Karlsson, Lars; Bliddal, Henning

2018-01-01

The need for biomarkers which can predict disease course and treatment response in rheumatoid arthritis (RA) is evident. We explored whether clinical and imaging responses to biologic disease modifying anti-rheumatic drug treatment (bDMARD) were associated with the individual's mediator production in explants obtained at baseline. RA Patients were evaluated by disease activity score 28 joint C-reactive protein (DAS 28-)), colour Doppler ultrasound (CDUS) and 3 Tesla RA magnetic resonance imaging scores (RAMRIS). Explants were established from synovectomies from a needle arthroscopic procedure prior to initiation of bDMARD. Explants were incubated with the bDMARD in question, and the productions of interleukin-6 (IL-6), monocyte chemo-attractive protein-1 (MCP-1) and macrophage inflammatory protein-1-beta (MIP-1b) were measured by multiplex immunoassays. The changes in clinical and imaging variables following a minimum of 3 months bDMARD treatment were compared to the baseline explant results. Mixed models and Spearman's rank correlations were performed. P-values below 0.05 were considered statistically significant. 16 patients were included. IL-6 production in bDMARD-treated explants was significantly higher among clinical non-responders compared to responders (P = 0.04), and a lack of suppression of IL-6 by the bDMARDS correlated to a high DAS-28 (ρ = 0.57, P = 0.03), CDUS (ρ = 0.53, P = 0.04) and bone marrow oedema (ρ = 0.56, P = 0.03) at follow-up. No clinical association was found with explant MCP-1 production. MIP-1b could not be assessed due to a large number of samples below the detection limit. Synovial explants appear to deliver a disease-relevant output testing which when carried out in advance of bDMARD treatment can potentially pave the road for a more patient tailored treatment approach with better treatment effects.

Ultrastructural interaction between spermatozoon and human oviductal cells in vitro.

PubMed

Vigil, Pilar; Salgado, Ana María; Cortés, Manuel E

2012-04-01

The oviduct is an important organ for successful mammalian reproduction. In this work, human oviducts were inseminated and their explants analyzed using scanning electron microscopy in order to study, at a finer ultrastructual level, the interaction between spermatozoon and oviduct in vitro. Results show unequivocally a spermatozoon tightly attached through the acrosomal region of its head to several cilia of the human tubal epithelial cells. This finding proves that spermatozoa do indeed adhere to the endosalpinx, a fact of utmost relevance for the physiology of the reproductive process, since it supports the idea of a spermatozoa reservoir being formed in the oviduct, which is also briefly discussed.

Pasteurization of bone for tumour eradication prior to reimplantation – An in vitro & pre-clinical efficacy study

PubMed Central

Kode, Jyoti; Taur, Prasad; Gulia, Ashish; Jambhekar, Nirmala; Agarwal, Manish; Puri, Ajay

2014-01-01

Background & objectives: In current era of limb-salvage therapy, pasteurization of bone sarcomas is receiving growing attention as a potential extracorporeal treatment and cost-effective alternative to allografts and radiation before surgical reimplantation. Detailed in vitro and in vivo pre-clinical study to evaluate efficacy of pasteurization to eradicate malignant cells has not been reported yet. The present study was carried out to assess the efficacy of pasteurization to kill tumour cells both in vitro and in vivo. Methods: Surgically resected specimens of osteosarcomas (n=4) were cut into equal halves and one section was pasteurized by heating at 60°C to 65°C for 40 min. Paired samples before and after pasteurization were studied in vitro for DNA ploidy, evaluation of histological change and elimination of mitotic activity. These tissues were transplanted in immune-deficient NOD-SCID mice to evaluate effect on tumour-generating ability, presence of human nuclei, osteopontin and cytokine/chemokines released in tumour-transplanted mice. Results: Non-pasteurized tumour samples had viable tumour cells which exhibited significant growth in culture, increased proliferative ability and clonogenic potential while respective pasteurized tumour tissues did not grow in culture and did not exhibit clonogenicity. Flow cytometry revealed that propidium iodide positive dead cells increased significantly (P< 0.01) post pasteurization. Seven of 12 non-pasteurized tumour transplanted mice demonstrated tumour-forming ability as against 0 of 12 in pasteurized tumour transplanted mice. Solid tumour xenografts exhibited strong expression of anti-human nuclei and osteopontin by immunohistochemistry as well as secretary human interluekin-6 (IL-6) while pasteurized mice failed to express these markers. Interpretation & conclusions: This study has provided a basis to establish pasteurization as being efficacious in ensuring tumour eradication from resected bone tumour specimens. Pasteurized tumour bearing bone can thus safely be used to reconstruct large defects after tumour resection. PMID:24927346

Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor.

PubMed

Bolnick, Alan D; Bolnick, Jay M; Kohan-Ghadr, Hamid-Reza; Kilburn, Brian A; Pasalodos, Omar J; Singhal, Pankaj K; Dai, Jing; Diamond, Michael P; Armant, D Randall; Drewlo, Sascha

2017-06-01

Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress? LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress. Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness. First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF. Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB) inhibitor, anti-ERBB1 or ERBB4 blocking antibodies, or pretreatment of cells with heparitinase I. Extravillous differentiation was assessed by immunocytochemistry to determine the relative levels of integrins α6β4 and α1β1. Trophoblast invasiveness was assessed in villous explants by measuring outgrowth from villous tips cultured on Matrigel, and by invasion assays with HTR-8/SVneo cells cultured on Matrigel-coated transwell insert. Placental explants and HTR-8/SVneo cells were exposed to oxidative stress in a hypoxia-reoxygenation (H-R) model, measuring cell death by TUNEL assay, caspase 3 cleavage, and BCL-2α expression. LMWH induced extravillous differentiation, according to trophoblast invasion assays and integrin (α6β4-α1β1) switching. Treatment with LMWH rescued cytotrophoblasts and HTR-8/SVneo cells from apoptosis during exposure to reoxygenation injury, based on TUNEL, caspase 3 cleavage and BCL-2α expression. Experiments using CRM197, ERBB1 and ERBB4 blocking antibodies, pan-ERBB inhibitor and removal of cell surface heparin demonstrated that the effects of LMWH on trophoblast invasion and survival were dependent upon HBEGF signaling. N/A. The primary limitation of this study was the use of only in vitro experiments. Patient demographics from elective terminations were not available. These data provide new insights into the non-coagulation-related aspects of perinatal LMWH treatment in the management of placental insufficiency disorders. This research was supported by grants from the National Institutes of Health (HD071408 and HL128628), the March of Dimes, and the W. K. Kellogg Foundation. There were no conflicts or competing interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates

PubMed Central

Bajpai, Vivek K.; Mistriotis, Panagiotis; Loh, Yuin-Han; Daley, George Q.; Andreadis, Stelios T.

2012-01-01

Aims Smooth muscle cells (SMC) play an important role in vascular homeostasis and disease. Although adult mesenchymal stem cells (MSC) have been used as a source of contractile SMC, they suffer from limited proliferation potential and culture senescence, particularly when originating from older donors. By comparison, human induced pluripotent stem cells (hiPSC) can provide an unlimited source of functional SMC for autologous cell-based therapies and for creating models of vascular disease. Our goal was to develop an efficient strategy to derive functional, contractile SMC from hiPSC. Methods and results We developed a robust, stage-wise, feeder-free strategy for hiPSC differentiation into functional SMC through an intermediate stage of multipotent MSC, which could be coaxed to differentiate into fat, bone, cartilage, and muscle. At this stage, the cells were highly proliferative and displayed higher clonogenic potential and reduced senescence when compared with parental hair follicle mesenchymal stem cells. In addition, when exposed to differentiation medium, the myogenic proteins such as α-smooth muscle actin, calponin, and myosin heavy chain were significantly upregulated and displayed robust fibrillar organization, suggesting the development of a contractile phenotype. Indeed, tissue constructs prepared from these cells exhibited high levels of contractility in response to receptor- and non-receptor-mediated agonists. Conclusion We developed an efficient stage-wise strategy that enabled hiPSC differentiation into contractile SMC through an intermediate population of clonogenic and multipotent MSC. The high yield of MSC and SMC derivation suggests that our strategy may facilitate an acquisition of the large numbers of cells required for regenerative medicine or for studying vascular disease pathophysiology. PMID:22941255

Down-regulation of let-7 microRNA increased K-ras expression in lung damage induced by radon.

PubMed

Chen, Zhihai; Wang, Dapeng; Gu, Chao; Liu, Xing; Pei, Weiwei; Li, Jianxiang; Cao, Yi; Jiao, Yang; Tong, Jian; Nie, Jihua

2015-09-01

Radon has long been recognized as a human carcinogen leading to lung cancer, but the underlying mechanisms remain obscure. Recent studies have shown that the let-7 microRNA and K-ras play an important role in the development of various cancers. However, the exact role between let-7 and K-ras in radon induced lung damage has not been explored so far. In the present study, wistar rats and human bronchial epithelial (HBE) cells were long-term exposed to radon, and then alterations in histological pathology of rat lung tissue, ROS, antioxidant enzymes activities and clonogenic formation in HBE cells, as well as changes in let-7 and K-ras expression were determined to observe the adverse effects induced by radon. The results showed that long-term exposure to radon produced severe lung damage in rats, significantly increased ROS production and clonogenic formation ratios and decreased SOD activities in HBE cells. In addition, an obvious down-regulation of let-7 and up-regulation of K-ras were also revealed both in mRNA and in protein level in lung tissue of rats and HBE cells exposed to radon. Furthermore, a significant down-regulation of K-ras was then confirmed in both let-7b-3p and let-7a-2-3p transfected HBE cells. Taken together, the present results propose an involvement of let-7 microRNA and K-ras in radon induced lung damage both in vivo and in vitro, which may thus be of potential value in early diagnosis and therapy of radon-induced lung tumorgenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

Synergistic Effect of Ionizing Radiation and β-lapachone against RKO Human Colon Adenocarcinoma Cells

PubMed Central

Kim, Eun Jung; Ji, In-Mi; Ahn, Ki-Jung; Choi, Eun Kyung; Park, Heon-Jin; Lim, Byung Uk; Song, Chang W.

2005-01-01

Purpose To reveal the interaction between β-Lapachone (β-lap) and ionizing radiation in causing cell death in RKO human colon adenocarcinoma cells, and to elucidate the potential usefulness of combined β-lap treatment and radiotherapy for cancer treatment. Materials and Methods The cytotoxicities of various treatments were determined in vitro using clonogenic and apoptotic cell death. The changes in cell cycle distribution were studied using flow cytometry and an in vitro kinase assay. The tumor growth was studied using RKO tumors grown s.c. in the hind leg BALB/c- nuslc nude mice. Results β-lap caused clonogenic cell death and rapid apoptosis in RKO cells in vitro, in a dose dependent manner. The repair of sublethal radiation damage was almost completely inhibited when cells were maintained in β-lap during the interval between the two-dose irradiation. Flow cytometry study demonstrated that β-lap induced apoptosis, independent of the cell cycle phase, and completely prohibited the induction of radiation-induced G2 arrest in irradiated cells. The prohibition of radiation-induced G2 arrest is unclear, but may be related to the profound suppression of the p53, p21 and cyclin B1-Cdc2 kinase activities observed in cells treated with β-lap. The combination of β-lap and radiation markedly enhanced the radiation-induced growth suppression of tumors. Conclusion β-lap is cytotoxic against RKO cells, both in vitro and in vivo, and also sensitized cells to ionizing radiation by inhibiting sublethal radiation damage repair. β-lap is potentially useful as a potent anti-cancer chemotherapy drug and potent radiosensitizer against caner cells. PMID:19956501

The Delayed Effects of Acute Radiation Syndrome: Evidence of Long-Term Functional Changes in the Clonogenic Cells of the Small Intestine.

PubMed

Booth, Catherine; Tudor, Gregory L; Katz, Barry P; MacVittie, Thomas J

2015-11-01

Long term or residual damage post-irradiation has been described for many tissues. In hematopoietic stem cells (HSC), this is only revealed when the HSC are stressed and required to regenerate and repopulate a myeloablated host. Such an assay cannot be used to assess the recovery potential of previously irradiated intestinal stem cells (ISC) due to their incompatibility with transplantation. The best approximation to the HSC assay is the crypt microcolony assay, also based on clonogen survival. In the current study, the regenerative capacity of intestinal clonogenic cells in mice that had survived 13 Gy irradiation (with 5% bone marrow shielding to allow survival through the hematopoietic syndrome) and were then aged for 200 d was compared to previously unirradiated age-matched controls. Interestingly, at 200 d following 13 Gy, there remained a statistically significant reduction in crypts present in the various small intestinal regions (illustrating that the gastrointestinal epithelium had not fully recovered despite the 200-d interval). However, upon re-irradiation on day 196, those mice previously irradiated had improved crypt survival and regeneration compared to the age-matched controls. This was evident in all regions of the small intestine following 11-13 Gy re-exposure. Thus, there were either more clonogens per crypt within those previously irradiated and/or those that were present were more radioresistant (possibly because a subpopulation was more quiescent). This is contrary to the popular belief that previously irradiated animals may have an impaired/delayed regenerative response and be more radiosensitive.

Mutations in CIC and IDH1 cooperatively regulate 2-hydroxyglutarate levels and cell clonogenicity

PubMed Central

Chittaranjan, Suganthi; Chan, Susanna; Yang, Cindy; Yang, Kevin C.; Chen, Vincent; Moradian, Annie; Firme, Marlo; Song, Jungeun; Go, Nancy E.; Blough, Michael D.; Chan, Jennifer A.; Cairncross, J. Gregory; Gorski, Sharon M.; Morin, Gregg B.; Yip, Stephen; Marra, Marco A.

2014-01-01

The majority of oligodendrogliomas (ODGs) exhibit combined losses of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH1-R132H or IDH2-R172K). Approximately 70% of ODGs with 1p19q co-deletions harbor somatic mutations in the Capicua Transcriptional Repressor (CIC) gene on chromosome 19q13.2. Here we show that endogenous long (CIC-L) and short (CIC-S) CIC proteins are predominantly localized to the nucleus or cytoplasm, respectively. Cytoplasmic CIC-S is found in close proximity to the mitochondria. To study wild type and mutant CIC function and motivated by the paucity of 1p19q co-deleted ODG lines, we created HEK293 and HOG stable cell lines ectopically co-expressing CIC and IDH1. Non-mutant lines displayed increased clonogenicity, but cells co-expressing the mutant IDH1-R132H with either CIC-S-R201W or -R1515H showed reduced clonogenicity in an additive manner, demonstrating cooperative effects in our assays. Expression of mutant CIC-R1515H increased cellular 2-Hydroxyglutarate (2HG) levels compared to wild type CIC in IDH1-R132H background. Levels of phosphorylated ATP-citrate Lyase (ACLY) were lower in cell lines expressing mutant CIC-S proteins compared to cells expressing wild type CIC-S, supporting a cytosolic citrate metabolism-related mechanism of reduced clonogenicity in our in vitro model systems. ACLY or phospho-ACLY were similarly reduced in CIC-mutant 1p19q co-deleted oligodendroglioma patient samples. PMID:25277207

Somatic embryogenesis from flower explants of cocoa (Theobroma cacao L.).

PubMed

Silva, J J; Debergh, P

2001-01-01

Two types of flower explants, staminoides and petals, were used for in vitro induction of somatic embryos in cocoa. After 14 days in culture, we observed globular structures and callus formation on both types of explants. However, the better results were obtained on staminoides: 98.3% formed callus and 86.2% somatic embryos on Murashige and Skoog (1962) medium supplemented with sucrose, coconut water, 2,4-D, kinetin and agar.

Assessment of Adeno-Associated Virus Serotype Tropism in Human Retinal Explants.

PubMed

Wiley, Luke A; Burnight, Erin R; Kaalberg, Emily E; Jiao, Chunhua; Riker, Megan J; Halder, Jennifer A; Luse, Meagan A; Han, Ian C; Russell, Stephen R; Sohn, Elliott H; Stone, Edwin M; Tucker, Budd A; Mullins, Robert F

2018-04-01

Advances in the discovery of the causes of monogenic retinal disorders, combined with technologies for the delivery of DNA to the retina, offer enormous opportunities for the treatment of previously untreatable blinding diseases. However, for gene augmentation to be most effective, vectors that have the correct cell-type specificity are needed. While animal models are very useful, they often exhibit differences in retinal cell surface receptors compared to the human retina. This study evaluated the use of an ex vivo organotypic explant system to test the transduction efficiency and tropism of seven different adeno-associated virus type 2 (AAV2) serotypes in the human retina and retinal pigment epithelium-choroid-AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/8, and AAV2/9-all driving expression of GFP under control of the cytomegalovirus promoter. After 7 days in culture, it was found that AAV2/4 and AAV2/5 were particularly efficient at transducing photoreceptor cells and that AAV2/5 was highly specific to the outer nuclear layer, whereas AAV2/8 displayed consistently low transduction of photoreceptors. To validate the authenticity of the organotypic culture system, the transduction of the same set of AAVs was also compared in a pig model, in which sub-retinal injections in vivo were compared to cultured and transduced organotypic cultures ex vivo. This study shows how different AAV serotypes behave in the human retina and provides insight for further investigation of each of these serotypes for gene augmentation-based treatment of inherited retinal degeneration.

Targeting and crossing of the human maternofetal barrier by Listeria monocytogenes: role of internalin interaction with trophoblast E-cadherin.

PubMed

Lecuit, Marc; Nelson, D Michael; Smith, Steve D; Khun, Huot; Huerre, Michel; Vacher-Lavenu, Marie-Cécile; Gordon, Jeffrey I; Cossart, Pascale

2004-04-20

Listeria monocytogenes produces severe fetoplacental infections in humans. How it targets and crosses the maternofetal barrier is unknown. We used immunohistochemistry to examine the location of L. monocytogenes in placental and amniotic tissue samples obtained from women with fetoplacental listeriosis. The results raised the possibility that L. monocytogenes crosses the maternofetal barrier through the villous syncytiotrophoblast, with secondary infection occurring via the amniotic epithelium. Because epidemiological studies indicate that the bacterial surface protein, internalin (InlA), may play a role in human fetoplacental listeriosis, we investigated the cellular patterns of expression of its host receptor, E-cadherin, at the maternofetal interface. E-cadherin was found on the basal and apical plasma membranes of syncytiotrophoblasts and in villous cytotrophoblasts. Established trophoblastic cell lines, primary trophoblast cultures, and placental villous explants were each exposed to isogenic InlA+ or InlA- strains of L. monocytogenes, and to L. innocua expressing or not InlA. Quantitative assays of cellular invasion demonstrated that bacterial entry into syncytiotrophoblasts occurs via the apical membrane in an InlA-E-cadherin dependent manner. In human placental villous explants, bacterial invasion of the syncytiotrophoblast barrier and underlying villous tissue and subsequent replication produces histopathological lesions that mimic those seen in placentas of women with listeriosis. Thus, the InlA-E-cadherin interaction that plays a key role in the crossing of the intestinal barrier in humans is also exploited by L. monocytogenes to target and cross the placental barrier. Such a ligand-receptor interaction allowing a pathogen to specifically cross the placental villous trophoblast barrier has not been reported previously.

Targeting and crossing of the human maternofetal barrier by Listeria monocytogenes: Role of internalin interaction with trophoblast E-cadherin

PubMed Central

Lecuit, Marc; Nelson, D. Michael; Smith, Steve D.; Khun, Huot; Huerre, Michel; Vacher-Lavenu, Marie-Cécile; Gordon, Jeffrey I.; Cossart, Pascale

2004-01-01

Listeria monocytogenes produces severe fetoplacental infections in humans. How it targets and crosses the maternofetal barrier is unknown. We used immunohistochemistry to examine the location of L. monocytogenes in placental and amniotic tissue samples obtained from women with fetoplacental listeriosis. The results raised the possibility that L. monocytogenes crosses the maternofetal barrier through the villous syncytiotrophoblast, with secondary infection occurring via the amniotic epithelium. Because epidemiological studies indicate that the bacterial surface protein, internalin (InlA), may play a role in human fetoplacental listeriosis, we investigated the cellular patterns of expression of its host receptor, E-cadherin, at the maternofetal interface. E-cadherin was found on the basal and apical plasma membranes of syncytiotrophoblasts and in villous cytotrophoblasts. Established trophoblastic cell lines, primary trophoblast cultures, and placental villous explants were each exposed to isogenic InlA+ or InlA- strains of L. monocytogenes, and to L. innocua expressing or not InlA. Quantitative assays of cellular invasion demonstrated that bacterial entry into syncytiotrophoblasts occurs via the apical membrane in an InlA–E-cadherin dependent manner. In human placental villous explants, bacterial invasion of the syncytiotrophoblast barrier and underlying villous tissue and subsequent replication produces histopathological lesions that mimic those seen in placentas of women with listeriosis. Thus, the InlA–E-cadherin interaction that plays a key role in the crossing of the intestinal barrier in humans is also exploited by L. monocytogenes to target and cross the placental barrier. Such a ligand–receptor interaction allowing a pathogen to specifically cross the placental villous trophoblast barrier has not been reported previously. PMID:15073336

Hydrocortisone in culture protects the blast cells in acute myeloblastic leukemia from the lethal effects of cytosine arabinoside

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

Yang, G.S.; Wang, C.; Minkin, S.

The blast cells in acute myeloblastic leukemia (AML) respond to many of the same regulatory mechanisms that control normal hemopoiesis. These include the growth factors that bind to membrane receptors and steroid hormones or vitamins that have intracellular receptors. The authors report the effects in culture of the steroid glucocorticoid hydrocortisone on freshly explanted AML blasts from patients and on two continuous AML cell lines. Only small changes in clonogenic cell numbers in suspension cultures were seen in the presence of hydrocortisone. The most striking effect of the hormone was on the sensitivity of blasts cells to cytosine arabinoside (ara-C).more » In contrast to the response of AML blast cells to retinoic acid, a ligand for intracellular steroid receptors that sensitizes some blast populations to ara-C, hydrocortisone reduced the toxic effects of the drug. The protective action of hydrocortisone was not mediated through the cell cycle since exposure of blasts to hydrocortisone did not affect the percentage of cells in DNA synthesis as measured with the tritiated thymidine (3HTdR) suicide technique. The hydrocortisone effect could be demonstrated using a pulse (20 min) exposure protocol. Blasts pulsed with increasing specific activities of 3HTdR showed the usual response pattern with an initial loss in plating efficiency to about 50% of control, followed by a plateau, regardless of whether the cells had been exposed to hydrocortisone. Control blasts exposed to increasing ara-C concentrations gave very similar dose-response curves; in striking contrast, blast cells cultured in hydrocortisone, then pulsed with ara-C did not lose colony-forming ability even though the same population was sensitive to 3HTdR.« less

Early LC3 lipidation induced by d-limonene does not rely on mTOR inhibition, ERK activation and ROS production and it is associated with reduced clonogenic capacity of SH-SY5Y neuroblastoma cells.

PubMed

Berliocchi, Laura; Chiappini, Carlotta; Adornetto, Annagrazia; Gentile, Debora; Cerri, Silvia; Russo, Rossella; Bagetta, Giacinto; Corasaniti, Maria Tiziana

2018-02-01

d-Limonene is a natural monoterpene abundant in Citrus essential oils. It is endowed with several biological activities, including inhibition of carcinogenesis and promotion of tumour regression. Recently, d-limonene has been shown to modulate autophagic markers in vitro at concentrations found in vivo, in clinical pharmacokinetic studies. Autophagy is an intracellular catabolic process serving as both an adaptive metabolic response and a quality control mechanism. Because autophagy defects have been linked to a wide range of human pathologies, including neurodegeneration and cancer, there is a need for new pharmacological tools to control deregulated autophagy. To better understand the effects of d-limonene on autophagy, to identify the molecular mechanisms through which this monoterpene rapidly triggers LC3 lipidation and to evaluate the role for autophagy in long-term effects of d-limonene. Human SH-SY5Y neuroblastoma, HepG2 hepatocellular carcinoma and MCF7 breast cancer cells were used. Endogenous LC3-II levels were evaluated by western blotting. Autophagic flux assay was performed using bafilomycin A1 and chloroquine. Intracellular distribution of LC3 protein was studied by confocal microscopy analysis of LC3B-GFP transduced cells. Expression of lysosomal-membrane protein LAMP-1 was assessed by immunofluorescence analysis. Phosphorylated levels of downstream substrates of mTOR kinase (p70S6 kinase, 4E-BP1, and ULK1) and ERK were analyzed by western blotting. Production of reactive oxygen species (ROS) was assessed by live confocal microscopy of cells loaded with CellROX ® Green Reagent. Clonogenic assay was used to evaluate the ability of treated cells to proliferate and form colonies. LC3 lipidation promoted by d-limonene correlates with autophagosome formation and stimulation of basal autophagy. LC3 lipidation does not rely on inhibition of mTOR kinase, which instead appears to be transiently activated. In addition, d-limonene rapidly activates ERK and stimulates ROS generation, yet none of these events is implicated in lipidation of LC3, which was only partly reduced by chelation of intracellular calcium. The early LC3 lipidation induced by d-limonene is associated with inhibition of clonogenic capacity which is reverted by the autophagy inhibitor chloroquine. d-Limonene rapidly stimulates the autophagic flux in cultured cancer cells, which could be usefully exploited for therapeutic purposes. Copyright © 2018 Elsevier GmbH. All rights reserved.

Characterization of In Vitro Engineered Human Adipose Tissues: Relevant Adipokine Secretion and Impact of TNF-α

PubMed Central

Aubin, Kim; Safoine, Meryem; Proulx, Maryse; Audet-Casgrain, Marie-Alice; Côté, Jean-François; Têtu, Félix-André; Roy, Alphonse; Fradette, Julie

2015-01-01

Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells, extracellular matrix and differentiated adipocytes, in addition to compounds modulating adipogenesis from precursor cells. PMID:26367137

Effects of High Hydrostatic Pressure on Bacterial Growth on Human Ossicles Explanted from Cholesteatoma Patients

PubMed Central

Ostwald, Jürgen; Lindner, Tobias; Zautner, Andreas Erich; Arndt, Kathleen; Pau, Hans Wilhelm; Podbielski, Andreas

2012-01-01

Background High hydrostatic pressure (HHP) treatment can eliminate cholesteatoma cells from explanted human ossicles prior to re-insertion. We analyzed the effects of HHP treatment on the microbial flora on ossicles and on the planktonic and biofilm states of selected isolates. Methodology Twenty-six ossicles were explanted from cholesteatoma patients. Five ossicles were directly analyzed for microbial growth without further treatment. Fifteen ossicles were cut into two pieces. One piece was exposed to HHP of 350 MPa for 10 minutes. Both the treated and untreated (control) pieces were then assessed semi-quantitatively. Three ossicles were cut into two pieces and exposed to identical pressure conditions with or without the addition of one of two different combinations of antibiotics to the medium. Differential effects of 10-minute in vitro exposure of planktonic and biofilm bacteria to pressures of 100 MPa, 250 MPa, 400 MPa and 540 MPa in isotonic and hypotonic media were analyzed using two patient isolates of Staphylococcus epidermidis and Neisseria subflava. Bacterial cell inactivation and biofilm destruction were assessed by colony counting and electron microscopy. Principal Findings A variety of microorganisms were isolated from the ossicles. Irrespective of the medium, HHP treatment at 350 MPa for 10 minutes led to satisfying but incomplete inactivation especially of Gram-negative bacteria. The addition of antibiotics increased the efficacy of elimination. A comparison of HHP treatment of planktonic and biofilm cells showed that the effects of HPP were reduced by about one decadic logarithmic unit when HPP was applied to biofilms. High hydrostatic pressure conditions that are suitable to inactivate cholesteatoma cells fail to completely sterilize ossicles even if antibiotics are added. As a result of the reduced microbial load and the viability loss of surviving bacteria, however, there is a lower risk of re-infection after re-insertion. PMID:22291908

Btk inhibition suppresses agonist-induced human macrophage activation and inflammatory gene expression in RA synovial tissue explants.

PubMed

Hartkamp, Linda M; Fine, Jay S; van Es, Inge E; Tang, Man Wai; Smith, Michael; Woods, John; Narula, Satwant; DeMartino, Julie; Tak, Paul P; Reedquist, Kris A

2015-08-01

Bruton's tyrosine kinase (Btk) is required for B lymphocyte and myeloid cell contributions to pathology in murine models of arthritis. Here, we examined the potential contributions of synovial Btk expression and activation to inflammation in rheumatoid arthritis (RA). Btk was detected by immunohistochemistry and digital image analysis in synovial tissue from biologically naive RA (n=16) and psoriatic arthritis (PsA) (n=12) patients. Cell populations expressing Btk were identified by immunofluorescent double labelling confocal microscopy, quantitative (q-) PCR and immunoblotting. The effects of a Btk-specific inhibitor, RN486, on gene expression in human macrophages and RA synovial tissue explants (n=8) were assessed by qPCR, ELISA and single-plex assays. Btk was expressed at equivalent levels in RA and PsA synovial tissue, restricted to B lymphocytes, monocytes, macrophages and mast cells. RN486 significantly inhibited macrophage IL-6 production induced by Fc receptor and CD40 ligation. RN486 also reduced mRNA expression of overlapping gene sets induced by IgG, CD40 ligand (CD40L) and RA synovial fluid, and significantly suppressed macrophage production of CD40L-induced IL-8, TNF, MMP-1 and MMP-10, LPS-induced MMP-1, MMP-7 and MMP-10 production, and spontaneous production of IL-6, PDGF, CXCL-9 and MMP-1 by RA synovial explants. Btk is expressed equivalently in RA and PsA synovial tissue, primarily in macrophages. Btk activity is needed to drive macrophage activation in response to multiple agonists relevant to inflammatory arthritis, and promotes RA synovial tissue cytokine and MMP production. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA and other inflammatory diseases. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

The gallium complex KP46 exerts strong activity against primary explanted melanoma cells and induces apoptosis in melanoma cell lines

PubMed Central

Valiahdi, Seied Mojtaba; Heffeter, Petra; Jakupec, Michael A.; Marculescu, Rodrig; Berger, Walter; Rappersberger, Klemens; Keppler, Bernhard K.

2012-01-01

The antineoplastic properties of gallium are well documented. Owing to their robust accumulation of gallium, melanoma cells should be amenable to gallium-based anticancer drugs. With the aim of improving the disappointingly low activity of inorganic gallium salts, we have developed the orally bioavailable gallium complex KP46 [tris(8-quinolinolato)gallium(III)] that was already successfully studied in a phase I clinical trial. To assess its therapeutic potential in malignant melanoma, its antiproliferative effects were investigated in series of human cell lines and primary explanted melanoma samples by means of the MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay and the Human Tumor Cloning Assay, respectively. When compared with other cell lines, the majority of melanoma cells rank among the KP46-sensitive cell lines (50% inhibitory concentration values: 0.8–3.7 μmol/l). Clinically achievable concentrations of KP46 proved to be highly effective in melanoma cells from primary explants of cutaneous and lymph node metastases. Colony growth was inhibited in 10 of 10 specimens by 5 lmol/l KP46 (corresponding to the steady-state plasma concentration measured earlier in a study patient) and in four of 10 specimens by 0.5 μmol/l KP46. In-vitro potency of KP46 is higher than that of dacarbazine or fotemustine and comparable with that of cisplatin. The effects induced by KP46 in melanoma cell lines involve cell cycle perturbations (S-phase arrest) and apoptosis (activation of caspase-9, PARP [poly(ADP-ribose) polymerase] cleavage, formation of apoptotic bodies). No effects on DNA secondary structure could be observed in an electrophoretic mobility shift assay using double-stranded plasmid DNA. Thus, further studies on the therapeutic applicability of KP46 in malignant melanoma are warranted. PMID:19584767

Micropropagation of Araucaria excelsa R. Br. var. glauca Carrière from orthotropic stem explants.

PubMed

Sarmast, Mostafa Khoshhal; Salehi, Hassan; Khosh-Khui, Morteza

2012-07-01

The objectives of the present work were in vitro propagation of Araucaria excelsa R. Br. var. glauca Carrière (Norfolk Island pine) with focus on the evaluation of the mean number of shoots per explant (MNS/E) and mean length of shoots per explants (MLS/E) produced by different parts of the orthotropic stem of A. excelsa R. Br. var. glauca in response to plant growth regulators. Norfolk Island pine axillary meristems responded very well to the 2-iso-pentenyl adenine (2iP) and thidiazuron (TDZ) levels. Explants taken from stem upper segments in the media containing 2iP had a higher MNS/E (3.47) and MLS/E (6.27 mm) in comparison to those taken from stem lower segments, which were 0.71 and 0.51 mm, respectively. Using 0.045 μM TDZ in the MS medium not only resulted in 4.60 MNS/E with 7.08 mm MLS/E but proliferated shoots showed a good performance as well. Investigating the best position of stem explant on mother plant as well as the best concentrations of growth regulators were performed which were useful for efficient micropropagation of this plant. Thirty three percent of explants were rooted in the MS medium containing 3 % sucrose, supplemented with 7.5 μM of both NAA and IBA for 2 weeks before transferring to a half strength MS medium without any growth regulator. Plantlets obtained were acclimatized and transferred to the greenhouse with less than 20 % mortality. This procedure considered the first successful report for regeneration and acclimatization of A. excelsa R. Br. var. glauca plantlet through main stem explants.

Preculturing effect of thidiazuron on in vitro shoot multiplication and micropropagation round in Capparis decidua (Forsk.) an important multipurpose plant.

PubMed

Bukhari, Najat A W; Siddique, Iram; Perveen, Kahkashan

2016-09-01

An efficient protocol was developed for clonal multiplication of an important shrub: Capparis decidua (Forsk.) Edgew, through in vitro shoot induction and multiplication from nodal explants. Pretreatment of nodal explants in a liquid Murashige and Skoog (MS) medium augmented with various thidiazuron (TDZ) concentrations at relatively high levels (5-100 μM) for different time duration (4, 8, 12 and 16 d), proved a significant approach for in vitro shoot production. After an initial exposure time to TDZ, nodal explants were inoculated onto a MS basal medium devoid of TDZ for further induction and proliferation. The highest regeneration rate (85%), average number of shoots/explant (8.7 ± 0.22) and maximum shoot length (3.9 ± 0.33 cm) were obtained from the nodal explants exposed to 50 μM TDZ for 8 d. The nodal explants excised from the proliferated cultures of TDZ (50 μM) for 8 d were used as explants and showed an enhancement rate after next three round of in vitro propagation. Best results for rooting was obtained by ex vitro treatment of shoots with 200 μM indole-3-butyric acid (IBA) for 20 min. as it produced an average of 5.7 ± 0.41 roots per microshoot with 4.4 ± 0.39 cm root length in 84% shoots. Different planting substrates was tested for maximum survival of hardening off micropropagated plantlets and soilrite proved most effective than others as 97.1 ± 7.21 plantlets survived. All micropropagated plants grew well in natural conditions and showed similar morphology to the mother plant.

Comparative effects of plant growth regulators on leaf and stem explants of Labisia pumila var. alata

PubMed Central

Ling, Anna Pick Kiong; Tan, Kinn Poay; Hussein, Sobri

2013-01-01

Objective: Labisia pumila var. alata, commonly known as ‘Kacip Fatimah’ or ‘Selusuh Fatimah’ in Southeast Asia, is traditionally used by members of the Malay community because of its post-partum medicinal properties. Its various pharmaceutical applications cause an excessive harvesting and lead to serious shortage in natural habitat. Thus, this in vitro propagation study investigated the effects of different plant growth regulators (PGRs) on in vitro leaf and stem explants of L. pumila. Methods: The capabilities of callus, shoot, and root formation were evaluated by culturing both explants on Murashige and Skoog (MS) medium supplemented with various PGRs at the concentrations of 0, 1, 3, 5, and 7 mg/L. Results: Medium supplemented with 3 mg/L indole-3-butyric acid (IBA) showed the optimal callogenesis from both leaf and stem explants with (72.34±19.55)% and (70.40±14.14)% efficacy, respectively. IBA was also found to be the most efficient PGR for root induction. A total of (50.00±7.07)% and (77.78±16.47)% of root formation were obtained from the in vitro stem and leaf explants after being cultured for (26.5±5.0) and (30.0±8.5) d in the medium supplemented with 1 and 3 mg/L of IBA, respectively. Shoot formation was only observed in stem explant, with the maximum percentage of formation ((100.00±0.00)%) that was obtained in 1 mg/L zeatin after (11.0±2.8) d of culture. Conclusions: Callus, roots, and shoots can be induced from in vitro leaf and stem explants of L. pumila through the manipulation of types and concentrations of PGRs. PMID:23825148

l-N-acetylcysteine protects outer hair cells against TNFα initiated ototoxicity in vitro.

PubMed

Tillinger, Joshua A; Gupta, Chhavi; Ila, Kadri; Ahmed, Jamal; Mittal, Jeenu; Van De Water, Thomas R; Eshraghi, Adrien A

2018-08-01

The present study is aimed at determining the efficacy and exploring the mechanisms by which l-N-acetylcysteine (l-NAC) provides protection against tumor necrosis factor-alpha (TNFα)-induced oxidative stress damage and hair cell loss in 3-day-old rat organ of Corti (OC) explants. Previous work has demonstrated a high level of oxidative stress in TNFα-challenged OC explants. TNFα can potentially play a significant role in hair cell loss following an insult to the inner ear. l-NAC has shown to provide effective protection against noise-induced hearing loss in laboratory animals but mechanisms of this otoprotective effect are not well-defined. Rat OC explants were exposed to either: (1) saline control (N = 12); (2) TNFα (2 μg/ml, N = 12); (3) TNFα+l-NAC (5 mM, N = 12); (4) TNFα+l-NAC (10 mM, N = 12); or (5) l-NAC (10 mM, N = 12). Outer hair cell (OHC) density, levels of reactive oxygen species (ROS), lipid peroxidation of cell membranes, gluthathione activity, and mitochondrial viability were assayed. l-NAC (5 and 10 mM) provided protection for OHCs from ototoxic level of TNFα in OC explants. Groups treated with TNFα+l-NAC (5 mM) showed a highly significant reduction of both ROS (p < 0.01) and 4-hydroxy-2-nonenal immunostaining (p < 0.001) compared to TNFα-challenged explants. Total glutathione levels were low in TNFα-challenged explants compared to control and TNFα+l-NAC (5 mM) treated explants (p < 0.001). l-NAC is a promising treatment for protecting auditory HCs from TNFα-induced oxidative stress and subsequent loss via programmed cell death.

Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

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

Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves

The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression.more » Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. - Highlights: • Recycled fin explants outgrow cells bearing stable mesenchymal traits. • Cell production and quality is enhanced in the recycled explant culture system. • Fresh fin primary culture is highly variable and loose epithelial traits over time.« less

Lactobacillus plantarum culture supernatants improve intestinal tissue exposed to deoxynivalenol.

PubMed

Maidana, L G; Gerez, J; Pinho, F; Garcia, S; Bracarense, A P F L

2017-10-02

In the present study, histological, morphometrical and ultrastructural analysis were performed to investigate intestinal mucosa changes in piglets exposed to deoxynivalenol alone or associated with two strains of Lactobacillus plantarum and the respective culture supernatants. Jejunal explants were incubated for 4h in culture medium with a) only culture medium (DMEM, control group), b) deoxynivalenol (DON, 10μM), c) heat-inactivated Lactobacillus plantarum strain1 - LP1 (1.1×10 8 CFU/ml) plus DON, d) heat-inactivated Lactobacillus plantarum strain2-LP2 (2.0×10 9 CFU/ml) plus DON, e) heat-inactivated Lactobacillus plantarum strain1 culture supernatant (CS1) plus DON, and f) heat-inactivated Lactobacillus plantarum strain1 culture supernatant (CS1) plus DON. Explants exposed to DON and DON plus LP1 and LP2 showed a significant increase in histological changes (mainly villi atrophy and apical necrosis) and a significant decrease in villi height when compared to unexposed explants. However, explants treated with CS1+DON and CS2+DON remained similar to the control group both in histological and morphometrical aspects. DON also induced a significant decrease in goblet cell density compared to control whereas CS1+DON treatment induced an increase in the number of goblet cells in comparison to DON explants. In addition, ultrastructural assessment showed control, CS1+DON and CS2+DON explants with well delineated finger shape villi, meanwhile DON-treated, LP1+DON and LP2+DON explants showed a severe villi atrophy with leukocytes exudation on the intestinal surface. Taken together, our results indicate that the culture supernatant treatment reduced the toxic effects induced by DON on intestinal tissue and may contribute as an alternative strategy to reduce mycotoxin toxicity. Copyright © 2017 Elsevier GmbH. All rights reserved.

Genetic and functional studies of the intervertebral disc: a novel murine intervertebral disc model.

PubMed

Pelle, Dominic W; Peacock, Jacqueline D; Schmidt, Courtney L; Kampfschulte, Kevin; Scholten, Donald J; Russo, Scott S; Easton, Kenneth J; Steensma, Matthew R

2014-01-01

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration.

Genetic and Functional Studies of the Intervertebral Disc: A Novel Murine Intervertebral Disc Model

PubMed Central

Pelle, Dominic W.; Peacock, Jacqueline D.; Schmidt, Courtney L.; Kampfschulte, Kevin; Scholten, Donald J.; Russo, Scott S.; Easton, Kenneth J.; Steensma, Matthew R.

2014-01-01

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration. PMID:25474689

Collaborative Model for Acceleration of Individualized Therapy of Colon Cancer

DTIC Science & Technology

2012-10-01

will be analyzed by one of two methods. The human CRC explants will be assessed (in our CLIA-certified UCCC Pathology Core) using the DxS Scorpion ...also developing ways to select patients for those treatments. Unfortunately the lack of such strategies is what led to thousands of CRC patients with...individualized therapy for patients with KRAS mutant colorectal cancer (CRC) using a comprehensive bioinformatics approach and novel preclinical

Highly Human Immunodeficiency Virus-Exposed Seronegative Men Have Lower Mucosal Innate Immune Reactivity.

PubMed

Fulcher, Jennifer A; Romas, Laura; Hoffman, Jennifer C; Elliott, Julie; Saunders, Terry; Burgener, Adam D; Anton, Peter A; Yang, Otto O

2017-08-01

Risk of HIV acquisition varies, and some individuals are highly HIV-1-exposed, yet, persistently seronegative (HESN). The immunologic mechanisms contributing to this phenomenon are an area of intense interest. As immune activation and inflammation facilitate disease progression in HIV-1-infected persons and gastrointestinal-associated lymphoid tissue is a highly susceptible site for transmission, we hypothesized that reduced gut mucosal immune reactivity may contribute to reduced HIV-1 susceptibility in HESN men with a history of numerous rectal sexual exposures. To test this, we used ex vivo mucosal explants from freshly acquired colorectal biopsies from healthy control and HESN subjects who were stimulated with specific innate immune ligands and inactivated whole pathogens. Immune reactivity was then assessed via cytokine arrays and proteomic analysis. Mucosal immune cell compositions were quantified via immunohistochemistry. We found that explants from HESN subjects produced less proinflammatory cytokines compared with controls following innate immune stimulation; while noninflammatory cytokines were similar between groups. Proteomic analysis identified several immune response proteins to be differentially expressed between HIV-1-stimulated HESN and control explants. Immunohistochemical examination of colorectal mucosa showed similar amounts of T cells, macrophages, and dendritic cells between groups. The results of this pilot study suggest that mucosal innate immune reactivity is dampened in HESN versus control groups, despite presence of similar densities of immune cells in the colorectal mucosa. This observed modulation of the rectal mucosal immune response may contribute to lower risk of mucosal HIV-1 transmission in these individuals.

Mathematics as a conduit for translational research in post-traumatic osteoarthritis.

PubMed

Ayati, Bruce P; Kapitanov, Georgi I; Coleman, Mitchell C; Anderson, Donald D; Martin, James A

2017-03-01

Biomathematical models offer a powerful method of clarifying complex temporal interactions and the relationships among multiple variables in a system. We present a coupled in silico biomathematical model of articular cartilage degeneration in response to impact and/or aberrant loading such as would be associated with injury to an articular joint. The model incorporates fundamental biological and mechanical information obtained from explant and small animal studies to predict post-traumatic osteoarthritis (PTOA) progression, with an eye toward eventual application in human patients. In this sense, we refer to the mathematics as a "conduit of translation." The new in silico framework presented in this paper involves a biomathematical model for the cellular and biochemical response to strains computed using finite element analysis. The model predicts qualitative responses presently, utilizing system parameter values largely taken from the literature. To contribute to accurate predictions, models need to be accurately parameterized with values that are based on solid science. We discuss a parameter identification protocol that will enable us to make increasingly accurate predictions of PTOA progression using additional data from smaller scale explant and small animal assays as they become available. By distilling the data from the explant and animal assays into parameters for biomathematical models, mathematics can translate experimental data to clinically relevant knowledge. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:566-572, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Osteopontin and splice variant expression level in human malignant glioma: radiobiologic effects and prognosis after radiotherapy.

PubMed

Güttler, Antje; Giebler, Maria; Cuno, Peter; Wichmann, Henri; Keßler, Jacqueline; Ostheimer, Christian; Söling, Ariane; Strauss, Christian; Illert, Jörg; Kappler, Matthias; Vordermark, Dirk; Bache, Matthias

2013-09-01

We investigated the role of the hypoxia-associated secreted glycoprotein osteopontin (OPN) in the response of malignant glioma to radiotherapy by characterizing OPN and its splice variants in vitro and in patient material. The effect of siRNA knockdown of OPN splice variants on cellular and radiobiologic behavior was analyzed in U251MG cells using OpnS siRNA (inhibition of all OPN splice variants) and OpnAC siRNA (knockdown only of OPNa and OPNc). OPN and splice variant mRNA levels were quantified in archival material of 41 glioblastoma tumor samples. Plasma OPN was prospectively measured in 33 malignant glioma patients. Inhibition of OPNa and OPNc (OpnAC) reduced clonogenic survival in U251MG cells but did not affect proliferation, migration or apoptosis. Knockdown of all OPN splice variants (OpnS) resulted in an even stronger inhibition of clonogenic survival, while cell proliferation and migration were reduced and rate of apoptosis was increased. Additional irradiation had additive effects with both siRNAs. Plasma OPN increased continuously in malignant glioma patients and was associated with poor survival. OPNb is partially able to compensate the effects of OPNa and OPNc knockdown in U251MG cells. High OPN plasma levels at the end of radiotherapy are associated with poor survival. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Spatial and temporal characterization of endometrial mesenchymal stem-like cells activity during the menstrual cycle

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

Shan, Xu; Chan, Rachel W.S., E-mail: rwschan@hku.hk; Centre of Reproduction, Development of Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR

The human endometrium is a highly dynamic tissue with the ability to cyclically regenerate during the reproductive life. Endometrial mesenchymal stem-like cells (eMSCs) located throughout the endometrium have shown to functionally contribute to endometrial regeneration. In this study we examine whether the menstrual cycle stage and the location in the endometrial bilayer (superficial and deep portions of the endometrium) has an effect on stem cell activities of eMSCs (CD140b{sup +}CD146{sup +} cells). Here we show the percentage and clonogenic ability of eMSCs were constant in the various stages of the menstrual cycle (menstrual, proliferative and secretory). However, eMSCs from themore » menstrual endometrium underwent significantly more rounds of self-renewal and enabled a greater total cell output than those from the secretory phase. Significantly more eMSCs were detected in the deeper portion of the endometrium compared to the superficial layer but their clonogenic and self-renewal activities remained similar. Our findings suggest that eMSCs are activated in the menstrual phase for the cyclical regeneration of the endometrium. - Highlights: • The percentages of endometrial mesenchymal-like stem cells (eMSCs) were constant across the menstrual cycle. • Menstruation eMSCs display superior self-renewal and long-term proliferative activities. • More eMSCs reside in the deeper portion of the endometrium than the superficial layer.« less

Acid ceramidase in prostate cancer radiation therapy resistance and relapse

NASA Astrophysics Data System (ADS)

Cheng, Joseph C.

Prostate tumor cell escape from ionizing radiation (IR)-induced killing can lead to disease progression and relapse. Sphingolipids such as ceramide and sphingosine 1-phosphate influence signal transduction pathways that regulate stress response in cancer cells. In particular, metabolism of apoptotic ceramide constitutes an important survival adaptation. Assessments of enzyme activity, mRNA, and protein demonstrated preferential upregulation of the ceramide deacylating enzyme acid ceramidase (AC) in irradiated cancer cells. Promoter-reporter and ChIP-qPCR assays revealed AC transcription by activator protein 1 (AP-1) is sensitive to pharmacological inhibition of de novo ceramide biosynthesis, identifying a protective feedback mechanism that mitigates the effects of IR-induced ceramide. Deregulation of c-Jun, in particular, induced marked radiosensitization in vitro and in vivo, which was rescued by ectopic AC over-expression. AC over-expression in prostate cancer clonogens surviving 80 Gray fractionated irradiation was associated with increased radioresistance and proliferation, suggesting a role in radiotherapy failure and relapse. Indeed, immunohistochemical analysis of human prostate cancer tissues revealed higher levels of AC after radiotherapy failure than therapy-naive adenocarcinoma, PIN, or benign tissues. By genetically downregulating AC with small interfering RNA (siRNA), we observed radiosensitization of cells using clonogenic and cytotoxicity assays. Finally, treatment with lysosomotropic small molecule inhibitors of AC, LCL385 or LCL521, induced prostate cancer xenograft radiosensitization and long-term suppression, suggesting AC is a tractable target for adjuvant radiotherapy.

Effects of silibinin on growth and invasive properties of human ovarian carcinoma cells through suppression of heregulin/HER3 pathway.

PubMed

Momeny, Majid; Ghasemi, Reza; Valenti, Giovanni; Miranda, Mariska; Zekri, Ali; Zarrinrad, Ghazaleh; Javadikooshesh, Sepehr; Yaghmaie, Marjan; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Ghaffari, Seyed H

2016-03-01

Epithelial ovarian cancer (EOC) is the most fatal gynecological malignancy due to its high proliferative and invasive capacities. A heregulin (HRG)/HER3 autocrine loop increases proliferative and metastatic properties of EOC cells, suggesting that modulators of this signaling pathway may prove effective to trammel growth and motility of these cells. This study aimed to evaluate the effects of multi-tyrosine kinase inhibitor silibinin on proliferative and invasive characteristics of EOC cell lines OVCAR8 and SKOV3 through suppression of the HRG/HER3 pathway. To achieve this, the effects of silibinin on proliferation, DNA synthesis, clonogenicity, cell cycle progression, cathepsin B enzymatic activity, and migration and invasion were explored in vitro. Silibinin suppressed proliferation, DNA synthesis, and clonogenic abilities of OVCAR8 and SKOV3 cells through inhibition of the autocrine HRG/HER3 circuit. Silibinin-mediated attenuation of the HER3 signaling disabled the HER3/AKT/survivin axis and thereby, induced G1/S cell cycle arrest. Furthermore, silibinin reduced invasive potentials of the EOC cells through quelling the HRG/HER3 pathway and suppression of cathepsin B activity. Altogether, these results suggest that silibinin is a potential anti-cancer drug to inhibit proliferative and invasive characteristics of the EOC cells that exhibit an autocrine HRG/HER3 pathway.

Evaluation of the cytotoxic and genotoxic effects of benchmark multi-walled carbon nanotubes in relation to their physicochemical properties.

PubMed

Louro, Henriqueta; Pinhão, Mariana; Santos, Joana; Tavares, Ana; Vital, Nádia; Silva, Maria João

2016-11-16

To contribute with scientific evidence to the grouping strategy for the safety assessment of multi-walled carbon nanotubes (MWCNTs), this work describes the investigation of the cytotoxic and genotoxic effects of four benchmark MWCNTs in relation to their physicochemical characteristics, using two types of human respiratory cells. The cytotoxic effects were analysed using the clonogenic assay and replication index determination. A 48h-exposure of cells revealed that NM-401 was the only cytotoxic MWCNT in both cell lines, but after 8-days exposure, the clonogenic assay in A549 cells showed cytotoxic effects for all the tested MWCNTs. Correlation analysis suggested an association between the MWCNTs size in cell culture medium and cytotoxicity. No induction of DNA damage was observed after any MWCNTs in any cell line by the comet assay, while the micronucleus assay revealed that both NM-401 and NM-402 were genotoxic in A549 cells. NM-401 and NM-402 are the two longest MWCNTs analyzed in this work, suggesting that length may be determinant for genotoxicity. No induction of micronuclei was observed in BBEAS-2Beas-2B cell line and the different effect in both cell lines is explained in view of the size-distribution of MWCNTs in the cell culture medium, rather than cell's specificities. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Embryonic Explant Culture: Studying Effects of Regulatory Molecules on Gene Expression in Craniofacial Tissues.

PubMed

Närhi, Katja

2017-01-01

The ex vivo culture of embryonic tissue explants permits the continuous monitoring of growth and morphogenesis at specific embryonic stages. The functions of soluble regulatory molecules can be analyzed by introducing them into culture medium or locally with beads to the tissue. Gene expression in the manipulated tissue explants can be analyzed using in situ hybridization, quantitative PCR, and reporter constructs combined to organ culture to examine the functions of the signaling molecules.

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection.

PubMed

Fornaro, Michele; Sharthiya, Harsh; Tiwari, Vaibhav

2018-03-09

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

Altered Gravity and Early Heart Development in Culture

NASA Technical Reports Server (NTRS)

Wiens, Darrell J.; Lwigale, P.; Denning, J.

1996-01-01

The macromolecules comprising the cytoskeleton and extracellular matrix of cells may be sensitive to gravitation. Since early development of organs depends on dynamic interactions across cell surfaces, altered gravity may disturb development. We investigated this possibility for heart development. Previous studies showed that the extracellular matrix glycoprotein fibronectin (Fn) is necessary for normal heart development. We cultured precardiac tissue explants in a high aspect ratio bioreactor vessel (HARV) to simulate microgravity. We observed tissue morphology, contraction, and Fn distribution by immunolocalization in HARV rotated and control (lxg) explants, cultured 18 hr. We also measured Fn amount by immunoassay. Explants in HARV were rotated at 6 rpm to achieve continuous freefall. Thirty-five of 37 control, but only 1 of 37 matched rotated explants exhibited contractions. Tissue architecture was identical. Immunolocalization of Fn showed remarkable differences which may be related to the development of contractions. The Fn staining in the HARV explants was less intense in all areas. Areas of linear staining along epithelia were present but shorter, and there was less intercellular staining in both mesenchymal tissue and myocardium. Initial immunoassay results of 5 matched pairs of explants showed a 22% reduction in total tissue Fn in the HARV rotated samples. Our results indicate that altered gravity in the HARV reduced the amount and distribution of Fn, as assessed by two independent criteria. This was correlated with a reduction in the development of contractile activity.

Expression of human oxoguanine glycosylase 1 or formamidopyrimidine glycosylase in human embryonic kidney 293 cells exacerbates methylmercury toxicity in vitro

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

Ondovcik, Stephanie L.; Preston, Thomas J.; McCallum, Gordon P.

Exposure to methylmercury (MeHg) acutely at high levels, or via chronic low-level dietary exposure from daily fish consumption, can lead to adverse neurological effects in both the adult and developing conceptus. To determine the impact of variable DNA repair capacity, and the role of reactive oxygen species (ROS) and oxidatively damaged DNA in the mechanism of toxicity, transgenic human embryonic kidney (HEK) 293 cells that stably express either human oxoguanine glycosylase 1 (hOgg1) or its bacterial homolog, formamidopyrimidine glycosylase (Fpg), which primarily repair the oxidative lesion 8-oxo-2′-deoxyguanosine (8-oxodG), were used to assess the in vitro effects of MeHg. Western blottingmore » confirmed the expression of hOgg1 or Fpg in both the nuclear and mitochondrial compartments of their respective cell lines. Following acute (1–2 h) incubations with 0–10 μM MeHg, concentration-dependent decreases in clonogenic survival and cell growth accompanied concentration-dependent increases in lactate dehydrogenase (LDH) release, ROS formation, 8-oxodG levels and apurinic/apyrimidinic (AP) sites, consistent with the onset of cytotoxicity. Paradoxically, hOgg1- and Fpg-expressing HEK 293 cells were more sensitive than wild-type cells stably transfected with the empty vector control to MeHg across all cellular and biochemical parameters, exhibiting reduced clonogenic survival and cell growth, and increased LDH release and DNA damage. Accordingly, upregulation of specific components of the base excision repair (BER) pathway may prove deleterious potentially due to the absence of compensatory enhancement of downstream processes to repair toxic intermediary abasic sites. Thus, interindividual variability in DNA repair activity may constitute an important risk factor for environmentally-initiated, oxidatively damaged DNA and its pathological consequences. - Highlights: • hOgg1 and Fpg repair oxidatively damaged DNA. • hOgg1- and Fpg-expressing cells are more sensitive to MeHg toxicity. • Enhanced sensitivity is likely due to an accumulation of toxic repair intermediates. • Interindividual variability in DNA repair activity may modulate toxicological risk.« less

Identification of the optimal therapeutic antibody for fluorescent imaging of cutaneous squamous cell carcinoma

PubMed Central

Day, Kristine E.; Beck, Lauren N.; Heath, C. Hope; Huang, Conway C.; Zinn, Kurt R.; Rosenthal, Eben L.

2013-01-01

Intraoperative, real-time fluorescence imaging may significantly improve tumor visualization and resection and postoperatively, in pathological assessment. To this end, we sought to determine the optimal FDA approved therapeutic monoclonal antibody for optical imaging of human cutaneous squamous cell carcinoma (cSCC). A near-infrared (NIR) fluorescent probe (IRDye800) was covalently linked to bevacizumab, panitumumab or tocilizumab and injected systemically into immunodeficient mice bearing either cutaneous tumor cell lines (SCC13) or cutaneous human tumor explants. Tumors were then imaged and resected under fluorescent guidance with the SPY, an FDA-approved intraoperative imaging system, and the Pearl Impulse small animal imaging system. All fluorescently labeled antibodies delineated normal tissue from tumor in SCC13 xenografts based on tumor-to-background (TBR) ratios. The conjugated antibodies produced TBRs of 1.2–2 using SPY and 1.6–3.6 using Pearl; in comparison, isotype control antibody IgG-IRDye produced TBRs of 1.0 (SPY) and 0.98 (Pearl). Comparison between antibodies revealed them to be roughly equivalent for imaging purposes with both the SPY and Pearl (p = 0.89 SPY, p = 0.99 Pearl; one way ANOVA). Human tumor explants were also imaged and tumor detection was highest with panitumumab-IRDye800 when using the SPY (TBR 3.0) and Pearl (TBR 4.0). These data suggest that FDA approved antibodies may be clinically used for intraoperative detection of cSCC. PMID:23298904

Inhibitory effect of mitomycin C on proliferation of primary cultured fibroblasts from human airway granulation tissues.

PubMed

Chen, Nan; Zhang, Jie; Xu, Min; Wang, Yu Ling; Pei, Ying Hua

2013-01-01

Airway granulation tissue and scar formation pose a challenge because of the high incidence of recurrence after treatment. As an emerging treatment modality, topical application of mitomycin C has potential value in delaying the recurrence of airway obstruction. Several animal and clinical studies have already proven its feasibility and efficacy. However, the ideal dosage has still not been determined. To establish a novel method for culturing primary fibroblasts isolated from human airway granulation tissue, and to investigate the dose-effect of mitomycin C on the fibroblast proliferation in vitro, so as to provide an experimental reference for clinical practitioners. Granulation tissues were collected during the routine bronchoscopy at our department. The primary fibroblasts were obtained by culturing the explanted tissues. The cells were treated with different concentrations of mitomycin C (0.1, 0.2, 0.4, 0.8 and 1.6 mg/ml) for 5 min followed by additional 48-hour culture before an MTT assay was performed to measure cell viability. MTT assay showed that mitomycin C reduced cell viability at all tested concentrations. The inhibitory ratios were 10.26, 26.77, 32.88, 64.91 and 80.45% for cells treated with mitomycin C at 0.1, 0.2, 0.4, 0.8 and 1.6 mg/ml, respectively. Explant culture is a reliable method for culturing primary fibroblasts from human airway granulation tissue, and mitomycin C can inhibit proliferation of the fibroblasts in vitro. Copyright © 2013 S. Karger AG, Basel.

[Explantation method of isolating a persistent tick-borne encephalitis virus from the organs of infected monkeys].

PubMed

Levina, L S; Pogodina, V V

1981-01-01

The method of explantation was used to examine 63 organs from M. rhesus monkeys 92-783 days after intracerebral and subcutaneous inoculation with the Vasilchenko, Aina/1448 and 41/65 strains of tick-borne encephalitis virus. The optimal time for examination of the explants by tests of the hemagglutinating, cytopathogenic activity of the virus and its pathogenicity for mice was found to be the 15th day of cultivation. A comparative study of the properties of 3 isolates obtained from explants of the spleen, liver and subcortical cerebral ganglia 202 and 307 days after inoculation of monkeys was carried out. The isolates differed from the parental TBE virus strains by their capacity to form small plaques in PEKV cell cultures (pig embryo kidney cells in versen medium).

Variability of Powassan virus cultured in tissue explants and organism of Hyalomma anatolicum ticks.

PubMed

Khozinskaya, G A; Chunikhin, S P; Khozinsky, V V; Stefutkina, L F

1985-07-01

The variability of Powassan virus was studied during successive passages in Hyalomma anatolicum ticks or prolonged reproduction in their tissue explants. It had been shown that in the course of tick passages and during reproduction in the explants, pathogenicity of the virus in respect to causing acute disease in mice after peripheral inoculation was decreased, while virus ability to cause death after intracerebral (i.c.) inoculation remained unchanged. In mice infected with the original strain P-40 of Powassan virus damaging effect of the immune response prevailed, while in mice infected with the strains passaged for a long time in ticks (strain P-40Hat) or in tick tissue explants (strain P-40Haex), the protective effect of the immune response was prominent.

Explanting the Nellix Endovascular Aortic Sealing Endoprosthesis for Proximal Aortic Neck Failure.

PubMed

Lee, Cheong Jun; Cuff, Robert

2018-05-17

Open conversion following endovascular aortic repair (EVAR) has inherent challenges particular to the device being explanted. The Nellix endograft is unlike any other device as it utilizes polymer filling of endobags within the aorto-iliac lumen to seal the AAA sac; a developing concept known as endovascular aortic sealing (EVAS). Conversion to open repair of AAA treated with the Nellix endograft have rarely been discussed. Explants that have been previously reported were for graft infection. We present two Nellix graft explants that were required for device migration and subsequent development of a type IA endoleak. The technique and nuances observed during open conversion of this novel endograft for proximal aortic neck failure is described in this report. Copyright © 2018. Published by Elsevier Inc.

Radiation-induced biologic bystander effect elicited in vitro by targeted radiopharmaceuticals labeled with alpha-, beta-, and auger electron-emitting radionuclides.

PubMed

Boyd, Marie; Ross, Susan C; Dorrens, Jennifer; Fullerton, Natasha E; Tan, Ker Wei; Zalutsky, Michael R; Mairs, Robert J

2006-06-01

Recent studies have shown that indirect effects of ionizing radiation may contribute significantly to the effectiveness of radiotherapy by sterilizing malignant cells that are not directly hit by the radiation. However, there have been few investigations of the importance of indirect effects in targeted radionuclide treatment. Our purpose was to compare the induction of bystander effects by external beam gamma-radiation with those resultant from exposure to 3 radiohaloanalogs of metaiodobenzylguanidine (MIBG): (131)I-MIBG (low-linear-energy-transfer [LET] beta-emitter), (123)I-MIBG (potentially high-LET Auger electron emitter), and meta-(211)At-astatobenzylguanidine ((211)At-MABG) (high-LET alpha-emitter). Two human tumor cell lines-UVW (glioma) and EJ138 (transitional cell carcinoma of bladder)-were transfected with the noradrenaline transporter (NAT) gene to enable active uptake of MIBG. Medium from cells that accumulated the radiopharmaceuticals or were treated with external beam radiation was transferred to cells that had not been exposed to radioactivity, and clonogenic survival was determined in donor and recipient cultures. Over the dose range 0-9 Gy of external beam radiation of donor cells, 2 Gy caused 30%-40% clonogenic cell kill in recipient cultures. This potency was maintained but not increased by higher dosage. In contrast, no corresponding saturation of bystander cell kill was observed after treatment with a range of activity concentrations of (131)I-MIBG, which resulted in up to 97% death of donor cells. Cellular uptake of (123)I-MIBG and (211)At-MABG induced increasing recipient cell kill up to levels that resulted in direct kill of 35%-70% of clonogens. Thereafter, the administration of higher activity concentrations of these high-LET emitters was inversely related to the kill of recipient cells. Over the range of activity concentrations examined, neither direct nor indirect kill was observed in cultures of cells not expressing the NAT and, thus, incapable of active uptake of MIBG. Potent toxins are generated specifically by cells that concentrate radiohalogenated MIBG. These may be LET dependent and distinct from those elicited by conventional radiotherapy.

Toward a clinical application of ex situ boron neutron capture therapy for lung tumors at the RA-3 reactor in Argentina

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

Farías, R. O.; Trivillin, V. A.; Portu, A. M.

Purpose: Many types of lung tumors have a very poor prognosis due to their spread in the whole organ volume. The fact that boron neutron capture therapy (BNCT) would allow for selective targeting of all the nodules regardless of their position, prompted a preclinical feasibility study of ex situ BNCT at the thermal neutron facility of RA-3 reactor in the province of Buenos Aires, Argentina. (L)-4p-dihydroxy-borylphenylalanine fructose complex (BPA-F) biodistribution studies in an adult sheep model and computational dosimetry for a human explanted lung were performed to evaluate the feasibility and the therapeutic potential of ex situ BNCT. Methods: Twomore » kinds of boron biodistribution studies were carried out in the healthy sheep: a set of pharmacokinetic studies without lung excision, and a set that consisted of evaluation of boron concentration in the explanted and perfused lung. In order to assess the feasibility of the clinical application of ex situ BNCT at RA-3, a case of multiple lung metastases was analyzed. A detailed computational representation of the geometry of the lung was built based on a real collapsed human lung. Dosimetric calculations and dose limiting considerations were based on the experimental results from the adult sheep, and on the most suitable information published in the literature. In addition, a workable treatment plan was considered to assess the clinical application in a realistic scenario. Results: Concentration-time profiles for the normal sheep showed that the boron kinetics in blood, lung, and skin would adequately represent the boron behavior and absolute uptake expected in human tissues. Results strongly suggest that the distribution of the boron compound is spatially homogeneous in the lung. A constant lung-to-blood ratio of 1.3 ± 0.1 was observed from 80 min after the end of BPA-F infusion. The fact that this ratio remains constant during time would allow the blood boron concentration to be used as a surrogate and indirect quantification of the estimated value in the explanted healthy lung. The proposed preclinical animal model allowed for the study of the explanted lung. As expected, the boron concentration values fell as a result of the application of the preservation protocol required to preserve the lung function. The distribution of the boron concentration retention factor was obtained for healthy lung, with a mean value of 0.46 ± 0.14 consistent with that reported for metastatic colon carcinoma model in rat perfused lung. Considering the human lung model and suitable tumor control probability for lung cancer, a promising average fraction of controlled lesions higher than 85% was obtained even for a low tumor-to-normal boron concentration ratio of 2. Conclusions: This work reports for the first time data supporting the validity of the ovine model as an adequate human surrogate in terms of boron kinetics and uptake in clinically relevant tissues. Collectively, the results and analysis presented would strongly suggest that ex situ whole lung BNCT irradiation is a feasible and highly promising technique that could greatly contribute to the treatment of metastatic lung disease in those patients without extrapulmonary spread, increasing not only the expected overall survival but also the resulting quality of life.« less

Toward a clinical application of ex situ boron neutron capture therapy for lung tumors at the RA-3 reactor in Argentina.

PubMed

Farías, R O; Garabalino, M A; Ferraris, S; Santa María, J; Rovati, O; Lange, F; Trivillin, V A; Monti Hughes, A; Pozzi, E C C; Thorp, S I; Curotto, P; Miller, M E; Santa Cruz, G A; Bortolussi, S; Altieri, S; Portu, A M; Saint Martin, G; Schwint, A E; González, S J

2015-07-01

Many types of lung tumors have a very poor prognosis due to their spread in the whole organ volume. The fact that boron neutron capture therapy (BNCT) would allow for selective targeting of all the nodules regardless of their position, prompted a preclinical feasibility study of ex situ BNCT at the thermal neutron facility of RA-3 reactor in the province of Buenos Aires, Argentina. (l)-4p-dihydroxy-borylphenylalanine fructose complex (BPA-F) biodistribution studies in an adult sheep model and computational dosimetry for a human explanted lung were performed to evaluate the feasibility and the therapeutic potential of ex situ BNCT. Two kinds of boron biodistribution studies were carried out in the healthy sheep: a set of pharmacokinetic studies without lung excision, and a set that consisted of evaluation of boron concentration in the explanted and perfused lung. In order to assess the feasibility of the clinical application of ex situ BNCT at RA-3, a case of multiple lung metastases was analyzed. A detailed computational representation of the geometry of the lung was built based on a real collapsed human lung. Dosimetric calculations and dose limiting considerations were based on the experimental results from the adult sheep, and on the most suitable information published in the literature. In addition, a workable treatment plan was considered to assess the clinical application in a realistic scenario. Concentration-time profiles for the normal sheep showed that the boron kinetics in blood, lung, and skin would adequately represent the boron behavior and absolute uptake expected in human tissues. Results strongly suggest that the distribution of the boron compound is spatially homogeneous in the lung. A constant lung-to-blood ratio of 1.3 ± 0.1 was observed from 80 min after the end of BPA-F infusion. The fact that this ratio remains constant during time would allow the blood boron concentration to be used as a surrogate and indirect quantification of the estimated value in the explanted healthy lung. The proposed preclinical animal model allowed for the study of the explanted lung. As expected, the boron concentration values fell as a result of the application of the preservation protocol required to preserve the lung function. The distribution of the boron concentration retention factor was obtained for healthy lung, with a mean value of 0.46 ± 0.14 consistent with that reported for metastatic colon carcinoma model in rat perfused lung. Considering the human lung model and suitable tumor control probability for lung cancer, a promising average fraction of controlled lesions higher than 85% was obtained even for a low tumor-to-normal boron concentration ratio of 2. This work reports for the first time data supporting the validity of the ovine model as an adequate human surrogate in terms of boron kinetics and uptake in clinically relevant tissues. Collectively, the results and analysis presented would strongly suggest that ex situ whole lung BNCT irradiation is a feasible and highly promising technique that could greatly contribute to the treatment of metastatic lung disease in those patients without extrapulmonary spread, increasing not only the expected overall survival but also the resulting quality of life.

Regeneration of Cuphea tolucana Peyr. in in vitro culture.

PubMed

Przybecki, Z; Olejniczak, J; Adamska, E

2001-01-01

In order to regenerate Cuphea tolucana from hypocotyl, cotyledon and root explants, a solid culture and 8 hormone combinations were used. Only the root explants did not react to any of the media. On most of the media, the other explants formed shoots, roots or callus, or their reaction was more complex. The regeneration probably occurred via direct organogenesis. The regenerants displayed a wide variety of morphological characteristics. However, their offspring did not show any differences from plants, which had not undergone culture.

Development of efficient plant regeneration and transformation system for impatiens using Agrobacterium tumefaciens and multiple bud cultures as explants.

PubMed

Dan, Yinghui; Baxter, Aaron; Zhang, Song; Pantazis, Christopher J; Veilleux, Richard E

2010-08-09

Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system has the advantages of 1) efficient, simple and rapid regeneration and transformation (with no need for sterilization or a greenhouse to grow stock plants), 2) flexibility (available all the time) for in vitro manipulation, 3) uniform and desirable green tissue explants for both nuclear and plastid transformation using Agrobacterium-mediated and biolistics methods, 4) no somaclonal variation and 5) resolution of necrosis of Agrobacterium-inoculated tissues.

Kinetics of depopulation, repopulation and host cell infiltration in the rhabdomyosarcoma R1H after 14 MeV neutron irradiation.

PubMed

Brammer, I; Zywietz, F; Beck-Bornholdt, H P; Jung, H

1992-05-01

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H in the rat was studied following irradiation with 5 Gy of 14 MeV neutrons. Several parameters were sequentially measured over a time period of 4 weeks after irradiation: the tumour volume was assessed by in situ caliper measurements; the numerical density of tumour cells was obtained by morphometry; the clonogenic fraction of tumour cells was derived from in vitro colony assay; and the numerical ratio of host to tumour cells was determined by flow cytometry. From these primary parameters the number of clonogenic tumour cells, non-clonogenic tumour cells, and nucleated host cells per tumour, as well as their variation with time, were derived. The results were compared with two sets of data obtained previously for the same tumour exposed to 15 Gy of 200 kVp X-rays. Survival of tumour cells was reduced to 5.5 +/- 0.5% by 5 Gy neutrons and to 4.5 +/- 0.5% by 15 Gy X-rays, i.e. an RBE of close to 3. There was a lag period before the onset of repopulation (4.9 +/- 0.4 days and 4.9 +/- 0.5 days, respectively), followed by a high initial rate of repopulation corresponding to a doubling time of 2.0 +/- 0.2 days for neutrons and 2.1 +/- 0.2 days for X-rays. The rate of depopulation was significantly different for the two treatment modalities; the halving time for the number of non-clonogenic tumour cells was 11 +/- 4 days for neutrons and 2.8 +/- 0.5 days for X-rays.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated level of human RPA interacting protein α (hRIPα) in cervical tumor cells is involved in cell proliferation through regulating RPA transport.

PubMed

Namkoong, Sim; Lee, Eun-Ju; Jang, Ik-Soon; Park, Junsoo

2012-10-19

Replication protein A (RPA) is a eukaryotic single-stranded DNA binding protein that is essential for DNA replication, repair, and recombination, and human RPA interacting protein α (hRIPα) is the nuclear transporter of RPA. Here, we report the regulatory role of hRIPα protein in cell proliferation. Western blot analysis revealed that the level of hRIPα was frequently elevated in cervical tumors tissues and hRIPα knockdown by siRNA inhibited cellular proliferation through deregulation of the cell cycle. In addition, overexpression of hRIPα resulted in increased clonogenicity. These results indicate that hRIPα is involved in cell proliferation through regulation of RPA transport. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Somatic embryogenesis from leaf explants of Australian fan flower, Scaevola aemula R. Br.

PubMed

Wang, Y-H; Bhalla, P L

2004-01-01

Somatic embryogenesis from leaf explants of Scaevola aemula R. Br. was achieved. Somatic embryos were induced from explants cultured on MS medium supplemented with 0.2 mg/ 2,4-dichlorophenoxyacetic acid and 0.2-0.5 mg/l 6-benzylaminopurine (BAP). Various developmental stages of somatic embryos were found on this medium-from globular embryos to germinated embryos. The transfer of globular embryos to MS medium containing 0.5 mg/l BAP resulted in a high frequency of shoot regeneration. Leaf explants cultured on MS medium containing different combinations of BAP and alpha-naphthaleneacetic acid formed adventitious shoots and roots. Histological examination confirmed the process of somatic embryogenesis. Induction of somatic embryogenesis in Scaevola provides a system for studying embryogenesis in Australian native plants and will facilitate the improvement of these plants using genetic transformation techniques.

Self-reported quality of life and health among Björk-Shiley convexo-concave prosthetic heart valve patients.

PubMed

Signorello, L B; Kennedy, J A; Richmond, R A; Sieu, K L; Blot, W J; Harrison, D C

2001-03-01

The risk of fracture of Björk-Shiley convexo-concave (BSCC) prosthetic heart valves has resulted in consideration of prophylactic explantation and replacement for patients with high-risk valves. Little information exists on perceived quality of life, health status, and serious morbidity among BSCC patients, including those who have undergone explantation. Self-administered questionnaires were completed by a cohort of 585 BSCC patients who participated in an X-ray imaging study to detect precursors to valve fracture up to seven years (average 3.9 years) previously. Responses from 31 explant patients were contrasted with those from 554 BSCC patients in whom explant surgery was not attempted. Perceived quality of life and health status and risk of hospitalization after participating in the imaging study varied considerably among patients, but on average tended not to differ significantly between those with and without explants. A slightly greater proportion of explantees tended to report both improved health status and high rates of heart attack and pacemaker implantation. The health status of these patients was, in general, considerably worse than previously reported among valve implant patients. Over half the cohort were hospitalized during follow up, and half were unable to walk up more than one flight of stairs without shortness of breath. The less than optimal health status of most BSCC patients and relatively high rates of morbidity should be taken into account when considering potential explantation of the valves.

Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins

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

Lone , Abdul G.; Atci, Erhan; Renslow, Ryan S.

A partial-thickness epidermal explant model was colonized with GFP-expressing S. aureus and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. Oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using MSE mass spectrometry. We found that S. aureus biofilm grows predominantly in sebum-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2-3 fold) inmore » these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after four days of culture. The colonized explants released significantly (P< 0.01) more anti-oxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentration found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in media from infected explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.« less

Long-term experimental in situ farming of Crambe crambe (Demospongiae: Poecilosclerida).

PubMed

Padiglia, Andrea; Ledda, Fabio D; Padedda, Bachisio M; Pronzato, Roberto; Manconi, Renata

2018-01-01

The marine sponge Crambe crambe was chosen as an experimental model of sustainable shallow-water mariculture in the Sardinian Sea (Western Mediterranean) to provide biomass with high potential in applied research. Explants were cultured in four long-term experiments (19 and 31 months at ca. 2.5 m depth), to determine the suitability of new culture techniques by testing substrata and seeding time (season), and monitoring survival and growth. Explants were excised and grown in an experimental plant close to the wild donor sponge population. Percentage growth rate (GR%) was measured in terms of surface cover area, and explant survival was monitored in situ by means of a digital photo camera. Explant survival was high throughout the trial, ranging from 78.57% to 92.85% on travertine tiles and from 50% to 71.42% on oyster shells. A few instances of sponge regression were observed. Explant cover area correlated positively with season on two substrata, i.e., tiles and shells. The surface cover area and GR% of explants were measured in the starting phase and monitored up to the end of the trial. High GR% values were observed both on tiles (>21%) and on oyster shells (>15%). The data on the behaviour and life-style of cultured fragments, together with an increase >2,400% in cover area, demonstrate that in situ aquaculture is a viable and sustainable method for the shallow-water biomass supply of Crambe crambe .

Long-term experimental in situ farming of Crambe crambe (Demospongiae: Poecilosclerida)

PubMed Central

Padiglia, Andrea; Ledda, Fabio D.; Padedda, Bachisio M.; Pronzato, Roberto

2018-01-01

Background The marine sponge Crambe crambe was chosen as an experimental model of sustainable shallow-water mariculture in the Sardinian Sea (Western Mediterranean) to provide biomass with high potential in applied research. Methods Explants were cultured in four long-term experiments (19 and 31 months at ca. 2.5 m depth), to determine the suitability of new culture techniques by testing substrata and seeding time (season), and monitoring survival and growth. Explants were excised and grown in an experimental plant close to the wild donor sponge population. Percentage growth rate (GR%) was measured in terms of surface cover area, and explant survival was monitored in situ by means of a digital photo camera. Results Explant survival was high throughout the trial, ranging from 78.57% to 92.85% on travertine tiles and from 50% to 71.42% on oyster shells. A few instances of sponge regression were observed. Explant cover area correlated positively with season on two substrata, i.e., tiles and shells. The surface cover area and GR% of explants were measured in the starting phase and monitored up to the end of the trial. High GR% values were observed both on tiles (>21%) and on oyster shells (>15%). Discussion The data on the behaviour and life-style of cultured fragments, together with an increase >2,400% in cover area, demonstrate that in situ aquaculture is a viable and sustainable method for the shallow-water biomass supply of Crambe crambe. PMID:29915695

High Concentration of Benzyladenine Solution Stimulates Anthers for Inducing Callus in Ricinus Communis L.

NASA Astrophysics Data System (ADS)

Liu, Ying; Yan, Shuying; Yang, Fuguang; Li, Dongliang; Tang, Jianian; Liu, Guoxuan; Lin, Shiwan; Niu, Sufang; Yang, Yali

2017-12-01

An high-frequency protocol for induction of callus from anther explants of Ricinus communis was described. When anther explants of R. communis was cultured directly onto medium containing 6-benzylaminopurine (BA) induced formation of only poor quality callus that had a low induction frequency of anther callus (10.67%). However, treating the anther explants with high concentrations (7.5-120 mg/L) of BA solution for short time periods (5-80 min) helped to improve the induction frequency and enhance the quality of the callus formation significantly. The best callus induction (41.25%) was observed when anther explants were treated with 15 mg/L BA solution for 10 min before being inoculated onto hormone-free Murashige and Skoog (MS) medium for 30 days. In order to further optimize the culture system, after treated with 15 mg/L BA for 10 min, anther explants were inoculated on the hormone-free MS medium contained concentrations of sodium nitroprusside (SNP). The results showed that SNP significantly promoted the response of callus induction, especially when 8 mg/L SNP was applied, the the highest percentage of callus induction (60.37%) were gained.

A microenvironment-mediated c-Myc/miR-548m/HDAC6 amplification loop in non-Hodgkin B cell lymphomas

PubMed Central

Lwin, Tint; Zhao, Xiaohong; Cheng, Fengdong; Zhang, Xinwei; Huang, Andy; Shah, Bijal; Zhang, Yizhuo; Moscinski, Lynn C.; Choi, Yong Sung; Kozikowski, Alan P.; Bradner, James E.; Dalton, William S.; Sotomayor, Eduardo; Tao, Jianguo

2013-01-01

A dynamic interaction occurs between the lymphoma cell and its microenvironment, with each profoundly influencing the behavior of the other. Here, using a clonogenic coculture growth system and a xenograft mouse model, we demonstrated that adhesion of mantle cell lymphoma (MCL) and other non-Hodgkin lymphoma cells to lymphoma stromal cells confers drug resistance, clonogenicity, and induction of histone deacetylase 6 (HDAC6). Furthermore, stroma triggered a c-Myc/miR-548m feed-forward loop, linking sustained c-Myc activation, miR-548m downregulation, and subsequent HDAC6 upregulation and stroma-mediated cell survival and lymphoma progression in lymphoma cell lines, primary MCL and other B cell lymphoma cell lines. Treatment with an HDAC6-selective inhibitor alone or in synergy with a c-Myc inhibitor enhanced cell death, abolished cell adhesion–mediated drug resistance, and suppressed clonogenicity and lymphoma growth ex vivo and in vivo. Together, these data suggest that the lymphoma-stroma interaction in the lymphoma microenvironment directly impacts the biology of lymphoma through genetic and epigenetic regulation, with HDAC6 and c-Myc as potential therapeutic targets. PMID:24216476

The microRNA-218~Survivin axis regulates migration, invasion, and lymph node metastasis in cervical cancer

PubMed Central

Kogo, Ryunosuke; How, Christine; Chaudary, Naz; Bruce, Jeff; Shi, Wei; Hill, Richard P.; Zahedi, Payam; Yip, Kenneth W.; Liu, Fei-Fei

2015-01-01

Cervical cancer is the third most common cancer in women worldwide. In the present study, global microRNA profiling for 79 cervical cancer patient samples led to the identification of miR-218 down-regulation in cervical cancer tissues compared to normal cervical tissues. Lower miR-218 expression was associated significantly with worse overall survival (OS), disease-free survival (DFS), and pelvic/aortic lymph node recurrence. In vitro, miR-218 over-expression decreased clonogenicity, migration, and invasion. Survivin (BIRC5) was subsequently identified as an important cervical cancer target of miR-218 using in silico prediction, mRNA profiling, and quantitative real-time PCR (qRT-PCR). Concordant with miR-218 over-expression, survivin knockdown by siRNA decreased clonogenicity, migration, and invasion. YM155, a small molecule survivin inhibitor, significantly suppressed tumor growth and lymph node metastasis in vivo. Our findings demonstrate that the miR-218~survivin axis inhibits cervical cancer progression by regulating clonogenicity, migration, and invasion, and suggest that the inhibition of survivin could be a potential therapeutic strategy to improve outcome in this disease. PMID:25473903

Matriptase Induction of Metalloproteinase‐Dependent Aggrecanolysis In Vitro and In Vivo: Promotion of Osteoarthritic Cartilage Damage by Multiple Mechanisms

PubMed Central

Wilkinson, David J.; Habgood, Angela; Lamb, Heather K.; Thompson, Paul; Hawkins, Alastair R.; Désilets, Antoine; Leduc, Richard; Steinmetzer, Torsten; Hammami, Maya; Lee, Melody S.; Craik, Charles S.; Watson, Sharon; Lin, Hua; Milner, Jennifer M.

2017-01-01

Objective To assess the ability of matriptase, a type II transmembrane serine proteinase, to promote aggrecan loss from the cartilage of patients with osteoarthritis (OA) and to determine whether its inhibition can prevent aggrecan loss and cartilage damage in experimental OA. Methods Aggrecan release from human OA cartilage explants and human stem cell–derived cartilage discs was evaluated, and cartilage‐conditioned media were used for Western blotting. Gene expression was analyzed by real‐time polymerase chain reaction. Murine OA was induced by surgical destabilization of the medial meniscus, and matriptase inhibitors were administered via osmotic minipump or intraarticular injection. Cartilage damage was scored histologically and aggrecan cleavage was visualized immunohistochemically using specific neoepitope antibodies. Results The addition of soluble recombinant matriptase promoted a time‐dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease‐activated receptor 2 antagonism. Although engineered human (normal) cartilage discs failed to release aggrecan following matriptase addition, both matrix metalloproteinase– and aggrecanase‐mediated cleavages of aggrecan were detected in human OA cartilage. Additionally, while matriptase did not directly degrade aggrecan, it promoted the accumulation of low‐density lipoprotein receptor–related protein 1 (LRP‐1) in conditioned media of the OA cartilage explants. Matriptase inhibition via neutralizing antibody or small molecule inhibitor significantly reduced cartilage damage scores in murine OA, which was associated with reduced generation of metalloproteinase‐mediated aggrecan cleavage. Conclusion Matriptase potently induces the release of metalloproteinase‐generated aggrecan fragments as well as soluble LRP‐1 from OA cartilage. Therapeutic targeting of matriptase proteolytic activity reduces metalloproteinase activity, further suggesting that this serine proteinase may have potential as a disease‐modifying therapy in OA. PMID:28464560

Solar-simulating irradiation of the skin of human subjects in vivo produces Langerhans cell responses distinct from irradiation ex vivo and in vitro.

PubMed

Laihia, J K; Jansen, C T

2000-08-01

It has been postulated that Langerhans cells (LC) provide tolerogenic signals in the local impairment of cutaneous immune functions and antigen-specific tolerance induced by UV radiation. Studies in vitro and ex vivo have indicated that UV radiation may down-regulate the expression of costimulatory molecules on LC, leading to reduced antigen-presenting function. In contrast, we recently observed an up-regulatory stage in the number of human epidermal LC with induced expression of B7 costimulatory molecules 12-24 h after solar-simulating UV radiation (SSR) in vivo. To examine the apparent discrepancy between the observed human LC responses in vitro, ex vivo and in vivo, we compared the three protocols in a parallel fashion. The intact skin as well as skin explants and epidermal cell suspensions from the same individuals were irradiated with a single erythematogenic dose of SSR. The expression of cell surface markers in the epidermal cells was analysed with flow cytometry 24 h later. The number of CD1a+/HLA-DR+ LC increased post-SSR in vivo by a factor of 2.8+/-0.4, whereas in irradiated skin explants ex vivo or in cell suspensions in vitro, reduced numbers were seen. HLA-DR expression intensities were found to have increased on DR+ and CD1a+/DR+ cells in vivo. Similarly, SSR induced B7-2 (CD86) expression in CD1a+ cells significantly in vivo (P=0.031) but reduced the expression ex vivo or in vitro. We conclude that the early up-regulatory stage of human LC number and membrane markers, recorded at 24 h after a single exposure to SSR, is exclusively an in vivo phenomenon.

Genetic and Dietary Regulation of Glyburide Efflux by the Human Placental Breast Cancer Resistance Protein Transporter

PubMed Central

Bircsak, Kristin M.; Gupta, Vivek; Yuen, Poi Yu Sofia; Gorczyca, Ludwik; Weinberger, Barry I.; Vetrano, Anna M.

2016-01-01

Glyburide is frequently used to treat gestational diabetes owing to its low fetal accumulation resulting from placental efflux by the breast cancer resistance protein (BCRP)/ABCG2 transporter. Here we sought to determine how exposure to the dietary phytoestrogen genistein and expression of a loss-of-function polymorphism in the ABCG2 gene (C421A) impacted the transport of glyburide by BCRP using stably transfected human embryonic kidney 293 (HEK) cells, human placental choriocarcinoma BeWo cells, and human placental explants. Genistein competitively inhibited the BCRP-mediated transport of 3H-glyburide in both wild-type (WT) and C421A-BCRP HEK-expressing cells, with greater accumulation of 3H-glyburide in cells expressing the C421A variant. In BeWo cells, exposure to genistein for 60 minutes increased the accumulation of 3H-glyburide 30%–70% at concentrations relevant to dietary exposure (IC50 ∼180 nM). Continuous exposure of BeWo cells to genistein for 48 hours reduced the expression of BCRP mRNA and protein by up to 40%, which impaired BCRP transport activity. Pharmacologic antagonism of the estrogen receptor attenuated the genistein-mediated downregulation of BCRP expression, suggesting that phytoestrogens may reduce BCRP levels through this hormone receptor pathway in BeWo cells. Interestingly, genistein treatment for 48 hours did not alter BCRP protein expression in explants dissected from healthy term placentas. These data suggest that whereas genistein can act as a competitive inhibitor of BCRP-mediated transport, its ability to downregulate placental BCRP expression may only occur in choriocarcinoma cells. Overall, this research provides important mechanistic data regarding how the environment (dietary genistein) and a frequent genetic variant (ABCG2, C421A) may alter the maternal-fetal disposition of glyburide. PMID:26850786

A regulator of ubiquitin-proteasome activity, 2-hexyldecanol, suppresses melanin synthesis and the appearance of facial hyperpigmented spots.

PubMed

Hakozaki, T; Laughlin, T; Zhao, S; Wang, J; Deng, D; Jewell-Motz, E; Elstun, L

2013-07-01

2-Hexyldecanol has long been used in skin-care products, but has not previously been reported as an active ingredient for skin benefits. To evaluate 2-hexyldecanol in in vitro and ex vivo systems and, if found to be active, progress it to topical clinical testing to determine effects on pigmentation in skin. 2-Hexyldecanol was tested in melanocyte cell culture systems (B16 mouse melanoma cells and normal human melanocytes) for its effect on proteolytic activity and melanin production, in the absence and presence of the proteasome-specific inhibitor, MG132. It was further tested in a human skin explant model for its effect on melanin production. Lastly, topically applied 2-hexyldecanol was evaluated for its effect on the appearance of facial pigmentation in an 8-week, randomized, double-blind, vehicle-controlled, split-face incomplete block design study in Chinese women. In submerged cell culture, 2-hexyldecanol upregulated proteolytic activity and decreased melanin synthesis. These effects were antagonized by the proteasome-specific inhibitor MG132. MG132, tested in the absence of 2-hexyldecanol, increased melanin production. In a human skin explant model, topical 2-hexyldecanol suppressed the production of melanin vs. a vehicle control. In a human clinical study in Chinese women (n = 110 observations per test material), a 2-hexyldecanol-containing formulation significantly reduced the appearance of facial hyperpigmented spots vs. its control. These data indicate that regulation of proteasome activity is a viable target for control of melanin production, that 2-hexyldecanol upregulates proteasomal activity in melanocytes, and that topical 2-hexyldecanol reduces the appearance of hyperpigmentation. © 2013 The Authors BJD © 2013 British Association of Dermatologists.

Responsiveness of mouse calvaria to parathyroid hormone after explant cryopreservation: 45Ca release in vitro

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

Wezeman, F.H.; Dungan, D.D.

1986-08-01

Newborn mouse calvaria prelabeled with /sup 45/Ca and cryopreserved at -196 degrees C in serum-free medium containing dimethylsulfoxide were compared to unpreserved explants for response to parathyroid hormone during subsequent culture. After short-term cryopreservation followed by rapid thawing, the viable explants continued to release /sup 45/Ca to the culture medium but additions of parathyroid hormone to the medium did not cause increased bone resorption. The data suggest that cryopreservation and thawing impairs mechanisms responsible for parathyroid hormone action on bone cells.

Severe pigment dispersion after iris-claw phakic intraocular lens implantation.

PubMed

Galvis, Virgilio; Carreño, Néstor I; Tello, Alejandro; Laiton, Andrea N

2017-12-01

A 23-year-old female patient presented 3 months after the implantation of an Artisan® phakic intraocular lens with a severe depigmentation of the iris and peripheral anterior synechiae. Explantation of the intraocular lens and goniosynechialysis were performed. Eleven months after the explantation appearance of the iris significantly improved. There was no loss of lines of corrected distance visual acuity. Severe pigment dispersion after the implantation of an Artisan® phakic intraocular lens may happen and may require explantation of the lens. Iris depigmentation may improve with time.

Insights on the antitumor effects of kahweol on human breast cancer: Decreased survival and increased production of reactive oxygen species and cytotoxicity

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

Cárdenas, Casimiro; IBIMA; Research Support Central Services

Highlights: • Kahweol inhibits growth and attachment-independent proliferation of tumor cells. • Kahweol induces apoptosis in MDA-MB231 human breast cancer cells. • Kahweol-induced apoptosis involves caspase activation and cytochrome c release. • Kahweol does not protect against hydrogen peroxide cytotoxicity. • Kahweol increases hydrogen peroxide production by human breast cancer cells. - Abstract: The present study aims to identify the modulatory effects of kahweol, an antioxidant diterpene present in coffee beans, on a panel of human tumor cell lines. Kahweol inhibits tumor cell proliferation and clonogenicity and induces apoptosis in several kinds of human tumor cells. In the estrogen receptor-negativemore » MDA-MB231 human breast cancer, the mentioned effects are accompanied by caspases 3/7 and 9 activation and cytochrome c release. On the other hand, kahweol increases the production of reactive oxygen species and their cytotoxicity in human breast cancer cells but not in normal cells. Taken together, our data suggest that kahweol is an antitumor compound with inhibitory effects on tumor cell growth and survival, especially against MDA-MB231 breast cancer cells.« less

Plant regeneration via direct somatic embryogenesis from leaf explants of Tolumnia Louise Elmore 'Elsa'.

PubMed

Shen, Hui-Ju; Chen, Jen-Tsung; Chung, Hsiao-Hang; Chang, Wei-Chin

2018-01-22

Tolumnia genus (equitant Oncidium) is a group of small orchids with vivid flower color. Thousands of hybrids have been registered on Royal Horticulture Society and showed great potential for ornamental plant market. The aim of this study is to establish an efficient method for in vitro propagation. Leaf explants taken from in vitro-grown plants were used to induce direct somatic embryogenesis on a modified 1/2 MS medium supplemented with five kinds of cytokinins, 2iP, BA, kinetin, TDZ and zeatin at 0.3, 1 and 3 mg l -1 in darkness. TDZ at 3 mg l -1 gave the highest percentage of explants with somatic globular embryos after 90 days of culture. It was found that 2,4-D and light regime highly retarded direct somatic embryogenesis and showed 95-100% of explant browning. Histological observations revealed that the leaf cells divided into meristematic cells firstly, followed by somatic proembryos, and then somatic globular embryos. Eventually, somatic embryos developed a bipolar structure with the shoot apical meristem and the root meristem. Scanning electron microscopy observations showed that the direct somatic embryogenesis from leaf explants was asynchronously. The somatic embryos were found on the leaf tip, the adaxial surface and also the mesophyll through a cleft, and it reflected the heterogeneity of the explant. The 90-day-old globular embryos were detached from the parent explants and transferred onto a hormone-free 1/2 MS medium in light condition for about 1 month to obtain 1-cm-height plantlets. After another 3 months for growth, the plantlets were potted with Sphagnum moss and were acclimatized in a shaded greenhouse. After 1 month of culture, the survival rate was 100%. In this report, a protocol for efficient regenerating a Tolumnia orchid, Louise Elmore 'Elsa', was established via direct somatic embryogenesis and might reveal an alternative approach for mass propagation of Tolumnia genus in orchid industry.

Presence of intrahepatic (total and ccc) HBV DNA is not predictive of HBV recurrence after liver transplantation.

PubMed

Hussain, Munira; Soldevila-Pico, Consuelo; Emre, Sukru; Luketic, Velimir; Lok, Anna S F

2007-08-01

Previous studies reported that hepatitis B virus (HBV) deoxyribonucleic acid (DNA) can be detected in livers of patients who received transplants for hepatitis B despite the absence of serological markers of HBV recurrence. Quantification of HBV DNA was not performed and presence of covalently closed circular (ccc) DNA was not analyzed in most studies. We aimed to quantify total and ccc HBV DNA in explant liver and post-orthotopic liver transplantation (OLT) biopsies and to correlate the values with HBV recurrence post-OLT. Frozen liver tissue from 34 patients (9 with explant liver only, 9 with explant liver and post-OLT liver biopsies, and 16 with post-OLT biopsies only) in the National Institutes of Health HBV-OLT study was examined using real-time polymerase chain reaction (PCR). Among the 18 patients with explant liver, 7 were hepatitis B e antigen (HBeAg)-positive, 8 had detectable serum HBV DNA, and 10 received antiviral therapy prior to OLT. Total and ccc HBV DNA was detected in explant livers of 17 and 16 patients, respectively. Of the 10 patients who received antiviral therapy pre-OLT, serum HBV DNA was undetectable in 8 at transplantation but 7 had detectable total and ccc HBV DNA in their explant liver. Of the 25 patients with post-OLT biopsies, total HBV DNA was detected in 83% and ccc DNA in 17% of 47 biopsies, although only 2 patients had HBV recurrence. In conclusion, total and ccc HBV DNA could be detected in explant livers of most patients despite antiviral therapy pre-OLT. Total but not ccc HBV DNA could be detected in post-OLT liver biopsies of most patients despite undetectable serum HBV DNA and hepatitis B surface antigen (HBsAg). Our findings suggest that occult HBV reinfection occurs in most HBV patients after OLT and continued administration of appropriate prophylactic therapy is important in preventing overt HBV recurrence. Copyright (c) 2007 AASLD.

[Influence of genotype, explant type and component of culture medium on in vitro callus induction and shoot organogenesis of tomato (Solanum lycopersicum L.)].

PubMed

Khaliluev, M R; Bogoutdinova, L R; Baranova, G B; Baranova, E N; Kharchenko, P N; Dolgov, S V

2014-01-01

The influence of explant type as well as of the type of growth regulators and concentration on callus induction processes and somatic organogenesis of shoots was studied in vitro on four tomato genotypes of Russian breeding. Cytological study of callus tissue was conducted. It was established that tomato varieties possess a substantially greater ability to indirect shoot organogenesis compared with the F1 hybrid. The highest frequency of somatic organogenesis of shoots, as well as their number per explant, was observed for most of the genotypes studied during the cultivation of cotyledons on Murashige-Skoog culture medium containing 2 mg/l of zeatin in combination with 0.1 mg/l of 3-indoleacetic acid. An effective protocol of indirect somatic organogenesis of shoots from different explants of tomato varieties with a frequency of more than 80% was developed.

Differentiation of presumptive primordial germ cell (pPGC)-like cells in explants into PGCs in experimental tadpoles

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

Ikenishi, K.; Okuda, T.; Nakazato, S.

1984-05-01

A single blastomere containing the ''germ plasm'' of 32-cell stage Xenopus embryos was cultured with (/sup 3/H)thymidine until the control embryos developed to the neurula stage. The explants, showing a spherical mass in which the nuclei of all cells were labeled, were implanted into the prospective place of presumptive primordial germ cells (pPGCs) in the endodermal cell mass of unlabeled host embryos of the neurula stage. Labeled PGCs as well as unlabeled, host PGCs were found in the genital ridges of experimental tadpoles. This indicates that the precursor of germ cells, corresponding to pPGCs in normal embryos of the neurulamore » stage, in the explants migrated to genital ridges just at the right moment to become PGCs, and suggests that the developmental process progressed normally, even in the explants, as far as the differentiation of pPGCs is concerned.« less

Effect of a Herbal-Leucine mix on the IL-1β-induced cartilage degradation and inflammatory gene expression in human chondrocytes

PubMed Central

2011-01-01

Background Conventional treatments for the articular diseases are often effective for symptom relief, but can also cause significant side effects and do not slow the progression of the disease. Several natural substances have been shown to be effective at relieving the symptoms of osteoarthritis (OA), and preliminary evidence suggests that some of these compounds may exert a favorable influence on the course of the disease. The objective of this study was to investigate the anti-inflammatory/chondroprotective potential of a Herbal and amino acid mixture containing extract of the Uncaria tomentosa, Boswellia spp., Lepidium meyenii and L-Leucine on the IL-1β-induced production of nitric oxide (NO), glycosaminoglycan (GAG), matrix metalloproteinases (MMPs), aggrecan (ACAN) and type II collagen (COL2A1) in human OA chondrocytes and OA cartilage explants. Methods Primary OA chondrocytes or OA cartilage explants were pretreated with Herbal-Leucine mixture (HLM, 1-10 μg/ml) and then stimulated with IL-1β (5 ng/ml). Effect of HLM on IL-1β-induced gene expression of iNOS, MMP-9, MMP-13, ACAN and COL2A1 was verified by real time-PCR. Estimation of NO and GAG release in culture supernatant was done using commercially available kits. Results HLM tested in these in vitro studies was found to be an effective anti-inflammatory agent, as evidenced by strong inhibition of iNOS, MMP-9 and MMP-13 expression and NO production in IL-1β-stimulated OA chondrocytes (p < 0.05). Supporting these gene expression results, IL-1β-induced cartilage matrix breakdown, as evidenced by GAG release from cartilage explants, was also significantly blocked (p < 0.05). Moreover, in the presence of herbal-Leucine mixture (HLM) up-regulation of ACAN and COL2A1 expression in IL-1β-stimulated OA chondrocytes was also noted (p < 0.05). The inhibitory effects of HLM were mediated by inhibiting the activation of nuclear factor (NF)-kB in human OA chondrocytes in presence of IL-1β. Conclusion Our data suggests that HLM could be chondroprotective and anti-inflammatory agent in arthritis, switching chondrocyte gene expression from catabolic direction towards anabolic and regenerative, and consequently this approach may be potentially useful as a new adjunct therapeutic/preventive agent for OA or injury recovery. PMID:21854562

Intraocular Lens Fragmentation Using Femtosecond Laser: An In Vitro Study

PubMed Central

Bala, Chandra; Shi, Jeffrey; Meades, Kerrie

2015-01-01

Purpose: To transect intraocular lenses (IOLs) using a femtosecond laser in cadaveric human eyes. To determine the optimal in vitro settings, to detect and characterize gasses or particles generated during this process. Methods: A femtosecond laser was used to transect hydrophobic and hydrophilic acrylic lenses. The settings required to enable easy separation of the lens fragment were determined. The gasses and particles generated were analysed using gas chromatography mass spectrometer (GC-MS) and total organic carbon analyzer (TOC), respectively. Results: In vitro the IOL fragments easily separated at the lowest commercially available energy setting of 1 μJ, 8-μm spot, and 2-μm line separation. No particles were detected in the 0.5- to 900-μm range. No significant gasses or other organic breakdown by products were detected at this setting. At much higher energy levels 12 μJ (4 × 6 μm spot and line separation) significant pyrolytic products were detected, which could be harmful to the eye. In cadaveric explanted IOL capsule complex the laser pulses could be applied through the capsule to the IOL and successfully fragment the IOL. Conclusion: IOL transection is feasible with femtosecond lasers. Further in vivo animal studies are required to confirm safety. Translational Relevance: In clinical practice there are a number of large intraocular lenses that can be difficult to explant. This in-vitro study examines the possibility of transecting the lasers quickly using femtosecond lasers. If in-vivo studies are successful, then this innovation could help ophthalmic surgeons in IOL explantation. PMID:26101721

In vitro antiretroviral activity and in vivo toxicity of the potential topical microbicide copper phthalocyanine sulfate.

PubMed

Styczynski, Ashley R; Anwar, Khandaker N; Sultana, Habiba; Ghanem, Abdelhamid; Lurain, Nell; Chua, Aishi; Ghassemi, Mahmood; Novak, Richard M

2015-08-30

Copper has antimicrobial properties and has been studied for its activity against viruses, including HIV. Copper complexed within a phthalocyanine ring, forming copper (II) phthalocyanine sulfate (CuPcS), may have a role in microbicide development when used intravaginally. CuPcS toxicity was tested against cervical epithelial cells, TZM-BL cells, peripheral blood mononuclear cells (PBMC), and cervical explant tissues using cell viability assays. In vivo toxicity was assessed following intravaginal administration of CuPcS in female BALB/C mice and measured using a standardized histology grading system on reproductive tract tissues. Efficacy studies for preventing infection with HIV in the presence of various non-toxic concentrations of CuPcS were carried out in TZM-BL, PBMC, and cervical explant cultures using HIV-1BAL and various pseudovirus subtypes. Non-linear regression was applied to the data to determine the EC50/90 and CC50/90. CuPcS demonstrated inhibition of HIV infection in PBMCs at concentrations that were non-toxic in cervical epithelial cells and PBMCs with EC50 values of approximately 50 μg/mL. Reproductive tract tissue analysis revealed no toxicity at 100 mg/mL. Human cervical explant tissues challenged with HIV in the presence of CuPcS also revealed a dose-response effect at preventing HIV infection at non-toxic concentrations with an EC50 value of 65 μg/mL. These results suggest that CuPcS may be useful as a topical microbicide in concentrations that can be achieved in the female genital tract.

The effect of short G-CSF administration on the numbers and clonogenic efficiency of hematopoietic progenitor cells in bone marrow and peripheral blood of normal donors.

PubMed

Zaucha, J M; Knopińska-Posłuszny, W; Bieniaszewska, M; Myśliwski, A; Hellmann, A

2000-01-01

We have analysed the cellularity, the number of clonogenic cells and their clonogenic efficiency (the number of clonogenic cells/2 x 10(5) MNC) in peripheral blood (PB) and bone marrow (BM) during and after filgrastim (rhG-CSF) mobilization of CD34+ cells in 12 healthy donors for allogeneic stem cell donation. G-CSF was administrated subcutaneously for 5 consecutive days at a dose of 10 micrograms/kg/day. WBC, MNC, CD34+ cell counts, CFU-GM and BFU-E assays in PB were performed at baseline and then daily 12 hours after each G-CSF dose. BM was assayed before start (day 1) and after the last dose (day 6) of G-CSF. Results are given as medians, with ranges in parentheses. In PB the total WBC and MNC increased 7.4-fold (6.0-12.3) and 3.3-fold (1.5-9.4), respectively, reaching a peak of 49.4 x 10(9)/l (32.5-66.6) on day 6 for WBC and 6.28 x 10(9)/l (4.7-13.3) for MNC on day 5. CD34+ cell number reached a peak value of 48.0 x 10(6)/l (45.6-285) on day 6 whereas CFU-GM and BFU-E reached their peaks on day 5, 0.95 x 10(4)/ml (0.05-6.08) and 1.04 x 10(4)/ml, respectively. CFU-MIX, not detectable at baseline, reached a peak of 0.95 x 10(4)/ml (0.006-0.51) on day 5 as well. This was accompanied by an increase in CFU-GM, BFU-E and CFU-MIX clonogenic efficiency: 23-fold (3-150), 9.75-fold (2.2-27.8) and 20-fold (2.5-210), respectively. In BM the total WBC number increased 2.5-fold (1.3-4.9) from the baseline value of 52.6 x 10(9)/l (7.9-137.0) whereas the MNC count increased 2.0-fold (0.81-3.7) from a baseline of 13.6 x 10(9)/l (3.5-54.8). This was, however, not significant. The number of CD34+ cells increased significantly 2.9-fold (0.8-8.3). In 8 donors CFU-MIX were detectable before but not after G-CSF treatment. A similar decrease in CFU-GM and BFU-E clonogenic efficiency occurred but was not significant. CFU-GM and BFU-E numbers did not change. We conclude that the total body numbers of lineage committed progenitors increased during G-CSF administration, which indicate their proliferation in addition to mobilization. The effect of G-CSF on the number of more primitive progenitors in BM is less clear and needs further investigation.

Magnetic resonance imaging screening results compared with explantation results in poly implant prothèse silicone breast implants, recalled from the European market in 2010.

PubMed

Maijers, Maria C; Niessen, Francisus B; Veldhuizen, Jacob F H; Ritt, Marco J P F; Manoliu, Radu A

2014-02-01

In a prospective cohort study, the authors followed 112 women whose Poly Implant Prothèse silicone breast implants were recalled. Magnetic resonance imaging results and clinical consequences were previously published. The authors compared magnetic resonance imaging screening with explantation results to study the diagnostic value of magnetic resonance imaging in this unique unselected and nonbiased group. women with 224 proven Poly Implant Prothèse implants after a mean implantation time of 10 years were enrolled in 2011. All women underwent magnetic resonance imaging screening and were offered explantation. The explantation details of 107 women could be compared with magnetic resonance imaging results. Of 107 women, 29 (27 percent) had at least one ruptured implant at explantation, and 44 of 214 explanted implants (21 percent) were ruptured. The magnetic resonance imaging results correctly diagnosed 154 intact and 35 ruptured implants. Sensitivity and specificity were 80 percent and 91 percent, respectively. The positive predictive value was 69 percent, and the negative predictive value was 95 percent. The accuracy of magnetic resonance imaging is comparable to previously published data from other manufacturers of modern silicone implants but lower than that of some recent validation studies in selected symptomatic women. The authors believe that this study is representative of common daily practice as they followed normal day-to-day magnetic resonance imaging protocol without using multiple independent readers. The authors hope that this study will contribute to the ongoing discussion to screen asymptomatic women with modern silicone breast implants. Diagnostic, II.

Failure analysis of explanted sternal wires.

PubMed

Shih, Chun-Ming; Su, Yea-Yang; Lin, Shing-Jong; Shih, Chun-Che

2005-05-01

To classify and understand the mechanisms of surface damages and fracture mechanisms of sternal wires, explanted stainless steel sternal wires were collected from patients with sternal dehiscence following open-heart surgery. Surface alterations and fractured ends of sternal wires were examined and analyzed. Eighty fractured wires extracted from 25 patients from January 1999 to December 2003, with mean implantation interval of 55+/-149 days (range 5-729 days) after cardiac surgery, were studied by various techniques. The extracted wires were cleaned and the fibrotic tissues were removed. Irregularities and fractured ends were assayed by a scanning electron microscopy. After stereomicroscopy and documentation, the explants were cleaned with 1% sodium hypochlorite to remove the blood and tissues and was followed by cleaned with deionized water and alcohol. The explants were examined by stereomicroscopy, and irregularities on surface and fracture surfaces of sternal wires were assayed by scanning electron microscopy, energy dispersive X-ray analysis (EDAX) and X-ray mapping. The explants with surrounding fibrotic tissue were stained and examined with stereomicroscopy and transmission electronic microscopy. Corrosion pits were found on the surface of explanted sternal wires. EDAX and X-ray mapping examinations revealed diminution of nickel concentration in the severely corroded pits on sternal wires. A feature of transgranular cracking was observed for stress corrosion cracking and striation character for typical corrosion fatigue was also identified. TEM examination of tissue showed the metallic particles in phagolysosomes of macrophages inside the surrounding sternal tissue. The synergic effect of hostile environment and the stress could be the precursors of failures for sternal wires.

Effects of dexamethasone and meloxicam on Borrelia burgdorferi-induced inflammation in glial and neuronal cells of the central nervous system.

PubMed

Ramesh, Geeta; Martinez, Alejandra N; Martin, Dale S; Philipp, Mario T

2017-02-02

Lyme neuroborreliosis (LNB), caused by the spirochete Borrelia burgdorferi (Bb), affects both the central and peripheral nervous systems. Previously, we reported that in a model of acute LNB in rhesus monkeys, treatment with the anti-inflammatory drug dexamethasone significantly reduced both pleocytosis and levels of cerebrospinal fluid (CSF) immune mediators that were induced by Bb. Dexamethasone also inhibited the formation of inflammatory, neurodegenerative, and demyelinating lesions in the brain and spinal cord of these animals. In contrast, these signs were evident in the infected animals that were left untreated or in those that were treated with meloxicam, a non-steroidal anti-inflammatory drug. To address the differential anti-inflammatory effects of dexamethasone and meloxicam in the central nervous system (CNS), we evaluated the potential of these drugs to alter the levels of Bb-induced inflammatory mediators in culture supernatants of rhesus frontal cortex (FC) explants, primary rhesus astrocytes and microglia, and human oligodendrocytes. We also ascertained the potential of dexamethasone to modulate Bb-induced apoptosis in rhesus FC explants. As meloxicam is a known COX-2 inhibitor, we evaluated whether meloxicam altered the levels of COX-2 as induced by live Bb in cell lysates of primary rhesus astrocytes and microglia. Dexamethasone but not meloxicam significantly reduced the levels of several Bb-induced immune mediators in culture supernatants of FC explants, astrocytes, microglia, and oligodendrocytes. Dexamethasone also had a protective effect on Bb-induced neuronal and oligodendrocyte apoptosis in rhesus FC explants. Further, meloxicam significantly reduced the levels of Bb-induced COX-2 in microglia, while both Bb and meloxicam were unable to alter the constitutive levels of COX-2 in astrocytes. These data indicate that dexamethasone and meloxicam have differential anti-inflammatory effects on Bb-induced inflammation in glial and neuronal cells of the CNS and help explain the in vivo findings of significantly reduced inflammatory mediators in the CSF and lack of inflammatory neurodegenerative lesions in the brain and spinal cord of Bb-infected animals that were treated with dexamethasone but not meloxicam. Signaling cascades altered by dexamethasone could serve as possible therapeutic targets for limiting CNS inflammation and tissue damage in LNB.

Plant cell transformation with Agrobacterium tumefaciens under simulated microgravity

NASA Astrophysics Data System (ADS)

Sarnatska, Veresa; Gladun, Hanna; Padalko, Svetlana

To investigate simulated microgravity (clinorotation) effect on plant cell transformation with Agrobacterium tumefaciens and crown gall formation, the culture of primary explants of potato and Jerusalem artichoke tubers was used. It is found that the efficiency of tumor formation and development in clinorotated explants are considerably reduced. When using the explants isolated from potato tubers clinorotated for 3, 5 and 19 days, drastic reduction of formation and development of crown gall tumors was observed. Conversely, the tumor number and their development increased when potato tubers were clinorotated for one day. As was estimated by us previously, cells of Jerusalem artichoke explants are the most sensitive to agrobacteria on 4-5 h of in vitro culturing and this time corresponds to the certain period of G1-stage of the cell cycle. We have also estimated that this period is characterized by the increase of binding of acridine orange by nuclear chromatin and increase in activity of RNA-polymerase I and II. Inoculation of explants with agrobacteria in this period was the most optimal for transformation and crown gall induction. We estimated that at four - hour clinorotation of explants the intensity of acridine orange binding to nuclei was considerably lower than on 4h in the control. At one-day clinorotation of potato tubers, a considerable increase in template accessibility of chromatin and in activity of RNA-polymerase I and II occurred. These results may serve as an evidence for the ability of plant dormant tissues to respond to microgravity. Another demonstration of dormant tissue response to changed gravity we obtained when investigating pathogenesis-related proteins (PR-proteins). PR-proteins were subjected to nondenaturing PAGE.and we have not found any effect of microgravity on PR-proteins of potato explants with normal or tumorous growth. We may suggest that such response derives from the common effects of two stress factors - wounding and changed gravity. Investigation of the effect of microgravity on PR-proteins of dormant potato tubers showed that an intensity of several electrophoretic fractions of these proteins with middle electrophoretic mobility increased and appeared two new minor fractions with high electrophoretic mobility under clinorotation of tubers. We discuss the possibility to use short term clinorotation of plant organs, from which the explants for the transformation with A. tumefaciens will be isolated, for an increase in the transformation efficiency of recalcitrant plants.

Scaleable manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component

PubMed Central

O'Keefe, Barry R.; Vojdani, Fakhrieh; Buffa, Viviana; Shattock, Robin J.; Montefiori, David C.; Bakke, James; Mirsalis, Jon; d'Andrea, Anna-Lisa; Hume, Steven D.; Bratcher, Barry; Saucedo, Carrie J.; McMahon, James B.; Pogue, Gregory P.; Palmer, Kenneth E.

2009-01-01

To prevent sexually transmitted HIV, the most desirable active ingredients of microbicides are antiretrovirals (ARVs) that directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals, which are costly to manufacture and deliver to resource-poor areas where effective microbicides are urgently needed. Here, we report a manufacturing breakthrough for griffithsin (GRFT), one of the most potent HIV entry inhibitors. This red algal protein was produced in multigram quantities after extraction from Nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing GRFT. Plant-produced GRFT (GRFT-P) was shown as active against HIV at picomolar concentrations, directly virucidal via binding to HIV envelope glycoproteins, and capable of blocking cell-to-cell HIV transmission. GRFT-P has broad-spectrum activity against HIV clades A, B, and C, with utility as a microbicide component for HIV prevention in established epidemics in sub-Saharan Africa, South Asia, China, and the industrialized West. Cognizant of the imperative that microbicides not induce epithelial damage or inflammatory responses, we also show that GRFT-P is nonirritating and noninflammatory in human cervical explants and in vivo in the rabbit vaginal irritation model. Moreover, GRFT-P is potently active in preventing infection of cervical explants by HIV-1 and has no mitogenic activity on cultured human lymphocytes. PMID:19332801

PPARγ controls pregnancy outcome through activation of EG-VEGF: new insights into the mechanism of placental development.

PubMed

Garnier, Vanessa; Traboulsi, Wael; Salomon, Aude; Brouillet, Sophie; Fournier, Thierry; Winkler, Carine; Desvergne, Beatrice; Hoffmann, Pascale; Zhou, Qun-Yong; Congiu, Cenzo; Onnis, Valentina; Benharouga, Mohamed; Feige, Jean-Jacques; Alfaidy, Nadia

2015-08-15

PPARγ-deficient mice die at E9.5 due to placental abnormalities. The mechanism by which this occurs is unknown. We demonstrated that the new endocrine factor EG-VEGF controls the same processes as those described for PPARγ, suggesting potential regulation of EG-VEGF by PPARγ. EG-VEGF exerts its functions via prokineticin receptor 1 (PROKR1) and 2 (PROKR2). This study sought to investigate whether EG-VEGF mediates part of PPARγ effects on placental development. Three approaches were used: 1) in vitro, using human primary isolated cytotrophoblasts and the extravillous trophoblast cell line (HTR-8/SVneo); 2) ex vivo, using human placental explants (n = 46 placentas); and 3) in vivo, using gravid wild-type PPARγ(+/-) and PPARγ(-/-) mice. Major processes of placental development that are known to be controlled by PPARγ, such as trophoblast proliferation, migration, and invasion, were assessed in the absence or presence of PROKR1 and PROKR2 antagonists. In both human trophoblast cell and placental explants, we demonstrated that rosiglitazone, a PPARγ agonist, 1) increased EG-VEGF secretion, 2) increased EG-VEGF and its receptors mRNA and protein expression, 3) increased placental vascularization via PROKR1 and PROKR2, and 4) inhibited trophoblast migration and invasion via PROKR2. In the PPARγ(-/-) mouse placentas, EG-VEGF levels were significantly decreased, supporting an in vivo control of EG-VEGF/PROKRs system during pregnancy. The present data reveal EG-VEGF as a new mediator of PPARγ effects during pregnancy and bring new insights into the fine mechanism of trophoblast invasion. Copyright © 2015 the American Physiological Society.

Feasibility of the runt cow for in vivo testing of a spinal interbody prosthesis with preliminary results.

PubMed

Buttermann, Glenn R; Mendenhall, H Vincent

2012-04-01

The optimal lumbar spinal disc prosthesis has yet to be developed. Failures of clinical device studies may be minimized by appropriate large animal preclinical studies. The lumbar spine of the mature "runt" cow, Corrientes breed, has been shown to have a number of desirable characteristics to the human. This study assessed the feasibility of the "runt" cow for in vivo testing of human-sized lumbar interbody implants and the ability to perform common analyses of explants. Eight cows (four experimental and four controls) were compared. The experimental animals had transosseous implantation of the disc prosthesis at L4-L5, and their spines were harvested at four or six months. They were evaluated for the ease of surgical access and healing, motion segment mobility, ability to remove the implant nondestructively, and microradiography and histomorphology. All animals had successful implantation. All explantations were performed without alteration to the devices. All animals had surgical healing and intended device motion, and histology found device stability by demonstrating bone ingrowth into the device's porous plates. There was a significant 46% increase in the amount of trabecular bone adjacent to the implants. The mature runt cow allows for implantation of human-sized interbody and intrabody spinal prostheses. This animal model allowed for macro- and histological analysis of the implant and surrounding tissues. In vivo stability was demonstrated for the disc prosthesis while also allowing for evaluation of intended mobility. Additionally, this is the first study to suggest increased bone density supporting an interbody prosthesis.

A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

PubMed

Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

2015-08-01

At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

Targeting HSP70 and GRP78 in canine osteosarcoma cells in combination with doxorubicin chemotherapy.

PubMed

Asling, Jonathan; Morrison, Jodi; Mutsaers, Anthony J

2016-11-01

Heat shock proteins (HSPs) are molecular chaperones subdivided into several families based on their molecular weight. Due to their cytoprotective roles, these proteins may help protect cancer cells against chemotherapy-induced cell death. Investigation into the biologic activity of HSPs in a variety of cancers including primary bone tumors, such as osteosarcoma (OSA), is of great interest. Both human and canine OSA tumor samples have aberrant production of HSP70. This study assessed the response of canine OSA cells to inhibition of HSP70 and GRP78 by the ATP-mimetic VER-155008 and whether this treatment strategy could sensitize cells to doxorubicin chemotherapy. Single-agent VER-155008 treatment decreased cellular viability and clonogenic survival and increased apoptosis in canine OSA cell lines. However, combination schedules with doxorubicin after pretreatment with VER-155008 did not improve inhibition of cellular viability, apoptosis, or clonogenic survival. Treatment with VER-155008 prior to chemotherapy resulted in an upregulation of target proteins HSP70 and GRP78 in addition to the co-chaperone proteins Herp, C/EBP homologous transcription protein (CHOP), and BAG-1. The increased GRP78 was more cytoplasmic in location compared to untreated cells. Single-agent treatment also revealed a dose-dependent reduction in activated and total Akt. Based on these results, targeting GRP78 and HSP70 may have biologic activity in canine osteosarcoma. Further studies are required to determine if and how this strategy may impact the response of osteosarcoma cells to chemotherapy.

Saquinavir inhibits early events associated with establishment of HIV-1 infection: potential role for protease inhibitors in prevention.

PubMed

Stefanidou, Martha; Herrera, Carolina; Armanasco, Naomi; Shattock, Robin J

2012-08-01

The maturation of newly formed human immunodeficiency virus type 1 (HIV-1) virions is a critical step for the establishment of productive infection. We investigated the potential of saquinavir (SQV), a protease inhibitor (PI) used in highly active antiretroviral therapy (HAART), as a candidate microbicide. SQV inhibited replication of clade B and clade C isolates in a dose-dependent manner in all cellular models tested: PM-1 CD4 T cells, peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages (MDMs), and immature monocyte-derived dendritic cells (iMDDCs). SQV also inhibited production of infectious virus in cervical, penile, and colorectal explants cocultured with T cells. Moreover, SQV demonstrated inhibitory potency against trans infection of T cells by in vitro-derived dendritic cells and by primary dendritic cells that emigrate from penile and cervical tissue explants. No cellular or tissue toxicity was detected in the presence of SQV, suggesting that this drug could be considered for development as a component of an effective microbicide, capable of blocking viral maturation and transmission of HIV-1 at mucosal surfaces.

Deterministic Encapsulation of Human Cardiac Stem Cells in Variable Composition Nanoporous Gel Cocoons To Enhance Therapeutic Repair of Injured Myocardium.

PubMed

Kanda, Pushpinder; Alarcon, Emilio I; Yeuchyk, Tanya; Parent, Sandrine; de Kemp, Robert A; Variola, Fabio; Courtman, David; Stewart, Duncan J; Davis, Darryl R

2018-04-20

Although cocooning explant-derived cardiac stem cells (EDCs) in protective nanoporous gels (NPGs) prior to intramyocardial injection boosts long-term cell retention, the number of EDCs that finally engraft is trivial and unlikely to account for salutary effects on myocardial function and scar size. As such, we investigated the effect of varying the NPG content within capsules to alter the physical properties of cocoons without influencing cocoon dimensions. Increasing NPG concentration enhanced cell migration and viability while improving cell-mediated repair of injured myocardium. Given that the latter occurred with NPG content having no detectable effect on the long-term engraftment of transplanted cells, we found that changing the physical properties of cocoons prompted explant-derived cardiac stem cells to produce greater amounts of cytokines, nanovesicles, and microRNAs that boosted the generation of new blood vessels and new cardiomyocytes. Thus, by altering the physical properties of cocoons by varying NPG content, the paracrine signature of encapsulated cells can be enhanced to promote greater endogenous repair of injured myocardium.

Regeneration of Solanum nigrum by somatic embryogenesis, involving frog egg-like body, a novel structure.

PubMed

Xu, Kedong; Chang, Yunxia; Liu, Kun; Wang, Feige; Liu, Zhongyuan; Zhang, Ting; Li, Tong; Zhang, Yi; Zhang, Fuli; Zhang, Ju; Wang, Yan; Niu, Wei; Jia, Shuzhao; Xie, Hengchang; Tan, Guangxuan; Li, Chengwei

2014-01-01

A new protocol was established for the regeneration of Solanum nigrum by frog egg-like bodies (FELBs), which are novel somatic embryogenesis (SE) structures induced from the root, stem, and leaf explants. The root, stem, and leaf explants (93.33%, 85.10%, and 100.00%, respectively) were induced to form special embryonic calli on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid, under dark condition. Further, special embryonic calli from the root, stem, and leaf explants (86.97%, 83.30%, and 99.47%, respectively) were developed into FELBs. Plantlets of FELBs from the three explants were induced in vitro on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine and 0.1 mg/L gibberellic acid, and 100.00% plantlet induction rates were noted. However, plantlet induction in vivo on MS medium supplemented with 20 mg/L thidiazuron showed rates of 38.63%, 15.63%, and 61.30% for the root, stem, and leaf explants, respectively, which were lower than those of the in vitro culture. Morphological and histological analyses of FELBs at different development stages revealed that they are a novel type of SE structure that developed from the mesophyll (leaf) or cortex (stem and root) cells of S. nigrum.

In Vitro Plant Regeneration from Commercial Cultivars of Soybean

PubMed Central

Raza, Ghulam; Singh, Mohan B.

2017-01-01

Soybean, a major legume crop, is the source of vegetable oil and protein. There is a need for transgenic approaches to breeding superior soybean varieties to meet future climate challenges. Efficient plant regeneration is a prerequisite for successful application of genetic transformation technology. Soybean cultivars are classified into different maturity groups based on photoperiod requirements. In this study, nine soybean varieties belonging to different maturity group were regenerated successfully from three different explants: half split hypocotyl, complete hypocotyl, and cotyledonary node. All the genotypes and explant types responded by producing adventitious shoots. Shoot induction potential ranged within 60–87%, 50–100%, and 75–100%, and regeneration rate ranged within 4.2–10, 2.7–4.2, and 2.6–10.5 shoots per explant using half split hypocotyl, complete hypocotyl, and cotyledonary explants, respectively, among all the tested genotypes. Bunya variety showed the best regeneration response using half split and complete hypocotyl explants and the PNR791 with cotyledonary node. The regenerated shoots were successfully rooted and acclimatized to glasshouse conditions. This study shows that commercial varieties of soybean are amenable to shoot regeneration with high regeneration frequencies and could be exploited for genetic transformation. Further, our results show no correlation between shoots regeneration capacity with the maturity grouping of the soybean cultivars tested. PMID:28691031

In Vitro Plant Regeneration from Commercial Cultivars of Soybean.

PubMed

Raza, Ghulam; Singh, Mohan B; Bhalla, Prem L

2017-01-01

Soybean, a major legume crop, is the source of vegetable oil and protein. There is a need for transgenic approaches to breeding superior soybean varieties to meet future climate challenges. Efficient plant regeneration is a prerequisite for successful application of genetic transformation technology. Soybean cultivars are classified into different maturity groups based on photoperiod requirements. In this study, nine soybean varieties belonging to different maturity group were regenerated successfully from three different explants: half split hypocotyl, complete hypocotyl, and cotyledonary node. All the genotypes and explant types responded by producing adventitious shoots. Shoot induction potential ranged within 60-87%, 50-100%, and 75-100%, and regeneration rate ranged within 4.2-10, 2.7-4.2, and 2.6-10.5 shoots per explant using half split hypocotyl, complete hypocotyl, and cotyledonary explants, respectively, among all the tested genotypes. Bunya variety showed the best regeneration response using half split and complete hypocotyl explants and the PNR791 with cotyledonary node. The regenerated shoots were successfully rooted and acclimatized to glasshouse conditions. This study shows that commercial varieties of soybean are amenable to shoot regeneration with high regeneration frequencies and could be exploited for genetic transformation. Further, our results show no correlation between shoots regeneration capacity with the maturity grouping of the soybean cultivars tested.

Establishment of an efficient and rapid method of multiple shoot regeneration and a comparative phenolics profile in in vitro and greenhouse-grown plants of Psophocarpus tetragonolobus (L.) DC.

PubMed

Singh, Vinayak; Chauhan, Namita Singh; Singh, Mohit; Idris, Asif; Madanala, Raju; Pande, Veena; Mohanty, Chandra Sekhar

2014-01-01

An in vitro method of multiple shoot induction and plant regeneration in Psophocarpus tetragonolobus (L.) DC was developed. Cotyledons, hypocotyls, epicotyls, internodal and young seedling leaves were used as explants. MS media supplemented with various concentrations of either thidiazuron (TDZ) or N6-benzylaminopurine (BAP) along with NAA or IAA combinations were used to determine their influence on multiple shoot induction. MS media supplemented with TDZ induced direct shoot regeneration when epicotyls and internodal segments were used as explants. TDZ at 3 mg L(-1) induced highest rate (89.2 ± 3.28%) of regeneration with (13.4 ± 2.04) shoots per explant. MS media supplemented with BAP in combination with NAA or IAA induced callus mediated regeneration when cotyledons and hypocotyls were used as explants. BAP (2.5 mg L(-1)) and IAA (0.2 mg L(-1)) induced highest rate (100 ± 2.66%) of regeneration with (23.2 ± 2.66) shoots per explant. Mature plants produced from regenerated shoots were transferred successfully to the greenhouse. In a comparative study, the phenolics contents of various parts of greenhouse-grown plants with that of in vitro-raised plants showed significant variations.

Establishment of an efficient and rapid method of multiple shoot regeneration and a comparative phenolics profile in in vitro and greenhouse-grown plants of psophocarpus tetragonolobus (L.) DC

PubMed Central

Singh, Vinayak; Chauhan, Namita Singh; Singh, Mohit; Idris, Asif; Madanala, Raju; Pande, Veena; Mohanty, Chandra Sekhar

2014-01-01

An in vitro method of multiple shoot induction and plant regeneration in Psophocarpus tetragonolobus (L.) DC was developed. Cotyledons, hypocotyls, epicotyls, internodal and young seedling leaves were used as explants. MS media supplemented with various concentrations of either thidiazuron (TDZ) or N6-benzylaminopurine (BAP) along with NAA or IAA combinations were used to determine their influence on multiple shoot induction. MS media supplemented with TDZ induced direct shoot regeneration when epicotyls and internodal segments were used as explants. TDZ at 3 mg L−1 induced highest rate (89.2 ± 3.28%) of regeneration with (13.4 ± 2.04) shoots per explant. MS media supplemented with BAP in combination with NAA or IAA induced callus mediated regeneration when cotyledons and hypocotyls were used as explants. BAP (2.5 mg L−1) and IAA (0.2 mg L−1) induced highest rate (100 ± 2.66%) of regeneration with (23.2 ± 2.66) shoots per explant. Mature plants produced from regenerated shoots were transferred successfully to the greenhouse. In a comparative study, the phenolics contents of various parts of greenhouse-grown plants with that of in vitro-raised plants showed significant variations. PMID:25482808

Effects of La0.2Ce0.6Eu0.2F3 nanocrystals capped with polyethylene glycol on human pancreatic cancer cells in vitro

NASA Astrophysics Data System (ADS)

Withers, Nathan J.; Glazener, Natasha N.; Rivera, Antonio C.; Akins, Brian A.; Armijo, Leisha M.; Plumley, John B.; Cook, Nathaniel C.; Sugar, Jacqueline M.; Chan, Rana; Brandt, Yekaterina I.; Smolyakov, Gennady A.; Heintz, Philip H.; Osiński, Marek

2013-02-01

Lanthanide fluoride colloidal nanocrystals offer a way to improve the diagnosis and treatment of cancer through the enhanced absorption of ionizing radiation, in addition to providing visible luminescence. In order to explore this possibility, tests with a kilovoltage therapy unit manufactured by the Universal X-Ray Company were performed to estimate the energy sensitivity of this technique. La0.2Ce0.6Eu0.2F3 nanocrystals capped with polyethylene glycol of molecular weight 6000 were synthesized, suspended in deionized water, and made tolerant to biological ionic pressures by incubation with fetal bovine serum. These nanocrystals were characterized by dynamic light scattering, muffle furnace ashing, and photoluminescence spectroscopy. Clonogenic assays were performed on the cells to assay the cytotoxicity and radiotoxicity of the nanocrystals on the human pancreatic cancer cell line PANC-1, purchased from ATCC.

Synthesis, crystal structure and effect of indeno[1,2-b]indole derivatives on prostate cancer in vitro. Potential effect against MMP-9.

PubMed

Lobo, Gricela; Monasterios, Melina; Rodrigues, Juan; Gamboa, Neira; Capparelli, Mario V; Martínez-Cuevas, Javier; Lein, Michael; Jung, Klaus; Abramjuk, Claudia; Charris, Jaime

2015-01-01

A highly regiospecific synthesis of a series of indenoindoles is reported, together with X-ray studies and their activity against human prostate cancer cells PC-3 and LNCaP in vitro. The most effective compound 7,7-dimethyl-5-[(3,4-dichlorophenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9bhexahydro-indeno[1,2-b]indole-9,10-dione 7q reduced the viability in both cell lines in a time and dose-dependent manner. Inhibitory effects were also observed on the adhesion, migration, and invasion of the prostate cancer cells as well as on clonogenic possibly by inhibition of MMP-9 activity. Molecular docking of 7q and 6k into MMP-9 human active site was also performed to determine the probable binding mode. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Studies of rhodamine-123: effect on rat prostate cancer and human prostate cancer cells in vitro.

PubMed

Arcadi, J A; Narayan, K S; Techy, G; Ng, C P; Saroufeem, R M; Jones, L W

1995-06-01

The effect of the lipophilic, cationic dye, Rhodamine-123 (Rh-123), on prostate cancer in rats, and on three tumor cell lines in vitro is reported here. The general toxicity of Rh-123 in mice has been found to be minimal. Lobund-Wistar (L-W) rats with the autochthonous prostate cancer of Pollard were treated for six doses with Rh-123 at a dose of 15 mg/kg subcutaneously every other day. Microscopic examination of the tumors revealed cellular and acinar destruction. The effectiveness of Rh-123 as a cytotoxic agent was tested by clonogenic and viability assays in vitro with three human prostate cancer cell lines. Severe (60-95%) growth inhibition was observed following Rh-123 exposure for 2-5 days at doses as low as 1.6 micrograms/ml in all three prostate cancer cell lines.

Characterization of Epithelial Progenitors in Normal Human Palatine Tonsils and Their HPV16 E6/E7-Induced Perturbation

PubMed Central

Kang, Sung Yoon Catherine; Kannan, Nagarajan; Zhang, Lewei; Martinez, Victor; Rosin, Miriam P.; Eaves, Connie J.

2015-01-01

Summary Human palatine tonsils are oropharyngeal lymphoid tissues containing multiple invaginations (crypts) in which the continuity of the outer surface epithelium is disrupted and the isolated epithelial cells intermingle with other cell types. We now show that primitive epithelial cells detectable in vitro in 2D colony assays and in a 3D culture system are CD44+NGFR+ and present in both surface and crypt regions. Transcriptome analysis indicated a high similarity between CD44+NGFR+ cells in both regions, although those isolated from the crypt contained a higher proportion of the most primitive (holo)clonogenic cells. Lentiviral transduction of CD44+NGFR+ cells from both regions with human papillomavirus 16-encoded E6/E7 prolonged their growth in 2D cultures and caused aberrant differentiation in 3D cultures. Our findings therefore reveal a shared, site-independent, hierarchical organization, differentiation potential, and transcriptional profile of normal human tonsillar epithelial progenitor cells. They also introduce a new model for investigating the mechanisms of their transformation. PMID:26527383

COMPOSITION OF GLYCOPROTEINS SECRETED BY TRACHEAL EXPLANTS FROM VARIOUS ANIMAL SPECIES

EPA Science Inventory

The acidic and neutral glycoproteins secreted by cultured tracheal explants from pigs, sheep, rats, mice, monkeys, guinea pigs, dogs, and chickens were purified and fractionated by column chromatography on DEAE-cellulose and by electrophoresis on cellulose acetate. The ratios of ...

Milk Thistle Extract and Silymarin Inhibit Lipopolysaccharide Induced Lamellar Separation of Hoof Explants in Vitro

PubMed Central

Reisinger, Nicole; Schaumberger, Simone; Nagl, Veronika; Hessenberger, Sabine; Schatzmayr, Gerd

2014-01-01

The pathogenesis of laminitis is not completely identified and the role of endotoxins (lipopolysaccharides, LPS) in this process remains unclear. Phytogenic substances, like milk thistle (MT) and silymarin, are known for their anti-inflammatory and antioxidant properties and might therefore have the potential to counteract endotoxin induced effects on the hoof lamellar tissue. The aim of our study was to investigate the influence of endotoxins on lamellar tissue integrity and to test if MT and silymarin are capable of inhibiting LPS-induced effects in an in vitro/ex vivo model. In preliminary tests, LPS neutralization efficiency of these phytogenics was determined in an in vitro neutralization assay. Furthermore, tissue explants gained from hooves of slaughter horses were tested for lamellar separation after incubation with different concentrations of LPS. By combined incubation of explants with LPS and either Polymyxin B (PMB; positive control), MT or silymarin, the influence of these substances on LPS-induced effects was assessed. In the in vitro neutralization assay, MT and silymarin reduced LPS concentrations by 64% and 75%, respectively, in comparison PMB reduced 98% of the LPS concentration. In hoof explants, LPS led to a concentration dependent separation. Accordantly, separation force was significantly decreased by 10 µg/mL LPS. PMB, MT and silymarin could significantly improve tissue integrity of explants incubated with 10 µg/mL LPS. This study showed that LPS had a negative influence on the structure of hoof explants in vitro. MT and silymarin reduced endotoxin activity and inhibited LPS-induced effects on the lamellar tissue. Hence, MT and silymarin might be used to support the prevention of laminitis and should be further evaluated for this application. PMID:25290524

Long-Term Durability of Bioprosthetic Aortic Valves: Implications From 12,569 Implants

PubMed Central

Johnston, Douglas R.; Soltesz, Edward G.; Vakil, Nakul; Rajeswaran, Jeevanantham; Roselli, Eric E.; Sabik, Joseph F.; Smedira, Nicholas G.; Svensson, Lars G.; Lytle, Bruce W.; Blackstone, Eugene H.

2016-01-01

Background Increased life expectancy and younger patients’ desire to avoid lifelong anticoagulation requires a better understanding of bioprosthetic valve failure. This study evaluates risk factors associated with explantation for structural valve deterioration (SVD) in a long-term series of Carpentier-Edwards PERIMOUNT aortic valves (AV). Methods From June 1982 to January 2011, 12,569 patients underwent AV replacement with Edwards Lifesciences Carpentier-Edwards PERIMOUNT stented bovine pericardial prostheses, models 2700PM (n = 310) or 2700 (n = 12,259). Mean age was 71 ± 11 years (range, 18 to 98 years). 93% had native AV disease, 48% underwent concomitant coronary artery bypass grafting, and 26% had additional valve surgery. There were 81,706 patient-years of systematic follow-up data available for analysis. Demographics, intraoperative variables, and 27,386 echocardiographic records were used to identify risks for explant for SVD and assess longitudinal changes in transprosthesis gradients using time-varying covariable analyses. Results Three hundred fifty-four explants were performed, with 41% related to endocarditis and 44% to SVD. Actuarial estimates of explant for SVD at 10 and 20 years were 1.9% and 15% overall, respectively, and in patients younger than 60 years, 5.6% and 46%, respectively. Younger age (p < 0.0001), lipid-lowering drugs (p = 0.002), prosthesis–patient mismatch (p = 0.001), and higher postoperative peak and mean AV gradients were associated with explant for SVD (p < 0.0001). The effect of gradient on SVD was greatest in patients younger than 60 years. Conclusions Durability of the Carpentier-Edwards PERIMOUNT aortic valve is excellent even in younger patients. Explant for SVD is related to gradient at implantation, especially in younger patients. Strategies to reduce early postoperative AV gradients, such as root enlargement or more efficient prostheses, should be considered. PMID:25662439

Characterization of somatic embryogenesis initiated from the Arabidopsis shoot apex.

PubMed

Kadokura, Satoshi; Sugimoto, Kaoru; Tarr, Paul; Suzuki, Takamasa; Matsunaga, Sachihiro

2018-04-28

Somatic embryogenesis is one of the best examples of the remarkable developmental plasticity of plants, in which committed somatic cells can dedifferentiate and acquire the ability to form an embryo and regenerate an entire plant. In Arabidopsis thaliana, the shoot apices of young seedlings have been reported as an alternative tissue source for somatic embryos (SEs) besides the widely studied zygotic embryos taken from siliques. Although SE induction from shoots demonstrates the plasticity of plants more clearly than the embryo-to-embryo induction system, the underlying developmental and molecular mechanisms involved are unknown. Here we characterized SE formation from shoot apex explants by establishing a system for time-lapse observation of explants during SE induction. We also established a method to distinguish SE-forming and non-SE-forming explants prior to anatomical SE formation, enabling us to identify distinct transcriptome profiles of these two explants at SE initiation. We show that embryonic fate commitment takes place at day 3 of SE induction and the SE arises directly, not through callus formation, from the base of leaf primordia just beside the shoot apical meristem (SAM), where auxin accumulates and shoot-root polarity is formed. The expression domain of a couple of key developmental genes for the SAM transiently expands at this stage. Our data demonstrate that SE-forming and non-SE-forming explants share mostly the same transcripts except for a limited number of embryonic genes and root genes that might trigger the SE-initiation program. Thus, SE-forming explants possess a mixed identity (SAM, root and embryo) at the time of SE specification. Copyright © 2018. Published by Elsevier Inc.

Milk thistle extract and silymarin inhibit lipopolysaccharide induced lamellar separation of hoof explants in vitro.

PubMed

Reisinger, Nicole; Schaumberger, Simone; Nagl, Veronika; Hessenberger, Sabine; Schatzmayr, Gerd

2014-10-06

The pathogenesis of laminitis is not completely identified and the role of endotoxins (lipopolysaccharides, LPS) in this process remains unclear. Phytogenic substances, like milk thistle (MT) and silymarin, are known for their anti-inflammatory and antioxidant properties and might therefore have the potential to counteract endotoxin induced effects on the hoof lamellar tissue. The aim of our study was to investigate the influence of endotoxins on lamellar tissue integrity and to test if MT and silymarin are capable of inhibiting LPS-induced effects in an in vitro/ex vivo model. In preliminary tests, LPS neutralization efficiency of these phytogenics was determined in an in vitro neutralization assay. Furthermore, tissue explants gained from hooves of slaughter horses were tested for lamellar separation after incubation with different concentrations of LPS. By combined incubation of explants with LPS and either Polymyxin B (PMB; positive control), MT or silymarin, the influence of these substances on LPS-induced effects was assessed. In the in vitro neutralization assay, MT and silymarin reduced LPS concentrations by 64% and 75%, respectively, in comparison PMB reduced 98% of the LPS concentration. In hoof explants, LPS led to a concentration dependent separation. Accordantly, separation force was significantly decreased by 10 µg/mL LPS. PMB, MT and silymarin could significantly improve tissue integrity of explants incubated with 10 µg/mL LPS. This study showed that LPS had a negative influence on the structure of hoof explants in vitro. MT and silymarin reduced endotoxin activity and inhibited LPS-induced effects on the lamellar tissue. Hence, MT and silymarin might be used to support the prevention of laminitis and should be further evaluated for this application.

Lactobacillus amylovorus Inhibits the TLR4 Inflammatory Signaling Triggered by Enterotoxigenic Escherichia coli via Modulation of the Negative Regulators and Involvement of TLR2 in Intestinal Caco-2 Cells and Pig Explants

PubMed Central

Finamore, Alberto; Roselli, Marianna; Imbinto, Ambra; Seeboth, Julie; Oswald, Isabelle P.; Mengheri, Elena

2014-01-01

Inflammation derived from pathogen infection involves the activation of toll-like receptor (TLR) signaling. Despite the established immunomodulatory activities of probiotics, studies relating the ability of such bacteria to inhibit the TLR signaling pathways are limited or controversial. In a previous study we showed that Lactobacillus amylovorus DSM 16698T, a novel lactobacillus isolated from unweaned pigs, protects the intestinal cells from enterotoxigenic Escherichia coli (ETEC) K88 infection through cytokine regulation. In the present study we investigated whether the ability of L. amylovorus to counteract the inflammatory status triggered by ETEC in intestine is elicited through inhibition of the TLR4 signaling pathway. We used the human intestinal Caco-2/TC7 cells and intestinal explants isolated from 5 week-old crossbreed Pietrain/Duroc/Large-White piglets, treated with ETEC, L. amylovorus or L. amylovorus cell free supernatant, either alone or simultaneously with ETEC. Western blot analysis showed that L. amylovorus and its cell free supernatant suppress the activation of the different steps of TLR4 signaling in Caco-2/TC7 cells and pig explants, by inhibiting the ETEC induced increase in the level of TLR4 and MyD88, the phosphorylation of the IKKα, IKKβ, IκBα and NF-κB subunit p65, as well as the over-production of inflammatory cytokines IL-8 and IL-1β. The immunofluorescence analysis confirms the lack of phospho-p65 translocation into the nucleus. These anti-inflammatory effects are achieved through modulation of the negative regulators Tollip and IRAK-M. We also found that L. amylovorus blocks the up-regulation of the extracellular heat shock protein (Hsp)72 and Hsp90, that are critical for TLR4 function. By using anti-TLR2 antibody, we demonstrate that TLR2 is required for the suppression of TLR4 signaling activation. These results may contribute to develop therapeutic interventions using L. amylovorus in intestinal disorders of piglets and humans. PMID:24733511

[Effect of cryopreservation on umbilical blood cells and its mechanism].

PubMed

Li, Xin; Chen, Fangping; Jiang, Tiebin; Wang, Erhua; Liu, Jing

2013-07-01

To evaluate the effect of cryopreservation on clonogenic ability and apoptosis rate of mono-nuclear cells and CD34+ cells in umbilical blood (UB), and to choose the index to present the freezing injury and optimize the cryopreservation of UB. The mono-nuclear cells (MNC) and CD34+ cells were separated from UB and frozen.After 30 days, they were thawed in warm water. Clonogenic capacity and clonogenic recovery before and after the cryopreservation was compared. We also used Annexin V-FITC-PI to investigate the apoptosis rate of the cells before and after the cryopreservation of these 2 types of cells. The number of colony forming unit-granulocyte/monocyte (CFU-GMs) was not changed after freezing and thawing in both MNCs and CD34+ cells, while the number of colony forming unit-granulocyte, erythrocyte, monocyte and megakaryocyte (CFU-GEMM) was obviously reduced after freezing in CD34+ cells. The 2 types of cryopreserved cells had certain degree of apoptosis before the cryopreservation. MNC-type cryopreservation increased the cells apoptosis a little, while CD34+-type cryopreservation increased more. The cells have certain degree of apoptosis before the cryopreservation. The freezing and thawing procedure does affect the early stage progenitor cells-CFU-GEMM in the CD34+- type cryopreserved cells in UB. The damage may be induced by the cell apoptosis.

MicroRNA-451 regulates stemness of side population cells via PI3K/Akt/mTOR signaling pathway in multiple myeloma.

PubMed

Du, Juan; Liu, Shuyan; He, Jie; Liu, Xi; Qu, Ying; Yan, Wenqing; Fan, Jianling; Li, Rong; Xi, Hao; Fu, Weijun; Zhang, Chunyang; Yang, Jing; Hou, Jian

2015-06-20

Side population (SP) cells are an enriched source of cancer-initiating cells with stemness characteristics, generated by increased ABC transporter activity, which has served as a unique hallmark for multiple myeloma (MM) stem cell studies. Here we isolated and identified MM SP cells via Hoechst 33342 staining. Furthermore, we demonstrate that SP cells possess abnormal cell cycle, clonogenicity, and high drug efflux characteristics-all of which are features commonly seen in stem cells. Interestingly, we found that bortezomib, As2O3, and melphalan all affected apoptosis and clonogenicity in SP cells. We followed by characterizing the miRNA signature of MM SP cells and validated the specific miR-451 target tuberous sclerosis 1 (TSC1) gene to reveal that it activates the PI3K/Akt/mTOR signaling in MM SP cells. Inhibition of miR-451 enhanced anti-myeloma novel agents' effectiveness, through increasing cells apoptosis, decreasing clonogenicity, and reducing MDR1 mRNA expression. Moreover, the novel specific PI3K/Akt/mTOR signaling inhibitor S14161 displayed its prowess as a potential therapeutic agent by targeting MM SP cells. Our findings offer insights into the mechanisms regulating MM SP cells and provide a novel strategy to overcome resistance to existing therapies against myeloma.

Goat serum as an alternative to establish cell culture from Indian major carp, Cirrhinus mrigala.

PubMed

Nanda, P K; Swain, P; Nayak, S K; Dash, S; Routray, P; Swain, S K; Patra, B C

2009-01-01

Serum from goat, calf, and chicken sources were evaluated in terms of attachment, growth, and proliferation of explants of Indian major carp, Cirrhinus mrigala. The attachment of explants viz. heart, liver, and kidney was directly proportional to the concentration of the serum. Among these sera, the highest percentage of attachment, growth, and proliferation was recorded for 10% goat serum and 15% newborn calf serum without affecting their cell morphology. On contrary to these sera, chicken serum at 15% concentration was found to be mildly toxic for all the explants. The cell count was significantly high for the kidney, liver, and heart at 10% goat serum among all the tested sera as well as concentration. Similarly, the liver, heart, and kidney explants were found to survive up to the tenth, seventh, and ninth passage, respectively. Therefore, the goat serum at 10% concentration can be used as effectively as newborn calf serum for routine culture of fish cells.

Pericardium Plug in the Repair of the Corneoscleral Fistula After Ahmed Glaucoma Valve Explantation

PubMed Central

Yoo, Chungkwon; Kwon, Sung Wook

2008-01-01

We report four cases in which a pericardium (Tutoplast®) plug was used to repair a corneoscleral fistula after Ahmed Glaucoma Valve (AGV) explantation. In four cases in which the AGV tube had been exposed, AGV explantation was performed using a pericardium (Tutoplast®) plug to seal the defect previously occupied by the tube. After debridement of the fistula, a piece of processed pericardium (Tutoplast®), measured 1 mm in width, was plugged into the fistula and secured with two interrupted 10-0 nylon sutures. To control intraocular pressure, a new AGV was implanted elsewhere in case 1, phaco-trabeculectomy was performed concurrently in case 2, cyclophotocoagulation was performed postoperatively in case 3 and anti-glaucomatous medication was added in case 4. No complication related to the fistula developed at the latest follow-up (range: 12~26 months). The pericardium (Tutoplast®) plug seems to be an effective method in the repair of corneoscleral fistulas resulting from explantation of glaucoma drainage implants. PMID:19096247

Influence of nerve growth factor on developing dorso-medial and ventro-lateral neurons of chick and mouse trigeminal ganglia.

PubMed

Davies, A; Lumsden, A

1983-01-01

Trigeminal ganglia have been removed from five, six, seven and eight day chick embryos and explants of the dorso-medial (DM) and ventro-lateral (VL) parts of the maxillomandibular lobe were grown in tissue culture. Quantitative methods were used to assess the influence of nerve growth factor (NGF) on fiber outgrowth from these explants. At all ages outgrowth from DM explants was significantly greater than from VL explants, the difference being most pronounced between the extreme DM and VL poles of the maxillomandibular lobe. These observations are interpreted as indicating the existence of two distinct populations of neurons in terms of their response to NGF rather than the consequence of the asynchronous differentiation and maturation of the VL and DM neurons. A similar study of 10, 11 and 12 day embryonic mouse trigeminal ganglia revealed no significant difference in neurite outgrowth between DM and VL regions grown in the presence or absence of NGF. Copyright © 1983. Published by Elsevier Ltd.

Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

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

Shin, Youn Ho; Lee, Seo Jin; Jung, Junyang, E-mail: jjung@khu.ac.kr

Highlights: Black-Right-Pointing-Pointer ATP-treated sciatic explants shows the decreased expression of p75NGFR. Black-Right-Pointing-Pointer Extracellular ATP inhibits the expression of phospho-ERK1/2. Black-Right-Pointing-Pointer Lysosomal exocytosis is involved in Schwann cell dedifferentiation. Black-Right-Pointing-Pointer Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibitsmore » Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.« less

Morphogenetic effects of 2,4-dichlorophenoxyacetic acid on pinto bean (Phaseolus vulgaris L.) leaf explants in vitro.

PubMed

Saunders, J W; Hosfield, G L; Levi, A

1987-02-01

Roots, callus and/or globular structures were produced on primary leaf and distal cotyledon explants of pinto bean (Phaseolus vulgaris L. cv. UI 114) cultured on semisolid MS medium with a wide range of 2,4-D concentrations (0.01 to 80 mg/L) with either 0 or 1.0 mg/L kinetin. Explants rooted at lower 2,4-D concentrations than at those favoring globule formation on callus, although roots, callus and globules often developed from the same explant. Isolated opaque green globular structures developed when callus initiated on media with 3 or more mg/L 2,4-D was subcultured in liquid MS + 30 mg/L 2,4-D. These structures multiplied with a fresh weight doubling time of 8-9 days in MS + 30 mg/L 2,4-D. Although this multiplicative behavior and opaque color were reminiscent of embryoids reported for other species, no cotyledons or roots were seen.

A549 Cells: Lung Carcinoma Cell Line for Adenovirus | NCI Technology Transfer Center | TTC

Cancer.gov

Scientists at the National Cancer Institute have developed a cell line designated A549 that was derived from explanted cultures of human lung cancer tissue. The A549 cell line has been tested under the guidance of the United States Food and Drug Administration (FDA) so, under current Good Manufacturing Practices (GMP), these cells may be suitable for use in manufacturing constructs for use in clinical trials. The National Cancer Institute seeks parties to non-exclusively license this research material.

In Utero Influences, Breast Stem Cells, and Breast Cancer Risk Factors

DTIC Science & Technology

2011-08-01

predictor of human breast cancer risk. Our hypothesis is that the in utero levels of mitogens, such as insulin -like growth factor-1 (IGF-1), drive an...by histological assays and in vivo and explant imaging. Histologically, we stained mammary glands from virgin and pregnant C57BL/6J mice using whole...that of a E16 pregnant (right panel); both glands are from 8 week-old mice. Second, we performed preliminary studies to quantitate and validate

Effects of carbon ion beam irradiation on the shoot regeneration from in vitro axillary bud explants of the Impatiens hawkeri

NASA Astrophysics Data System (ADS)

Zhou, Libin; Zhou, Libin; Li, Wenjian; Li, Ping; Dong, Xicun; Qu, Ying; Ma, Shuang; Li, Qiang

Accelerated ion beams is an excellent mutagen in plant breeding which can induce higher mutation frequencies and wider mutation spectrum than those of low linear energy transfer (LET) irradiations, such as X-rays (Okamura et al. 2003, Yamaguchi et al. 2003). Mutation breeding operation of two Saintpaulia ionahta cultivars using the method combining plant tissue culture technique and carbon ion beam irradiations were set out at Institute of Modern Physics from 2005 (Zhou et al. 2006). The effects of 960 MeV carbon ion beam and 8 MeV X-ray irradiations on regenerated shoots of Impatiens hawkeri from another kind of explants named in vitro axillary buds explants were studied recently. The biology endpoints in this study included relative number of roots (RNR), relative length of roots (RLR), relative height of shoots (RHS), relative number of nodes (RNN), survival fraction (SF) and morphology changes in the regenerated shoots. The experimental results showed that carbon ion beams inhibited the root and stem developments of axillary bud explants more severely than X-rays did. And the 50% lethal dose (LD50 ) is about 23.3 Gy for the carbon ion beam and 49.1 Gy for the X-rays, respectively. Relative biological effectiveness (RBE) of Impatiens hawkeri with respect to X-rays according to 50% SF was about two. Secondly, the percentage of shoots regenerated with malformed shoots including curliness, carnification, nicks in all Impatiens hawkeri axillary bud explants irradiated with carbon ion beam at 20 Gy accounted for 55.6%, while the highest number for the 40 Gy X-ray irradiation was 40%. Last, many regenerated shoots whose vascular bundle fused together were obtained only from explants irradiated with carbon ion beams. Based on the results above, it can be concluded that the effect of mutation induction by carbon ion beam irradiation on the axillary explants of Impatiens hawkeri is better than that by X-ray irradiation; and the optimal mutagenic dose varies from 20 Gy for carbon ion beam irradiation.

SU-E-T-352: Why Is the Survival Rate Low in Oropharyngeal Squamous Cell Carcinoma?

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

Huang, Z; Feng, Y; Rasmussen, K

2014-06-01

Purpose: Tumors are composed of a large number of clonogens that have the capability of indefinite reproduction. Even when there is complete clinical or radiographic regression of the gross tumor mass after treatment, tumor recurrence can occur if the clonogens are not completely eradicated by radiotherapy. This study was to investigate the colonogen number and its association with the tumor control probability (TCP) in oropharyngeal squamous cell carcinoma (OSCCA). Methods: A literature search was conducted to collect clinical information of patients with OSCCA, including the prescription dose, tumor volume and survival rate. The linear-quadratic (LQ) model was incorporated into TCPmore » model for clinical data analysis. The total dose ranged from 60 to 70 Gy and tumor volume ranged from 10 to 50 cc. The TCP was calculated for each group according to tumor size and dose. The least χ{sup 2} method was used to fit the TCP calculation to clinical data while other LQ model parameters (α, β) were adopted from the literature, due to the limited patient data. Results: A total of 190 patients with T2–T4 OSCCA were included. The association with HPV was not available for all the patients. The 3-year survival rate was about 82% for T2 squamous cell carcinoma and 40% for advanced tumors. Fitting the TCP model to the survival data, the average clonogen number was 1.56×10{sup 12}. For the prescription dose of 70 Gy, the calculated TCP ranged from 40% to 90% when the tumor volume varied from 10 to 50 cc. Conclusion: Our data suggests variation between the clonogen number and TCP in OSCCA. Tumors with larger colonogen number tend to have lower TCP and therefore dose escalation above 70 Gy may be indicated in order to improve the TCP and survival rate. Our result will require future confirmation with a large number of patients.« less

Age-Dependent Decline in Mouse Lung Regeneration with Loss of Lung Fibroblast Clonogenicity and Increased Myofibroblastic Differentiation

PubMed Central

Paxson, Julia A.; Gruntman, Alisha; Parkin, Christopher D.; Mazan, Melissa R.; Davis, Airiel; Ingenito, Edward P.; Hoffman, Andrew M.

2011-01-01

While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX) in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month) effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area by 21 days. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days), and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII) proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days) post-PNX in 9 versus 3 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a) and more alpha smooth muscle actin (αSMA) positive myofibroblasts were present after PNX in 9 month than 3 month mice. Isolated lung fibroblasts showed a significant age-dependent loss of clonogenicity. Moreover, lung fibroblasts isolated from 9 and 17 month mice exhibited higher αSMA, Col3A1, Fn1 and S100A expression, and lower expression of the survival gene Mdk consistent with terminal differentiation. These data show that concomitant loss of clonogenicity and progressive myofibroblastic differentiation contributes to the age-dependent decline in the rate of lung regeneration. PMID:21912590

Functional Role of CLIC1 Ion Channel in Glioblastoma-Derived Stem/Progenitor Cells

PubMed Central

2013-01-01

Background Chloride channels are physiologically involved in cell division and motility. Chloride intracellular channel 1 (CLIC1) is overexpressed in a variety of human solid tumors compared with normal tissues, suggesting a potential involvement of CLIC1 in the regulation of tumorigenesis. This led us to investigate the role of CLIC1 in gliomagenesis. Methods We used the neurosphere system to isolate stem/progenitor cells from human glioblastomas (GBMs). CLIC1 targeting in GBM neurospheres was achieved by both lentiviral-mediated short-hairpin RNA transduction and CLIC1 antibody treatment, and its effect on stem-like properties was analyzed in vitro by proliferation and clonogenic assays and in vivo by orthotopic injection in immunocompromised mice. Channel activity was studied by perforated patch clamp technique. Differences in expression were analyzed by analysis of variance with Tamhane’s multiple comparison test. Kaplan–Meier analyses and log-rank test were used to assess survival. All statistical tests were two-sided. Results CLIC1 was statistically significantly overexpressed in GBMs compared with normal brain tissues (P < .001) with a better survival of patients with CLIC1 low-expressing tumors (CLIC1low vs CLIC1high survival: χ2 = 74.35; degrees of freedom = 1; log-rank P < .001). CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment. CLIC1 silencing reduced proliferative (P < .01), clonogenic (P < .01), and tumorigenic capacity (P < .05) of stem/progenitor cells. The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient–derived neurospheres. Conclusions Reduced gliomagenesis after CLIC1 targeting in tumoral stem/progenitor cells and the finding that CLIC1 expression is inversely associated with patient survival suggest CLIC1 as a potential target and prognostic biomarker. PMID:24115360

Sulforaphane down-regulates SKP2 to stabilize p27(KIP1) for inducing antiproliferation in human colon adenocarcinoma cells.

PubMed

Chung, Yuan-Kai; Chi-Hung Or, Richard; Lu, Chien-Hsing; Ouyang, Wei-Ting; Yang, Shu-Yi; Chang, Chia-Che

2015-01-01

Sulforaphane is a cruciferous vegetable-derived isothiocyanate with promising chemopreventive and therapeutic activities. Induction of proliferation arrest and apoptosis principally contribute to sulforaphane's anticancer activity, but the precise molecular mechanisms remain elusive. The oncoprotein SKP2 is a key component of the SKP1-CULLIN1-F-box (SCF) E3 ligase complex and is responsible for directing SCF-mediated degradation of cyclin-dependent kinase inhibitor p27(KIP1) to promote cell proliferation. We herein provide the first evidence supporting the critical involvement of the SKP2-p27(KIP1) axis in sulforaphane-induced antiproliferation in various human colon adenocarcinoma cell lines. Specifically, sulforaphane markedly suppressed the levels of bromodeoxyuridine (BrdU) incorporation and clonogenicity in all tested cell lines, illustrating the antiproliferative effect of sulforaphane. Of note, sulforaphane-induced antiproliferation was accompanied with down-regulation of SKP2, leading to the stabilization and thus up-regulation of p27(KIP1). Additionally, sulforaphane was found to down-regulate SKP2 mainly through transcriptional repression, as sulforaphane lowered SKP2 mRNA expression and the SKP2 promoter activity. Furthermore, sulforaphane treatment led to the activation of both AKT and ERK, thus ruling out the possibility that sulforaphane down-regulates SKP2 by inhibiting AKT or ERK. Notably, sulforaphane-elicited suppression of BrdU incorporation and clonogenicity were significantly rescued in the context of SKP2 overexpression or p27(KIP1) depletion, therefore highlighting the important role of SKP2 down-regulation and the ensuing stabilization of p27(KIP1) in sulforaphane-induced antiproliferation. Collectively, these data expand our molecular understanding about how sulforaphane elicits proliferation arrest, but also implicate the application of sulforaphane in therapeutic modalities targeting SKP2. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Differential Efficacy of Combined Therapy With Radiation and AEE788 in High and Low EGFR-Expressing Androgen-Independent Prostate Tumor Models

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

Huamani, Jessica; Willey, Christopher; Thotala, Dinesh

2008-05-01

Purpose: To determine the efficacy of combining radiation (XRT) with a dual epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor inhibitor, AEE788, in prostate cancer models with different levels of EGFR expression. Methods and Materials: Immunoblotting was performed for EGFR, phosphorylated-EGFR, and phosphorylated-AKT in prostate cancer cells. Clonogenic assays were performed on DU145, PC-3, and human umbilical vein endothelial cells treated with XRT {+-} AEE788. Tumor xenografts were established for DU145 and PC-3 on hind limbs of athymic nude mice assigned to four treatment groups: (1) control, (2) AEE788, (3) XRT, and (4) AEE788 + XRT. Tumor blood flowmore » and growth measurements were performed using immunohistochemistry and imaging. Results: AEE788 effectively decreased phosphorylated-EGFR and phosphorylated-AKT levels in DU145 and PC-3 cells. Clonogenic assays showed no radiosensitization for DU145 and PC-3 colonies treated with AEE788 + XRT. However, AEE788 caused decreased proliferation in DU145 cells. AEE788 showed a radiosensitization effect in human umbilical vein endothelial cells and increased apoptosis susceptibility. Concurrent AEE788 + XRT compared with either alone led to significant tumor growth delay in DU145 tumors. Conversely, PC-3 tumors derived no added benefit from combined-modality therapy. In DU145 tumors, a significant decrease in tumor blood flow with combination therapy was shown by using power Doppler sonography and tumor blood vessel destruction on immunohistochemistry. Maldi-spectrometry (MS) imaging showed that AEE788 is bioavailable and heterogeneously distributed in DU145 tumors undergoing therapy. Conclusions: AEE788 + XRT showed efficacy in vitro/in vivo with DU145-based cell models, whereas PC-3-based models were adequately treated with XRT alone without added benefit from combination therapy. These findings correlated with differences in EGFR expression and showed effects on both tumor cell proliferation and vascular destruction.« less

Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific.

PubMed

Pathak, Bhakti R; Breed, Ananya A; Nakhawa, Vaishali H; Jagtap, Dhanashree D; Mahale, Smita D

2010-09-01

The prostate secretory protein of 94 amino acids (PSP94) has been shown to interact with cysteine-rich secretory protein 3 (CRISP-3) in human seminal plasma. Interestingly, PSP94 expression is reduced or lost in the majority of the prostate tumours, whereas CRISP-3 expression is upregulated in prostate cancer compared with normal prostate tissue. To obtain a better understanding of the individual roles these proteins have in prostate tumourigenesis and the functional relevance of their interaction, we ectopically expressed either PSP94 or CRISP-3 alone or PSP94 along with CRISP-3 in three prostate cell lines (PC3, WPE1-NB26 and LNCaP) and performed growth inhibition assays. Reverse transcription-polymerase chain reaction and Western blot analysis were used to screen prostate cell lines for PSP94 and CRISP-3 expression. Mammalian expression constructs for human PSP94 and CRISP-3 were also generated and the expression, localization and secretion of recombinant protein were assayed by transfection followed by Western blot analysis and immunofluorescence assay. The effect that ectopic expression of PSP94 or CRISP-3 had on cell growth was studied by clonogenic survival assay following transfection. To evaluate the effects of co-expression of the two proteins, stable clones of PC3 that expressed PSP94 were generated. They were subsequently transfected with a CRISP-3 expression construct and subjected to clonogenic survival assay. Our results showed that PSP94 and CRISP-3 could each induce growth inhibition in a cell line specific manner. Although the growth of CRISP-3-positive cell lines was inhibited by PSP94, growth inhibition mediated by CRISP-3 was not affected by the presence or absence of PSP94. This suggests that CRISP-3 may participate in PSP94-independent activities during prostate tumourigenesis.

Protein kinase A inhibitor, H89, enhances survival and clonogenicity of dissociated human embryonic stem cells through Rho-associated coiled-coil containing protein kinase (ROCK) inhibition.

PubMed

Zhang, Liang; Xu, Yanqing; Xu, Jiandong; Wei, Yuping; Xu, Xia

2016-04-01

Can cell survival of dissociated human embryonic stem cells (hESCs) be increased during culture? A protein kinase A (PKA) inhibitor, H89, can significantly enhance survival and clonogenicity of dissociated hESCs without affecting their pluripotency. hESCs are vulnerable to massive cell death upon cellular detachment and dissociation. hESCs were dissociated into single cells and then cultured in feeder-dependent and -independent manners. H89 was added to the culture medium at different concentrations for 1 day. The statistical results were obtained from at least three independent experiments (n ≥ 4). The group without treatment was used as the negative control. 4 µM H89 was added in the culture medium to promote cell survival and colony formation of dissociated hESCs. MTT method and propidium iodide (PI) staining were used to determine cell proliferation, cell death and cell cycle, respectively. To count colony formation, alkaline phosphatase (AP) staining was carried out. Western blot was performed to determine protein expression. Except AP staining, immunofluorescence, RT-PCR and karyotype analysis were used to confirm the pluripotent state of H89 treated hESCs. H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK), myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1), significantly increases cell survival and colony formation, and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro. Appropriate H89 concentration should be used and 1 day of H89 treatment is sufficient for promoting survival and colony formation of dissociated hESCs. These results provide an alternative for human pluripotent stem cell (hPSC) culture, broaden the scope of participants in the cell death of single hES cells after dissociation and further enlighten clues to understand the mechanism of dissociation-induced cell death. This research was supported by the National Natural Science Foundation of China (21176238, 21576266), and Chinese Academy of Sciences. There is no conflict of interest to declare. Nil. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

[Mono- and mixed infection by the tick-borne encephalitis and Powassan viruses of tissue explants from ticks of the genus Hyalomma].

PubMed

Chunikhin, S P; Khozinskaia, G A; Stefutkina, L F; Korolev, M B

1984-01-01

The paper presents results of virusological and electron microscope studies of the reproduction of viruses of tick-borne encephalitis and Povassan at mono- and mixed persistent infection of explants of imaginal tissues of Hyalomma anatolicum and H. dromedarii with these viruses. The virus reproduction in explants was observed within 208 to 217 days after the infection. Joint reproduction of two model viruses within 1-2 months after the infection can take place and after that the inhibition of the reproduction of one of the viruses. This inhibition can be of cyclic character.

Investigating the Skoog-Miller Model for Organogenesis Using Sweet Potato Root Explants.

ERIC Educational Resources Information Center

Delany, William; And Others

1994-01-01

Describes an experiment in which groups of students in a plant tissue culture course worked together to test application of the Skoog-Miller model (developed by Skoog and Miller in regeneration of tobacco experiments to demonstrate organogenesis) to sweet potato root explants. (ZWH)

Tissue-specific effects of peptides.

PubMed

Khavinson, V K

2001-08-01

Synthetic peptides (cytogens) Cortagen, Epithalon, Livagen, and Vilon stimulated the growth of explants from rat brain cortex, subcortical structures, liver, and thymus, respectively, in organotypic cultures. These peptides produced tissue-specific effects: they stimulated the growth of explants from tissues, whose cytomedins (peptide complexes) were used for chemical synthesis.

Fumonisin B₁ (FB₁) Induces Lamellar Separation and Alters Sphingolipid Metabolism of In Vitro Cultured Hoof Explants.

PubMed

Reisinger, Nicole; Dohnal, Ilse; Nagl, Veronika; Schaumberger, Simone; Schatzmayr, Gerd; Mayer, Elisabeth

2016-03-24

One of the most important hoof diseases is laminitis. Yet, the pathology of laminitis is not fully understood. Different bacterial toxins, e.g. endotoxins or exotoxins, seem to play an important role. Additionally, ingestion of mycotoxins, toxic secondary metabolites of fungi, might contribute to the onset of laminitis. In this respect, fumonsins are of special interest since horses are regarded as species most susceptible to this group of mycotoxins. The aim of our study was to investigate the influence of fumonisin B₁ (FB₁) on primary isolated epidermal and dermal hoof cells, as well as on the lamellar tissue integrity and sphingolipid metabolism of hoof explants in vitro. There was no effect of FB₁ at any concentration on dermal or epidermal cells. However, FB₁ significantly reduced the separation force of explants after 24 h of incubation. The Sa/So ratio was significantly increased in supernatants of explants incubated with FB₁ (2.5-10 µg/mL) after 24 h. Observed effects on Sa/So ratio were linked to significantly increased sphinganine concentrations. Our study showed that FB₁ impairs the sphingolipid metabolism of explants and reduces lamellar integrity at non-cytotoxic concentrations. FB₁ might, therefore, affect hoof health. Further in vitro and in vivo studies are necessary to elucidate the effects of FB₁ on the equine hoof in more detail.

Interleukin-1 β Modulates Melatonin Secretion in Ovine Pineal Gland: Ex Vivo Study.

PubMed

Herman, A P; Bochenek, J; Skipor, J; Król, K; Krawczyńska, A; Antushevich, H; Pawlina, B; Marciniak, E; Tomaszewska-Zaremba, D

2015-01-01

The study was designed to determine the effect of proinflammatory cytokine, interleukin- (IL-) 1β, on melatonin release and expression enzymes essential for this hormone synthesis: arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) in ovine pineal gland, taking into account the immune status of animals before sacrificing. Ewes were injected by lipopolysaccharide (LPS; 400 ng/kg) or saline, two hours after sunset during short day period (December). Animals were euthanized three hours after the injection. Next, the pineal glands were collected and divided into four explants. The explants were incubated with (1) medium 199 (control explants), (2) norepinephrine (NE; 10 µM), (3) IL-1β (75 pg/mL), or (4) NE + IL-1β. It was found that IL-1β abolished (P < 0.05) NE-induced increase in melatonin release. Treatment with IL-1β also reduced (P < 0.05) expression of AA-NAT enzyme compared to NE-treated explants. There was no effect of NE or IL-1β treatment on gene expression of HIOMT; however, the pineal fragments isolated from LPS-treated animals were characterized by elevated (P < 0.05) expression of HIOMT mRNA and protein compared to the explants from saline-treated ewes. Our study proves that IL-1β suppresses melatonin secretion and its action seems to be targeted on the reduction of pineal AA-NAT protein expression.

Interleukin-1β Modulates Melatonin Secretion in Ovine Pineal Gland: Ex Vivo Study

PubMed Central

Herman, A. P.; Bochenek, J.; Skipor, J.; Król, K.; Krawczyńska, A.; Antushevich, H.; Pawlina, B.; Marciniak, E.; Tomaszewska-Zaremba, D.

2015-01-01

The study was designed to determine the effect of proinflammatory cytokine, interleukin- (IL-) 1β, on melatonin release and expression enzymes essential for this hormone synthesis: arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) in ovine pineal gland, taking into account the immune status of animals before sacrificing. Ewes were injected by lipopolysaccharide (LPS; 400 ng/kg) or saline, two hours after sunset during short day period (December). Animals were euthanized three hours after the injection. Next, the pineal glands were collected and divided into four explants. The explants were incubated with (1) medium 199 (control explants), (2) norepinephrine (NE; 10 µM), (3) IL-1β (75 pg/mL), or (4) NE + IL-1β. It was found that IL-1β abolished (P < 0.05) NE-induced increase in melatonin release. Treatment with IL-1β also reduced (P < 0.05) expression of AA-NAT enzyme compared to NE-treated explants. There was no effect of NE or IL-1β treatment on gene expression of HIOMT; however, the pineal fragments isolated from LPS-treated animals were characterized by elevated (P < 0.05) expression of HIOMT mRNA and protein compared to the explants from saline-treated ewes. Our study proves that IL-1β suppresses melatonin secretion and its action seems to be targeted on the reduction of pineal AA-NAT protein expression. PMID:26339621

A lower pH value benefits regeneration of Trichosanthes kirilowii by somatic embryogenesis, involving rhizoid tubers (RTBs), a novel structure.

PubMed

Xu, Ke-dong; Chang, Yun-xia; Zhang, Ju; Wang, Pei-long; Wu, Jian-xin; Li, Yan-yan; Wang, Xiao-wen; Wang, Wei; Liu, Kun; Zhang, Yi; Yu, De-shui; Liao, Li-bing; Li, Yi; Ma, Shu-ya; Tan, Guang-xuan; Li, Cheng-wei

2015-03-06

A new approach was established for the regeneration of Trichosanthes kirilowii from root, stem, and leaf explants by somatic embryogenesis (SE), involving a previously unreported SE structure, rhizoid tubers (RTBs). During SE, special rhizoids were first induced from root, stem, and leaf explants with average rhizoid numbers of 62.33, 40.17, and 11.53 per explant, respectively, on Murashige and Skoog (MS) medium (pH 4.0) supplemented with 1.0 mg/L 1-naphthaleneacetic acid (NAA) under dark conditions. Further, one RTB was formed from each of the rhizoids on MS medium (pH 4.0) supplemented with 20 mg/L thidiazuron (TDZ) under light conditions. In the suitable range (pH 4.0-9.0), a lower pH value increased the induction of rhizoids and RTBs. Approximately 37.77, 33.47, and 31.07% of in vivo RTBs from root, stem, and leaf explants, respectively, spontaneously developed into multiple plantlets on the same MS medium (supplemented with 20 mg/L TDZ) for induction of RTBs, whereas >95.00% of in vitro RTBs from each kind of explant developed into multiple plantlets on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine (BAP). Morphological and histological analyses revealed that RTB is a novel type of SE structure that develops from the cortex cells of rhizoids.

Plant regeneration through protocorm-like bodies induced from rhizoids using leaf explants of Rosa spp.

PubMed

Tian, Chuanwei; Chen, Ying; Zhao, Xiaolan; Zhao, Liangjun

2008-05-01

A new protocol for plant regeneration via protocorm-like bodies (PLBs) induced from rhizoids that developed from leaf explants of Rosa spp. (R. canina L., R. multiflora var. cathayensis Rehd. et Wils., and R. multiflora f. carnea Thory.) has been established. Rhizoids were induced from calli of leaf explants incubated under dark conditions on Murashige and Skoog (MS) medium containing 1.5 mg/l 2, 4-D. PLBs developed from the tip of rhizoids cultured under light conditions on (1/2) MS medium containing 20 mg/l TDZ. About 90, 17 and 93% of rhizoid formation were achieved for the above-mentioned Rosa spp., respectively using this protocol. The frequency of PLB clusters formation and the number of PLB clusters per explant reached 50% and 5.1 for R. canina, 46.7% and 0.8 for R. multifolra var. cathayensis, 46.7% and 4.2 for R. multiflora f. carnea, respectively. PLB clusters regenerated on MS medium supplemented with 2 mg/l 6-BA, 0.1 mg/l IBA, and 0.1 mg/l GA(3). The best result of regenerated plantlets per leaf explant achieved via PLBs for the three Rosa spp. mentioned above was 3.6, 0.1, and 1.2, respectively. Environmental scanning electron microscope and histological studies revealed that rhizoids were structurally different from roots grown in vitro, and PLBs developed from proembryos.

A Lower pH Value Benefits Regeneration of Trichosanthes kirilowii by Somatic Embryogenesis, Involving Rhizoid Tubers (RTBs), a Novel Structure

PubMed Central

Xu, Ke-dong; Chang, Yun-xia; Zhang, Ju; Wang, Pei-long; Wu, Jian-xin; Li, Yan-yan; Wang, Xiao-wen; Wang, Wei; Liu, Kun; Zhang, Yi; Yu, De-shui; Liao, Li-bing; Li, Yi; Ma, Shu-ya; Tan, Guang-xuan; Li, Cheng-wei

2015-01-01

A new approach was established for the regeneration of Trichosanthes kirilowii from root, stem, and leaf explants by somatic embryogenesis (SE), involving a previously unreported SE structure, rhizoid tubers (RTBs). During SE, special rhizoids were first induced from root, stem, and leaf explants with average rhizoid numbers of 62.33, 40.17, and 11.53 per explant, respectively, on Murashige and Skoog (MS) medium (pH 4.0) supplemented with 1.0 mg/L 1-naphthaleneacetic acid (NAA) under dark conditions. Further, one RTB was formed from each of the rhizoids on MS medium (pH 4.0) supplemented with 20 mg/L thidiazuron (TDZ) under light conditions. In the suitable range (pH 4.0–9.0), a lower pH value increased the induction of rhizoids and RTBs. Approximately 37.77, 33.47, and 31.07% of in vivo RTBs from root, stem, and leaf explants, respectively, spontaneously developed into multiple plantlets on the same MS medium (supplemented with 20 mg/L TDZ) for induction of RTBs, whereas >95.00% of in vitro RTBs from each kind of explant developed into multiple plantlets on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine (BAP). Morphological and histological analyses revealed that RTB is a novel type of SE structure that develops from the cortex cells of rhizoids. PMID:25744384

The promoting effect of pentadecapeptide BPC 157 on tendon healing involves tendon outgrowth, cell survival, and cell migration.

PubMed

Chang, Chung-Hsun; Tsai, Wen-Chung; Lin, Miao-Sui; Hsu, Ya-Hui; Pang, Jong-Hwei Su

2011-03-01

Pentadecapeptide BPC 157, composed of 15 amino acids, is a partial sequence of body protection compound (BPC) that is discovered in and isolated from human gastric juice. Experimentally it has been demonstrated to accelerate the healing of many different wounds, including transected rat Achilles tendon. This study was designed to investigate the potential mechanism of BPC 157 to enhance healing of injured tendon. The outgrowth of tendon fibroblasts from tendon explants cultured with or without BPC 157 was examined. Results showed that BPC 157 significantly accelerated the outgrowth of tendon explants. Cell proliferation of cultured tendon fibroblasts derived from rat Achilles tendon was not directly affected by BPC 157 as evaluated by MTT assay. However, the survival of BPC 157-treated cells was significantly increased under the H(2)O(2) stress. BPC 157 markedly increased the in vitro migration of tendon fibroblasts in a dose-dependent manner as revealed by transwell filter migration assay. BPC 157 also dose dependently accelerated the spreading of tendon fibroblasts on culture dishes. The F-actin formation as detected by FITC-phalloidin staining was induced in BPC 157-treated fibroblasts. The protein expression and activation of FAK and paxillin were determined by Western blot analysis, and the phosphorylation levels of both FAK and paxillin were dose dependently increased by BPC 157 while the total amounts of protein was unaltered. In conclusion, BPC 157 promotes the ex vivo outgrowth of tendon fibroblasts from tendon explants, cell survival under stress, and the in vitro migration of tendon fibroblasts, which is likely mediated by the activation of the FAK-paxillin pathway.

Prediction of skin anti-aging clinical benefits of an association of ingredients from marine and maritime origins: Ex vivo evaluation using a label-free quantitative proteomic and customized data processing approach.

PubMed

Hameury, Sebastien; Borderie, Laurent; Monneuse, Jean-Marc; Skorski, Gilbert; Pradines, Dominique

2018-05-23

The application of ingredients from marine and maritime origins is increasingly common in skin care products, driven by consumer expectations for natural ingredients. However, these ingredients are typically studied for a few isolated in vitro activities. The purpose of this study was to carry out a comprehensive evaluation of the activity on the skin of an association of ingredients from marine and maritime origins using label-free quantitative proteomic analysis, in order to predict the clinical benefits if used in a skin care product. An aqueous gel containing 6.1% of ingredients from marine and maritime origins (amino acid-enriched giant kelp extract, trace element-enriched seawater, dedifferentiated sea fennel cells) was topically applied on human skin explants. The skin explants' proteome was analyzed in a label-free manner by high-performance liquid nano-chromatography coupled with tandem mass spectrometry. A specific data processing pipeline (CORAVALID) providing an objective and comprehensive interpretation of the statistically relevant biological activities processed the results. Compared to untreated skin explants, 64 proteins were significantly regulated by the gel treatment (q-value ≤ 0.05). Computer data processing revealed an activity of the ingredients on the epidermis and the dermis. These significantly regulated proteins are involved in gene expression, cell survival and metabolism, inflammatory processes, dermal extracellular matrix synthesis, melanogenesis and keratinocyte proliferation, migration, and differentiation. These results suggest that the tested ingredients could help to preserve a healthy epidermis and dermis, and possibly to prevent the visible signs of skin aging. © 2018 The Authors. Journal of Cosmetic Dermatology Published by Wiley Periodicals, Inc.

Ex-vivo dynamic 3-D culture of human tissues in the RCCS™ bioreactor allows the study of Multiple Myeloma biology and response to therapy.

PubMed

Ferrarini, Marina; Steimberg, Nathalie; Ponzoni, Maurilio; Belloni, Daniela; Berenzi, Angiola; Girlanda, Stefania; Caligaris-Cappio, Federico; Mazzoleni, Giovanna; Ferrero, Elisabetta

2013-01-01

Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients'sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo. Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy.

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

PubMed Central

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

2014-01-01

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

Ex-Vivo Dynamic 3-D Culture of Human Tissues in the RCCS™ Bioreactor Allows the Study of Multiple Myeloma Biology and Response to Therapy

PubMed Central

Ponzoni, Maurilio; Belloni, Daniela; Berenzi, Angiola; Girlanda, Stefania; Caligaris-Cappio, Federico; Mazzoleni, Giovanna; Ferrero, Elisabetta

2013-01-01

Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients'sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo. Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy. PMID:23990965

Prevalence of polyreactive innate clones among graft--infiltrating B cells in human cardiac allograft vasculopathy.

PubMed

Chatterjee, Debanjana; Moore, Carolina; Gao, Baoshan; Clerkin, Kevin J; See, Sarah B; Shaked, David; Rogers, Kortney; Nunez, Sarah; Veras, Yokarla; Addonizio, Linda; Givertz, Michael M; Naka, Yoshifumi; Mancini, Donna; Vasilescu, Rodica; Marboe, Charles; Restaino, Susan; Madsen, Joren C; Zorn, Emmanuel

2018-03-01

Cardiac allograft vasculopathy (CAV) has been associated with graft-infiltrating B cells, although their characteristics are still unclear. In this study we examined the frequency, localization and reactivity profile of graft-infiltrating B cells to determine their contribution to the pathophysiology of CAV. B cells, plasma cells and macrophages were examined by immunohistochemistry in 56 allografts with CAV, 49 native failed hearts and 25 autopsy specimens. A total of 102 B-cell clones were immortalized directly from the infiltrates of 3 fresh cardiac samples with CAV. Their secreted antibodies were assessed using enzyme-linked immunoassay and flow cytometry. B-cell infiltration was observed around coronary arteries in 93% of allograft explants with CAV. Comparatively, intragraft B cells were less frequent and less dense in the intraventricular myocardium from where routine biopsies are obtained. Plasma cells and macrophages were also detected in 85% and 95% of explants, respectively. Remarkably, B-cell infiltrates were not associated with circulating donor-specific antibodies (DSA) or prior episodes of antibody-mediated rejection (AMR). Among all B-cell clones generated from 3 explants with CAV, a majority secreted natural antibodies reactive to multiple autoantigens and apoptotic cells, a characteristic of innate B cells. Our study reveals a high frequency of infiltrating B cells around the coronary arteries of allografts with CAV, independent of DSA or AMR. These cells are enriched for innate B cells with a polyreactive profile. The findings shift the focus from conventional DSA-producing B cells to the potentially pathogenic polyreactive B cells in the development of clinical CAV. Copyright © 2018 International Society for the Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

In vitro propagation of northern red oak (Quercus rubra L.)

Treesearch

G. Vengadesan; Paula M. Pijut

2009-01-01

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 µM 6-benzylaminopurine (BA), 0.45 ...

Staphylococcus aureus induces hypoxia and cellular damage in porcine dermal explants

USDA-ARS?s Scientific Manuscript database

Methicillin-resistant Staphylococcus aureus (MRSA) can infect wounds and produce difficult-to- treat biofilms. To determine the extent that MRSA biofilms can deplete oxygen, change pH and damage host tissue, we developed a porcine dermal explant model on which we cultured GFP-labeled MRSA biofilms. ...

Populational equilibrium through exosome-mediated Wnt signaling in tumor progression of diffuse large B-cell lymphoma.

PubMed

Koch, Raphael; Demant, Martin; Aung, Thiha; Diering, Nina; Cicholas, Anna; Chapuy, Bjoern; Wenzel, Dirk; Lahmann, Marlen; Güntsch, Annemarie; Kiecke, Christina; Becker, Sabrina; Hupfeld, Timo; Venkataramani, Vivek; Ziepert, Marita; Opitz, Lennart; Klapper, Wolfram; Trümper, Lorenz; Wulf, Gerald G

2014-04-03

Tumors are composed of phenotypically heterogeneous cell populations. The nongenomic mechanisms underlying transitions and interactions between cell populations are largely unknown. Here, we show that diffuse large B-cell lymphomas possess a self-organized infrastructure comprising side population (SP) and non-SP cells, where transitions between clonogenic states are modulated by exosome-mediated Wnt signaling. DNA methylation modulated SP-non-SP transitions and was correlated with the reciprocal expressions of Wnt signaling pathway agonist Wnt3a in SP cells and the antagonist secreted frizzled-related protein 4 in non-SP cells. Lymphoma SP cells exhibited autonomous clonogenicity and exported Wnt3a via exosomes to neighboring cells, thus modulating population equilibrium in the tumor.

Parallel assessment of the effects of bisphenol A and several of its analogs on the adult human testis.

PubMed

Desdoits-Lethimonier, C; Lesné, L; Gaudriault, P; Zalko, D; Antignac, J P; Deceuninck, Y; Platel, C; Dejucq-Rainsford, N; Mazaud-Guittot, S; Jégou, B

2017-07-01

Are bisphenol A (BPA) and BPA analogs (BPA-A) safe for male human reproductive function? The endocrine function of human testes explants [assessed by measuring testosterone and insulin-like factor 3 (INSL3)] was impacted by exposure of the human adult testis explants to BPA/BPA-A. The few epidemiologic studies performed suggest that bisphenols have potential endocrine disruptive properties, but they did not identify clear and direct patterns of endocrine disruption. Adult human testis explants in culture were exposed to BPA and the analogs bisphenol F (BPF), bisphenol S (BPS), bisphenol E (BPE), bisphenol B (BPB) and bisphenol A diglycidyl ether (BADGE) at 10-9-10-5 M for 24 or 48 h. Human adult testes were obtained from prostate cancer patients who had no hormone therapy, or from multiorgan donors. After ex vivo exposure to the investigated bisphenols, the measured outcomes were related to histopathology (gross morphology and germ cell viability determined by anti-caspase three immunohistochemistry), and the levels of testosterone, INSL3 and inhibin B were measured using immunoassays. The levels of mRNA encoding key enzymes of bisphenol biotransformation were investigated by quantitative PCR: UGT2B15 UDP (glucuronosyltransferase two family, polypeptide B15), GUSB (glucuronidase beta), SULT1A1 and 3 (sulfotransferase family 1 A member 1 and 3) and STS (steroid sulfatase). A significant dose-dependent inhibition was found between testosterone levels measured in the culture medium and concentrations of BPA (P = 0.00778 at 24 h and P = 0.0291 at 48 h), BPE (P = 0.039) and BPF (P = 0.00663). The observed BPA and BPA-A-induced inhibition of testosterone production varied according to duration of exposure and BPA/BPA-A concentrations. BPA (10-9 M; P < 0.05), BPB (10-9 M; P < 0.05), BPS (10-9 and 10-8 M; P < 0.05) and BADGE (10-5 M; P < 0.05) increased Leydig cell INSL3 production. By contrast, BPE dose dependently inhibited INSL3 (P = 0.0372). Conversely, Sertoli cell function (inhibin B) and germ cell viability were not significantly affected by either bisphenols. N/A. Environmental compounds cannot be deliberately administered to men, justifying the use of an ex vivo approach. A relatively low number of testes samples were available for analysis (n = 3, except for testosterone secretion with n = 5). The active concentrations of BPA and BPA-A used in the study were higher than those found in human biological fluids. Under our experimental conditions, direct exposure to BPA or BPA-A can result in endocrine disturbance in the adult human testis. This study was funded by Inserm (Institut National de la Santé et de la Recherche Médicale), EHESP-School of Public Health, University of Rennes1, by grants from the Agence Nationale de la Recherche (ANR; grant#ANR-13-CESA-0012-03 NEWPLAST) and Agence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail (ANSES; grant#EST-2010/2/046 (BPATESTIS)). All authors declare they have no current or potential competing financial interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Avoidance of Maternal Cell Contamination and Overgrowth in Isolating Fetal Chorionic Villi Mesenchymal Stem Cells from Human Term Placenta

PubMed Central

Sardesai, Varda S.; Shafiee, Abbas; Fisk, Nicholas M.

2017-01-01

Abstract Human placenta is rich in mesenchymal stem/stromal cells (MSC), with their origin widely presumed fetal. Cultured placental MSCs are confounded by a high frequency of maternal cell contamination. Our recent systematic review concluded that only a small minority of placental MSC publications report fetal/maternal origin, and failed to discern a specific methodology for isolation of fetal MSC from term villi. We determined isolation conditions to yield fetal and separately maternal MSC during ex vivo expansion from human term placenta. MSCs were isolated via a range of methods in combination; selection from various chorionic regions, different commercial media, mononuclear cell digest and/or explant culture. Fetal and maternal cell identities were quantitated in gender‐discordant pregnancies by XY chromosome fluorescence in situ hybridization. We first demonstrated reproducible maternal cell contamination in MSC cultures from all chorionic anatomical locations tested. Cultures in standard media rapidly became composed entirely of maternal cells despite isolation from fetal villi. To isolate pure fetal cells, we validated a novel isolation procedure comprising focal dissection from the cotyledonary core, collagenase/dispase digestion and explant culture in endothelial growth media that selected, and provided a proliferative environment, for fetal MSC. Comparison of MSC populations within the same placenta confirmed fetal to be smaller, more osteogenic and proliferative than maternal MSC. We conclude that in standard media, fetal chorionic villi‐derived MSC (CV‐MSC) do not grow readily, whereas maternal MSC proliferate to result in maternal overgrowth during culture. Instead, fetal CV‐MSCs require isolation under specific conditions, which has implications for clinical trials using placental MSC. Stem Cells Translational Medicine 2017;6:1070–1084 PMID:28205414

Retosiban Prevents Stretch-Induced Human Myometrial Contractility and Delays Labor in Cynomolgus Monkeys.

PubMed

Aye, Irving L M H; Moraitis, Alexandros A; Stanislaus, Dinesh; Charnock-Jones, D Stephen; Smith, Gordon C S

2018-03-01

Stretch of the myometrium promotes its contractility and is believed to contribute to the control of parturition at term and to the increased risk of preterm birth in multiple pregnancies. To determine the effects of the putative oxytocin receptor (OTR) inverse agonist retosiban on (1) the contractility of human myometrial explants and (2) labor in nonhuman primates. Human myometrial biopsies were obtained at planned term cesarean, and explants were exposed to stretch in the presence and absence of a range of drugs, including retosiban. The in vivo effects of retosiban were determined in cynomolgus monkeys. Prolonged mechanical stretch promoted myometrial extracellular signal-regulated kinase (ERK)1/2 phosphorylation. Moreover, stretch-induced stimulation of myometrial contractility was prevented by ERK1/2 inhibitors. Retosiban (10 nM) prevented stretch-induced stimulation of myometrial contractility and phosphorylation of ERK1/2. Moreover, the inhibitory effect of retosiban on stretch-induced ERK1/2 phosphorylation was prevented by coincubation with a 100-fold excess of a peptide OTR antagonist, atosiban. Compared with vehicle-treated cynomolgus monkeys, treatment with oral retosiban (100 to 150 days of gestational age) reduced the risk of spontaneous delivery (hazard ratio = 0.07, 95% confidence interval 0.01 to 0.60, P = 0.015). The OTR acts as a uterine mechanosensor, whereby stretch increases myometrial contractility through agonist-free activation of the OTR. Retosiban prevents this through inverse agonism of the OTR and, in vivo, reduced the likelihood of spontaneous labor in nonhuman primates. We hypothesize that retosiban may be an effective preventative treatment of preterm birth in high-risk multiple pregnancies, an area of unmet clinical need.

PSC-RANTES Blocks R5 Human Immunodeficiency Virus Infection of Langerhans Cells Isolated from Individuals with a Variety of CCR5 Diplotypes

PubMed Central

Kawamura, Tatsuyoshi; Bruce, Shannon E.; Abraha, Awet; Sugaya, Makoto; Hartley, Oliver; Offord, Robin E.; Arts, Eric J.; Zimmerman, Peter A.; Blauvelt, Andrew

2004-01-01

Topical microbicides that effectively block interactions between CCR5+ immature Langerhans cells (LC) residing within genital epithelia and R5 human immunodeficiency virus (HIV) may decrease sexual transmission of HIV. Here, we investigated the ability of synthetic RANTES analogues (AOP-, NNY-, and PSC-RANTES) to block R5 HIV infection of human immature LC by using a skin explant model. In initial experiments using activated peripheral blood mononuclear cells, each analogue compound demonstrated marked antiviral activity against two R5 HIV isolates. Next, we found that 20-min preincubation of skin explants with each RANTES analogue blocked R5 HIV infection of LC in a dose-dependent manner (1 to 100 nM) and that PSC-RANTES was the most potent of these compounds. Similarly, preincubation of LC with each analogue was able to block LC-mediated infection of cocultured CD4+ T cells. Competition experiments between primary R5 and X4 HIV isolates showed blocking of R5 HIV by PSC-RANTES and no evidence of increased propagation of X4 HIV, data that are consistent with the specificity of PSC-RANTES for CCR5 and the CCR5+ CXCR4− phenotype of immature LC. Finally, when CCR5 genetic polymorphism data were integrated with results from the in vitro LC infection studies, PSC-RANTES was found to be equally effective in inhibiting R5 HIV in LC isolated from individuals with CCR5 diplotypes known to be associated with low, intermediate, and high cell surface levels of CCR5. In summary, PSC-RANTES is a potent inhibitor of R5 HIV infection in immature LC, suggesting that it may be useful as a topical microbicide to block sexual transmission of HIV. PMID:15220435

Interleukin-32 promotes detachment and activation of human Langerhans cells in a human skin explant model.

PubMed

Gonnet, J; Perrin, H; Hutton, A J; Boccara, D; Bonduelle, O; Mimoun, M; Atlan, M; Soria, A; Combadière, B

2018-05-28

Cross-talk between skin keratinocytes (KCs) and Langerhans cells (LCs) plays a fundamental role in the body's first line of immunological defences. However, the mechanism behind the interaction between these two major epidermal cells is unknown. Interleukin (IL)-32 is produced in inflammatory skin disorders. We questioned the role of IL-32 in the epidermis. We aimed to determine the role of IL-32 produced by KCs on surrounding LCs. We used an ex vivo human explant model from healthy donors and investigated the role of IL-32 on LC activation using imaging, flow cytometry, reverse transcriptase quantitative polymerase chain reaction and small interfering (si)RNA treatment. Modified vaccinia virus ankara (MVA) infection induced KC death alongside the early production of the proinflammatory cytokine IL-32. We demonstrated that IL-32 produced by MVA-infected KCs induced modest but significant morphological changes in LCs and downregulation of adhesion molecules, such as epithelial cell adhesion molecule and very late antigen-4, and CXCL10 production. The treatment of KCs with IL-32-specific siRNA, and anti-IL-32 blocking antibody significantly inhibited LC activation, demonstrating the role of IL-32 in LC activation. We also found that some Toll-like receptor ligands induced a very high level of IL-32 production by KCs, which initiated LC activation. We propose, for the first time, that IL-32 is a molecular link between KCs and LCs in healthy skin, provoking LC migration from the epidermis to the dermis prior to their migration to the draining lymph nodes. © 2018 The Authors. British Journal of Dermatology published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.

The Expression and Regulation of Adrenomedullin in the Human Endometrium: A Candidate for Endometrial Repair

PubMed Central

Maybin, Jacqueline A.; Battersby, Sharon; Hirani, Nikhil; Nikitenko, Leonid L.; Critchley, Hilary O. D.

2011-01-01

After menstruation, the endometrium has a remarkable capacity for repair, but the factors involved remain undefined. We hypothesize adrenomedullin (AM) plays a role in this process. Premenstrually progesterone levels decline, stimulating prostaglandin (PG) synthesis, vasoconstriction, and hypoxia. This study aimed to determine 1) AM expression throughout the menstrual (M) cycle and 2) its regulation by PG and hypoxia. Human endometrial biopsies (n = 51) were collected with ethical approval and consent. AM mRNA expression was examined by quantitative RT-PCR and was found to be selectively elevated in endometrium from the menstrual (M) phase (P < 0.001). AM immunohistochemical staining was maximal in M and proliferative (P) endometrium. Culture of secretory, but not P, explants with 100 nm PGF2α or hypoxia (0.5% O2) increased AM mRNA (P < 0.05). P explants were induced to increase AM expression using in vitro progesterone withdrawal but required the presence of hypoxia (P < 0.05). Short hairpin sequences against hypoxia-inducible factor-1α (HIF-1α) inhibited AM hypoxic up-regulation but did not alter PGF2α-induced expression. The AM receptor was immunolocalized to endothelial cells in both lymphatic and blood vessels. Conditioned medium from PGF2α-treated cells increased endothelial cell proliferation and branching (P < 0.05). This was abolished by AM receptor antagonists. In conclusion, AM is elevated at the time of endometrial repair and induces both angiogenesis and lymphangiogenesis by stimulating endothelial cell proliferation and tube formation. In the human endometrium, AM expression is up-regulated by two mechanisms: a HIF-1α-mediated hypoxic induction and a HIF-1α-independent PGF2α pathway. These physiological mechanisms may provide novel therapeutic targets for disorders such as heavy menstrual bleeding. PMID:21558311

Effects of Coal Fly Ash Particulate Matter on the Antimicrobial Activity of Airway Surface Liquid

PubMed Central

Vargas Buonfiglio, Luis G.; Mudunkotuwa, Imali A.; Abou Alaiwa, Mahmoud H.; Vanegas Calderón, Oriana G.; Borcherding, Jennifer A.; Gerke, Alicia K.; Zabner, Joseph; Grassian, Vicki H.

2017-01-01

Background: Sustained exposure to ambient particulate matter (PM) is a global cause of mortality. Coal fly ash (CFA) is a byproduct of coal combustion and is a source of anthropogenic PM with worldwide health relevance. The airway epithelia are lined with fluid called airway surface liquid (ASL), which contains antimicrobial proteins and peptides (AMPs). Cationic AMPs bind negatively charged bacteria to exert their antimicrobial activity. PM arriving in the airways could potentially interact with AMPs in the ASL to affect their antimicrobial activity. Objectives: We hypothesized that PM can interact with ASL AMPs to impair their antimicrobial activity. Methods: We exposed pig and human airway explants, pig and human ASL, and the human cationic AMPs β-defensin-3, LL-37, and lysozyme to CFA or control. Thereafter, we assessed the antimicrobial activity of exposed airway samples using both bioluminescence and standard colony-forming unit assays. We investigated PM-AMP electrostatic interaction by attenuated total reflection Fourier-transform infrared spectroscopy and measuring the zeta potential. We also studied the adsorption of AMPs on PM. Results: We found increased bacterial survival in CFA-exposed airway explants, ASL, and AMPs. In addition, we report that PM with a negative surface charge can adsorb cationic AMPs and form negative particle–protein complexes. Conclusion: We propose that when CFA arrives at the airway, it rapidly adsorbs AMPs and creates negative complexes, thereby decreasing the functional amount of AMPs capable of killing pathogens. These results provide a novel translational insight into an early mechanism for how ambient PM increases the susceptibility of the airways to bacterial infection. https://doi.org/10.1289/EHP876 PMID:28696208

APR-246/PRIMA-1Met Inhibits and Reverses Squamous Metaplasia in Human Conjunctival Epithelium.

PubMed

Li, Jing; Li, Cheng; Wang, Guoliang; Liu, Zhen; Chen, Pei; Yang, Qichen; Dong, Nuo; Wu, Huping; Liu, Zuguo; Li, Wei

2016-02-01

Squamous metaplasia is a common pathologic condition in ocular surface diseases for which there is no therapeutic medication in clinic. In this study, we investigated the effect of a small molecule, APR-246/PRIMA-1(Met), on squamous metaplasia in human conjunctival epithelium. Human conjunctival explants were cultured for up to 12 days under airlifting conditions. Epithelial cell differentiation and proliferation were assessed by Cytokeratin 10 (K10), K14, K19, Pax6, MUC5AC, and p63 immunostaining patterns. β-catenin and TCF-4 immunofluorescent staining and real-time PCR characterized Wnt signaling pathway involvement. Pterygium clinical samples were cultured under airlifting conditions with or without APR-246 for 4 days. p63, K10, β-catenin, and TCF-4 expression in pterygial epithelium was determined by immunofluorescent staining and real-time PCR. Airlift conjunctival explants resulted in increased stratification and intrastromal epithelial invagination. Such pathology was accompanied by increases in K10, K14, and p63 expression, whereas K19 and Pax6 levels declined when compared to those in freshly isolated tissue. On the other hand, APR-246 reversed all of these declines in K10, K14, and p63 expression. Furthermore, K19 and Pax6 increased along with rises in goblet cell density. These effects of APR-246 were accompanied by near restoration of normal conjunctival epithelial histology. APR-246 also reversed squamous metaplasia in pterygial epithelium that had developed after 4 days in ex vivo culture. Reductions in squamous metaplasia induced by APR-246 suggest it may provide a novel therapeutic approach in different squamous metaplasia-associated ocular surface diseases.

Future dentistry: cell therapy meets tooth and periodontal repair and regeneration

PubMed Central

Catón, Javier; Bostanci, Nagihan; Remboutsika, Eumorphia; De Bari, Cosimo; Mitsiadis, Thimios A

2011-01-01

Abstract Cell-based tissue repair of the tooth and – tooth-supporting – periodontal ligament (PDL) is a new attractive approach that complements traditional restorative or surgical techniques for replacement of injured or pathologically damaged tissues. In such therapeutic approaches, stem cells and/or progenitor cells are manipulated in vitro and administered to patients as living and dynamic biological agents. In this review, we discuss the clonogenic potential of human dental and periodontal tissues such as the dental pulp and the PDL and their potential for tooth and periodontal repair and/or regeneration. We propose novel therapeutic approaches using stem cells or progenitor cells, which are targeted to regenerate the lost dental or periodontal tissue. PMID:21199329

Paracrine Engineering of Human Explant-Derived Cardiac Stem Cells to Over-Express Stromal-Cell Derived Factor 1α Enhances Myocardial Repair.

PubMed

Tilokee, Everad L; Latham, Nicholas; Jackson, Robyn; Mayfield, Audrey E; Ye, Bin; Mount, Seth; Lam, Buu-Khanh; Suuronen, Erik J; Ruel, Marc; Stewart, Duncan J; Davis, Darryl R

2016-07-01

First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826-1835. © 2016 AlphaMed Press.

Long term organ culture of human prostate tissue in a NASA-designed rotating wall bioreactor

NASA Technical Reports Server (NTRS)

Margolis, L.; Hatfill, S.; Chuaqui, R.; Vocke, C.; Emmert-Buck, M.; Linehan, W. M.; Duray, P. H.

1999-01-01

PURPOSE: To maintain ex vivo integral prostatic tissue including intact stromal and ductal elements using the NASA-designed Rotating Wall Vessel (RWV) which maintains colocalized cells in an environment that promotes both three-dimensional cellular interactions together with the uniform mass transfer of nutrients and metabolic wastes. MATERIALS AND METHODS: Samples of normal prostate were obtained as a byproduct of transurethral prostatectomy or needle biopsy. Prostatic tissue dissected into small 1 x 1 mm. blocks was cultured in the Rotating Wall Vessel (RWV) Bioreactor for various time periods and analyzed using histological, immunochemical, and total cell RNA assays. RESULTS: We report the long term maintenance of benign explanted human prostate tissue grown in simple culture medium, under the simulated microgravity conditions afforded by the RWV bioreactor. Mesenchymal stromal elements including blood vessels and architecturally preserved tubuloglandular acini were maintained for a minimum of 28 days. Cytokeratins, vimentin and TGF-beta2 receptor and ligand were preserved through the entire culture period as revealed by immunocytochemistry. Prostatic acid phosphatase (PAP) was continuously expressed during the culture period, although somewhat decreased. Prostatic specific antigen (PSA) and its transcript were down regulated over time of culture. Prostatic carcinoma cells from the TSU cell line were able to invade RWV-cultured benign prostate tissue explants. CONCLUSIONS: The RWV bioreactor represents an additional new technology for culturing prostate tissue for further investigations concerning the basic physiology and pathobiology of this clinically important tissue.

Soybean (Glycine max) transformation using mature cotyledonary node explants.

PubMed

Olhoft, Paula M; Donovan, Christopher M; Somers, David A

2006-01-01

Agrobacterium tumefaciens-mediated transformation of soybeans has been steadily improved since its development in 1988. Soybean transformation is now possible in a range of genotypes from different maturity groups using different explants as sources of regenerable cells, various selectable marker genes and selective agents, and different A. tumefaciens strains. The cotyledonary-node method has been extensively investigated and across a number of laboratories yields on average greater than 1% transformation efficiency (one Southern-positive, independent event per 100 cotyledonary-node explants). Continued improvements in the cotyledonary-node method concomitant with further increases in transformation efficiency will enhance broader adoption of this already productive transformation method for use in crop improvement and functional genomics research efforts.

Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

PubMed Central

Concannon, Caoimhin G.; Rehm, Markus; Kögel, Donat; Prehn, Jochen H. M.

2008-01-01

Background The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases. Methodology/Principal Findings To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations. Conclusions/Significance Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling. PMID:18665234

The targeted histone deacetylase inhibitor tefinostat (CHR-2845) shows selective in vitro efficacy in monocytoid-lineage leukaemias

PubMed Central

Zabkiewicz, Joanna; Gilmour, Marie; Hills, Robert; Vyas, Pares; Bone, Elizabeth; Davidson, Alan; Burnett, Alan; Knapper, Steven

2016-01-01

Tefinostat (CHR-2845) is a novel monocyte/macrophage-targeted histone deacetylase (HDAC) inhibitor which is cleaved into its active acid by the intracellular esterase human carboxylesterase-1 (hCE-1). The in vitro efficacy of tefinostat was characterised in cell lines and in a cohort of 73 primary AML and CMML samples. Dose-dependent induction of apoptosis and significant growth inhibitory effects were seen in myelomonocytic (M4), monocytic/monoblastic (M5) and CMML samples in comparison to non-monocytoid AML sub-types (p = 0.007). Importantly, no growth inhibitory effects were seen in normal bone marrow CD34+ cells exposed to AML-toxic doses of tefinostat in clonogenic assays. Expression of hCE-1 was measured by intracellular flow cytometry and immunoblotting across the cohort, with highest levels seen in M5 AML patients. hCE-1 levels correlated with significantly increased tefinostat sensitivity (low EC50) as measured by growth inhibition assays (p = 0.001) and concomitant elevation of the mature monocytoid marker CD14+. Strong induction of intracellular histone protein acetylation was observed in tefinostat-responsive samples, as were high levels of the DNA damage sensor γ-H2A.X, highlighting potential biomarkers of patient responsiveness. Synergistic interaction between tefinostat and the current standard treatment cytarabine was demonstrated in dose response and clonogenic assays using simultaneous drug addition in primary samples (median Combination Index value = 0.51). These data provide a strong rationale for the further clinical evaluation of tefinostat in monocytoid-lineage haematological neoplasms including CMML and monocyte-lineage AMLs. PMID:26934551

Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery.

PubMed

Buckel, Lisa; Savariar, Elamprakash N; Crisp, Jessica L; Jones, Karra A; Hicks, Angel M; Scanderbeg, Daniel J; Nguyen, Quyen T; Sicklick, Jason K; Lowy, Andrew M; Tsien, Roger Y; Advani, Sunil J

2015-04-01

Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor-targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell-penetrating peptide targeting matrix metalloproteinases and RGD-binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low-passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule- and dose-dependent manner, correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double-strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with nontargeted free MMAE or tumor-targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor-targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell-penetrating peptides. ©2015 American Association for Cancer Research.

Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery

PubMed Central

Crisp, Jessica L.; Jones, Karra A.; Hicks, Angel M.; Scanderbeg, Daniel J.; Nguyen, Quyen T.; Sicklick, Jason K.; Lowy, Andrew M.; Tsien, Roger Y.; Advani, Sunil J.

2015-01-01

Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell penetrating peptide targeting matrix metalloproteinases and RGD binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule and dose dependent manner correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with non-targeted free MMAE or tumor targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell penetrating peptides. PMID:25681274

Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro.

PubMed

Wang, Z-M; Kang, Y-H; Yang, X; Wang, J-F; Zhang, Q; Yang, B-X; Zhao, K-L; Xu, L-P; Yang, L-P; Ma, J-X; Huang, G-H; Cai, J; Sun, X-C

2016-01-01

To explore the radiosensitivity of andrographolide on esophageal cancer cell line ECA109. The inhibition effects of andrographolide were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Clonogenic survival assay was used to evaluate the effects of andrographolide on the radiosensitivity of esophageal cancer cells. Immunofluorescence was employed to examine Bax expression. The changes in cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of NF-κb/Cleaved-Caspase3/Bax/Bcl-2 was measured using Western blot analysis. DNA damage was detected via γ-H2AX foci counting. With a clear dose and time effects, andrographolide was found to inhibit the proliferation of esophageal cell line ECA109. The results of the clonogenic survival assay show that andrographolide could markedly enhance radiosensitivity (P < 0.05) with a sensitizing enhancement ratio of 1.28. Andrographolide caused a dose-dependent increase in Cleaved-Caspase3/Bax protein expression and a decrease in Bcl-2/NF-κb expression. Apoptosis in andrographolide-treated ECA-109 increased significantly compared with the apoptosis in the simple drug and radiation combined with drug groups (P < 0.001; P < 0.05). Moreover, compared with the independent radiation group, the andrographolide combined with radiation group increased the number of DNA double chain breaks. Andrographolide can increase the radiosensitivity of esophageal cell line ECA109. This result may be associated with the decrease in the NF-κb level and the induced apoptosis of esophageal cancer cells. © 2014 International Society for Diseases of the Esophagus.

Adventitious shoot regeneration from in vitro leaf explants of Fraxinus nigra

Treesearch

Jun Hyung Lee; Paula M. Pijut

2017-01-01

Black ash (Fraxinus nigra) is an endangered hardwood tree species under threat of extirpation by the emerald ash borer (EAB), an aggressive exotic phloemfeeding beetle. We have developed an efficient regeneration system through adventitious shoot organogenesis in F. nigra using in vitro-derived leaf explants. Two types of leaf...

HSV-1 interaction to 3-O-sulfated heparan sulfate in mouse-derived DRG explant and profiles of inflammatory markers during virus infection.

PubMed

Sharthiya, Harsh; Seng, Chanmoly; Van Kuppevelt, T H; Tiwari, Vaibhav; Fornaro, Michele

2017-06-01

The molecular mechanism of herpes simplex virus (HSV) entry and the associated inflammatory response in the nervous system remain poorly understood. Using mouse-derived ex vivo dorsal root ganglia (DRG) explant model and single cell neurons (SCNs), in this study, we provided a visual evidence for the expression of heparan sulfate (HS) and 3-O-sulfated heparan sulfate (3-OS HS) followed by their interactions with HSV-1 glycoprotein B (gB) and glycoprotein D (gD) during cell entry. Upon heparanase treatment of DRG-derived SCN, a significant inhibition of HSV-1 entry was observed suggesting the involvement of HS role during viral entry. Finally, a cytokine array profile generated during HSV-1 infection in DRG explant indicated an enhanced expression of chemokines (LIX, TIMP-2, and M-CSF)-known regulators of HS. Taken together, these results highlight the significance of HS during HSV-1 entry in DRG explant. Further investigation is needed to understand which isoforms of 3-O-sulfotransferase (3-OST)-generated HS contributed during HSV-1 infection and associated cell damage.

Primary cell culture of LHRH neurones from embryonic olfactory placode in the sheep (Ovis aries).

PubMed

Duittoz, A H; Batailler, M; Caldani, M

1997-09-01

The aim of this study was to establish an in vitro model of ovine luteinizing hormone-releasing hormone (LHRH) neurones. Olfactory placodes from 26 day-old sheep embryos (E26) were used for explant culture. Cultures were maintained successfully up to 35 days, but were usually used at 17 days for immunocytochemistry. LHRH and neuronal markers such as neurofilament (NF) were detected by immunocytochemistry within and/or outside the explant. Three main types of LHRH positive cells are described: (1) neuroblastic LHRH and NF immunoreactive cells with round cell body and very short neurites found mainly within the explant, (2) migrating LHRH bipolar neurones with an fusiform cell body, found outside the explant, (3) network LHRH neuron, bipolar or multipolar with long neurites connecting other LHRH neurons. Cell morphology was very similar to that which has been described in the adult sheep brain. These results strongly suggest that LHRH neurones in the sheep originate from the olfactory placode. This mode may represent a useful tool to study LHRH neurones directly in the sheep.

Unfertilized ovary: a novel explant for coconut (Cocos nucifera L.) somatic embryogenesis.

PubMed

Perera, Prasanthi I P; Hocher, Valerie; Verdeil, Jean Luc; Doulbeau, Sylvie; Yakandawala, Deepthi M D; Weerakoon, L Kaushalya

2007-01-01

Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 microM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 microM abscisic acid, followed by plant regeneration medium (with 5 microM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon germination they gave rise to normal shoots. However, some abnormal developments were also observed. Flow cytometric analysis revealed that all the calli tested were diploid. Through histological studies, it was possible to study the sequence of the events that take place during somatic embryogenesis including orientation, polarization and elongation of the embryos.

Micropropagation and genetic transformation of Tylophora indica (Burm. f.) Merr.: a review.

PubMed

Teixeira da Silva, Jaime A; Jha, Sumita

2016-11-01

This review provides an in-depth and comprehensive overview of the in vitro culture of Tylophora species, which have medicinal properties. Tylophora indica (Burm. f.) Merr. is a climbing perennial vine with medicinal properties. The tissue culture and genetic transformation of T. indica, which has been extensively studied, is reviewed. Micropropagation using nodal explants has been reported in 25 % of all publications. Leaf explants from field-grown plants has been the explant of choice of independent research groups, which reported direct and callus-mediated organogenesis as well as callus-mediated somatic embryogenesis. Protoplast-mediated regeneration and callus-mediated shoot organogenesis has also been reported from stem explants, and to a lesser degree from root explants of micropropagated plants in vitro. Recent studies that used HPLC confirmed the potential of micropropagated plants to synthesize the major T. indica alkaloid tylophorine prior to and after transfer to field conditions. The genetic integrity of callus-regenerated plants was confirmed by RAPD in a few reports. Tissue culture is an essential base for genetic transformation studies. Hairy roots and transgenic T. indica plants have been shown to accumulate tylophorine suggesting that in vitro biology and transgenic methods are viable ways of clonally producing valuable germplasm and mass producing compounds of commercial value. Further studies that investigate the factors affecting the biosynthesis of Tylophora alkaloids and other secondary metabolites need to be conducted using non-transformed as well as transformed cell and organ cultures.

Spermatozoa with high mitochondrial membrane potential and low tyrosine phosphorylation preferentially bind to oviduct explants in the water buffalo (Bubalus bubalis).

PubMed

Saraf, Kaustubh Kishor; Kumaresan, Arumugam; Chhillar, Shivani; Nayak, Samiksha; Lathika, Sreela; Datta, Tirtha Kumar; Gahlot, Subhash Chand; Karan, Prabha; Verma, Kiran; Mohanty, Tushar Kumar

2017-05-01

Although it is understood that spermatozoa are subjected to selection processes to form a functional sperm reservoir in the oviduct, the mechanism remains obscure. With the aim to understand the sperm selection process in the oviduct, in the present in vitro study, we analyzed mitochondrial membrane potential and tyrosine phosphorylation status in oviduct-explants bound and unbound spermatozoa. Frozen semen from Murrah buffalo bulls (n=10) used under progeny testing programme were utilized for the study. Oviduct explants were prepared by overnight culture of epithelial cells in TCM- 199 and washed spermatozoa were added to the oviduct explants and incubated for 4h. Mitochondrial membrane potential (MMP) and tyrosine phosphorylation status of bound and unbound spermatozoa were assessed at 1h and 4h of incubation. The proportion of spermatozoa with high MMP was significantly higher (P<0.001) among the bound spermatozoa (range 84.67-96.56%) compared to unbound (range 8.70-21.03%) spermatozoa. The proportion of tyrosine phosphorylated spermatozoa was significantly higher (P<0.001) among unbound population as compared to bound population. The proportion of spermatozoa displaying tyrosine phosphorylation at acrosomal area was significantly (P<0.05) lower in bound sperm population compared to unbound population. It was inferred that spermatozoa with high MMP and low tyrosine phosphorylation were preferred for oviduct-explants binding in the buffalo. Copyright © 2017 Elsevier B.V. All rights reserved.

Location-specific expression of chemokines, TNF-α and S100 proteins in a teat explant model.

PubMed

Lind, Monique; Sipka, Anja S; Schuberth, Hans-Joachim; Blutke, Andreas; Wanke, Rüdiger; Sauter-Louis, Carola; Duda, Katarzyna A; Holst, Otto; Rainard, Pascal; Germon, Pierre; Zerbe, Holm; Petzl, Wolfram

2015-04-01

The distal compartments of the udder are the first to interact with invading pathogens. The regulatory and effector functions of two major teat regions [Fürstenberg's rosette (FR); teat cistern (TC)] are largely unknown. The objective of this study was to establish an in vitro model with explants of the FR and the TC to analyse their response towards Escherichia coli LPS and Staphylococcus aureus lipoteichoic acid (LTA). Quantitative stereological analysis confirmed differences in the cellular composition of FR and TC explants. Chemokine (CXCL8, CCL5, CCL20) and TNF-α mRNA were expressed at low levels in both locations. Explant stimulation with LPS increased the mRNA abundance of all tested chemokines and TNF-α. Stimulation with LTA only induced CCL20 and CXCL8. LPS- and LTA-stimulated explant supernatants contained CXCL8 and CXCL3. Supernatants significantly attracted neutrophils in vitro. Compared with TC, the FR showed high constitutive mRNA expression of S100 proteins (A8, A9, A12). In the TC, both LPS and LTA significantly induced S100A8, whereas S100A9 and S100A12 expression was only induced by LPS. The novel model system underpins the role of the teat for recognising pathogens and shaping a pathogen- and location-specific immune response. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Development of a 3D printed device to support long term intestinal culture as an alternative to hyperoxic chamber methods.

PubMed

Costa, Matheus O; Nosach, Roman; Harding, John C S

2017-01-01

Most interactions between pathogenic microorganisms and their target host occur on mucosal surfaces of internal organs such as the intestine. In vitro organ culture (IVOC) provides an unique tool for studying host-pathogen interactions in a controlled environment. However, this technique requires a complex laboratory setup and specialized apparatus. In addition, issues arise when anaerobic pathogens are exposed to the hyperoxic environment required for intestinal culture. The objective of this study was to develop an accessible 3D-printed device that would allow manipulation of the gas mixture used to supply the tissue culture media separately from the gas mixture exposed to the mucosal side of explants. Porcine colon explants from 2 pigs were prepared ( n  = 20) and cultured for 0h, 8h, 18h and 24h using the device. After the culture period, explants were fixed in formalin and H&E stained sections were evaluated for histological defects of the mucosa. At 8h, 66% of samples displayed no histological abnormalities, whereas samples collected at 18h and 24h displayed progressively increasing rates of superficial epithelial erosion and epithelial metaplasia. The 3D-design reported here allows investigators to setup intestinal culture explants while manipulating the gas media explants are exposed to, to support tissue viability for a minimal of 8h. The amount of media necessary and tissue contamination are potential issues associated with this apparatus.

Re-use of explanted DDD pacemakers as VDD- clinical utility and cost effectiveness.

PubMed

Namboodiri, K K N; Sharma, Y P; Bali, H K; Grover, A

2004-01-01

Re-use of DDD pulse generators explanted from patients died of unrelated causes is associated with an additional cost of two transvenous leads if implanted as DDD itself, and high rate of infection according to some studies. We studied the clinical and economical aspects of reutilization of explanted DDD pacemakers programmed to VDD mode. Out of 28 patients who received VDD pacemaker during the period, October 2000- September 2001 in the Department of Cardiology, PGIMER, Chandigarh, 5 poor patients were implanted with explanted DDD pulse generators programmed to VDD mode. Each implantation was planned and carried out according to a standard protocol. The age ranged from 45 to 75 (mean-61) years. The indications for pacing were complete heart block (4) and second degree AV block (1). The clinical profile, costs and complications, if any were noted and followed up at regular intervals. The results were compared with patients who received new DDD pulse generators during this period. The additional cost for the atrial lead was not required in these patients. None of these patients had any local site infection. Compared to the two-lead system, the single lead system provided more rapid implantation and minimized complications associated with placement of an atrial lead. The explanted DDD pacemaker can be safely reused as VDD mode with same efficacy in selected patient population. This is associated with lower cost and complications compared to reimplantation as DDD itself.

Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish.

PubMed

Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves; Labbe, Catherine

2015-07-01

The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. Copyright © 2015 Elsevier Inc. All rights reserved.

Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex.

PubMed

Schaefer, Alisa; Poluch, Sylvie; Juliano, Sharon

2008-04-01

Numerous functions related to neuronal migration are linked to the glycoprotein reelin. Reelin also elongates radial glia, which are disrupted in mutant reeler mice. Our lab developed a model of cortical dysplasia in ferrets that shares features with the reeler mouse, including impaired migration of neurons into the cerebral cortex and disrupted radial glia. Explants of normal ferret cortex in coculture with dysplastic ferret cortex restore the deficits in this model. To determine if reelin is integral to the repair, we used explants of P0 mouse cortex either of the wild type (WT) or heterozygous (het) for the reelin gene, as well as P0 reeler cortex (not containing reelin), in coculture with organotypic cultures of dysplastic ferret cortex. This arrangement revealed that all types of mouse cortical explants (WT, het, reeler) elongated radial glia in ferret cortical dysplasia, indicating that reelin is not required for proper radial glial morphology. Migration of cells into ferret neocortex, however, did not improve with explants of reeler cortex, but was almost normal after pairing with WT or het explants. We also placed an exogenous source of reelin in ferret cultures at the pial surface to reveal that migrating cells move toward the reelin source in dysplastic cortex; radial glia in these cultures were also improved toward normal. Our results demonstrate that the normotopic position of reelin is important for proper neuronal positioning, and that reelin is capable of elongating radial glial cells but is not the only radialization factor.

Molecular and functional interactions between AKT and SOX2 in breast carcinoma

PubMed Central

Mir, Perihan; Konantz, Martina; Pereboom, Tamara C.; Paczulla, Anna M.; Merz, Britta; Fehm, Tanja; Perner, Sven; Rothfuss, Oliver C.; Kanz, Lothar; Schulze-Osthoff, Klaus; Lengerke, Claudia

2015-01-01

The transcription factor SOX2 is a key regulator of pluripotency in embryonic stem cells and plays important roles in early organogenesis. Recently, SOX2 expression was documented in various cancers and suggested as a cancer stem cell (CSC) marker. Here we identify the Ser/Thr-kinase AKT as an upstream regulator of SOX2 protein turnover in breast carcinoma (BC). SOX2 and pAKT are co-expressed and co-regulated in breast CSCs and depletion of either reduces clonogenicity. Ectopic SOX2 expression restores clonogenicity and in vivo tumorigenicity of AKT-inhibited cells, suggesting that SOX2 acts as a functional downstream AKT target. Mechanistically, we show that AKT physically interacts with the SOX2 protein to modulate its subcellular distribution. AKT kinase inhibition results in enhanced cytoplasmic retention of SOX2, presumably via impaired nuclear import, and in successive cytoplasmic proteasomal degradation of the protein. In line, blockade of either nuclear transport or proteasomal degradation rescues SOX2 expression in AKT-inhibited BC cells. Finally, AKT inhibitors efficiently suppress the growth of SOX2-expressing putative cancer stem cells, whereas conventional chemotherapeutics select for this population. Together, our results suggest the AKT/SOX2 molecular axis as a regulator of BC clonogenicity and AKT inhibitors as promising drugs for the treatment of SOX2-positive BC. PMID:26498353

Characterization of bone marrow mesenchymal stromal cells in aplastic anaemia.

PubMed

Hamzic, Edita; Whiting, Karen; Gordon Smith, Edward; Pettengell, Ruth

2015-06-01

In aplastic anaemia (AA), haemopoietic activity is significantly reduced and generally attributed to failure of haemopoietic stem cells (HSC) within the bone marrow (BM). The regulation of haemopoiesis depends on the interaction between HSC and various cells of the BM microenvironment, including mesenchymal stromal cells (MSC). MSC involvement in the functional restriction of HSC in AA is largely unknown and therefore, the physical and functional properties of AA MSC were studied in vitro. MSC were characterized by their phenotype and ability to form adherent stromal layers. The functional properties of AA MSC were assessed through proliferative, clonogenic and cross-over culture assays. Results indicate that although AA MSC presented typical morphology and distinctive mesenchymal markers, stromal formation was reduced, with 50% of BM samples failing to produce adherent layers. Furthermore, their proliferative and clonogenic capacity was markedly decreased (P = 0·03 and P = 0·04 respectively) and the ability to sustain haemopoiesis was significantly reduced, as assessed by total cell proliferation (P = 0·032 and P = 0·019 at Week 5 and 6, respectively) and clonogenic potential of HSC (P = 0·02 at Week 6). It was concluded that the biological characteristics of AA MSC are different from those of control MSC and their in vitro haemopoiesis-supporting ability is significantly reduced. © 2015 John Wiley & Sons Ltd.

Incidence and predictors of myocardial recovery on long-term left ventricular assist device support: Results from the United Network for Organ Sharing database.

PubMed

Pan, Stephen; Aksut, Baran; Wever-Pinzon, Omar E; Rao, Shaline D; Levin, Allison P; Garan, Arthur R; Fried, Justin A; Takeda, Koji; Hiroo, Takayama; Yuzefpolskaya, Melana; Uriel, Nir; Jorde, Ulrich P; Mancini, Donna M; Naka, Yoshifumi; Colombo, Paolo C; Topkara, Veli K

2015-12-01

Mechanical circulatory support (MCS) leads to favorable changes in the failing heart at the molecular, cellular, and structural levels. However, myocardial recovery leading to device explantation is rare. We reasoned that the multicenter United Network for Organ Sharing (UNOS) registry might provide insights into clinical predictors and outcomes of the recovery process. The MCS device data set of the UNOS registry was queried for patients with long-term continuous-flow left ventricular assist devices (CF-LVADs) that were explanted for heart transplantation or indication of recovery. Analysis was restricted to adult patients (≥18 years old) who were listed for an initial heart transplantation. Patients with CF-LVADs that were explanted because of recovery were compared with patients with CF-LVADs who underwent transplantation. We identified 594 patients with HeartMate II devices and 92 patients with HeartWare devices. Duration of support was on average 500.4 ± 325.3 days. In 34 (5.0%) patients, devices were explanted secondary to myocardial recovery. Univariate predictors of recovery in patients with long-term LVADs included younger age (40 years vs 53 years), female sex, lower body mass index (25.7 kg/m(2) vs 27.9 kg/m(2)), non-ischemic etiology (91% vs 59%), lack of implantable cardioverter defibrillator at the time of listing (44% vs 79%), and lower serum creatinine (0.97 mg/dl vs 1.28 mg/dl) (all p < 0.05). In the post-explantation period, freedom from death or transplantation was 66% at 1 year. The incidence of recovery on device support is low in the current MCS era and limited to a select cohort of predominantly young patients with non-ischemic myopathy. Given the high incidence of disease recurrence, patients should be closely followed after device explantation. Copyright © 2015 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna × E. maidenii trees.

PubMed

Corredoira, E; Ballester, A; Ibarra, M; Vieitez, A M

2015-06-01

A reproducible procedure for induction of somatic embryogenesis (SE) from adult trees of Eucalyptus globulus Labill. and the hybrid E. saligna Smith × E. maidenii has been developed for the first time. Somatic embryos were obtained from both shoot apex and leaf explants of all three genotypes evaluated, although embryogenic frequencies were significantly influenced by the species/genotype, auxin and explant type. Picloram was more efficient for somatic embryo induction than naphthaleneacetic acid (NAA), with the highest frequency of induction being obtained in Murashige and Skoog medium containing 40 µM picloram and 40 mg l(-1) gum Arabic, in which 64% of the shoot apex explants and 68.8% of the leaf explants yielded somatic embryos. The embryogenic response of the hybrid was higher than that of the E. globulus, especially when NAA was used. The cultures initiated on picloram-containing medium consisted of nodular embryogenic structures surrounded by a mucilaginous coating layer that emerged from a watery callus developed from the initial explants. Cotyledonary somatic embryos were differentiated after subculture of these nodular embryogenic structures on a medium lacking plant growth regulators. Histological analysis confirmed the bipolar organization of the somatic embryos, with shoot and root meristems and closed procambial tissue that bifurcated into small cotyledons. The root pole was more differentiated than the shoot pole, which appeared to be formed by a few meristematic layers. Maintenance of the embryogenic lines by secondary SE was attained by subculturing individual cotyledonary embryos or small clusters of globular and torpedo embryos on medium with 16.11 µM NAA at 4- to 5-week intervals. Somatic embryos converted into plantlets after being transferred to liquid germination medium although plant regeneration remained poor. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Determination of hepatocellular carcinoma grade by needle biopsy is unreliable for liver transplant candidate selection.

PubMed

Court, Colin M; Harlander-Locke, Michael P; Markovic, Daniela; French, Samuel W; Naini, Bita V; Lu, David S; Raman, Steven S; Kaldas, Fady M; Zarrinpar, Ali; Farmer, Douglas G; Finn, Richard S; Sadeghi, Saeed; Tomlinson, James S; Busuttil, Ronald W; Agopian, Vatche G

2017-09-01

The objective of this article is to evaluate the utility of preoperative needle biopsy (PNB) grading of hepatocellular carcinoma (HCC) as a biomarker for liver transplantation (LT) candidate selection. Given the prognostic significance of HCC tumor grade, PNB grading has been proposed as a biomarker for LT candidate selection. Clinicopathologic characteristics of HCC LT recipients (1989-2014) with a PNB were analyzed, and the concordance of PNB grade to explant grade and vascular invasion was assessed to determine whether incorporation of PNB grade to accepted transplant criteria improved candidate selection. Of 965 patients undergoing LT for HCC, 234 (24%) underwent PNB at a median of 280 days prior to transplant. Grade by PNB had poor concordance to final explant pathology (κ = 0.22; P = 0.003), and low sensitivity (29%) and positive predictive value (35%) in identifying poorly differentiated tumors. Vascular invasion was predicted by explant pathologic grade (r s = 0.24; P < 0.001) but not PNB grade (r s = -0.05; P = 0.50). Increasing explant pathology grade (P = 0.02), but not PNB grade (P = 0.65), discriminated post-LT HCC recurrence risk. The incorporation of PNB grade to the established radiologic Milan criteria (MC) did not result in improved prognostication of post-LT recurrence (net reclassification index [NRI] = 0%), whereas grade by explant pathology resulted in significantly improved reclassification of risk (NRI = 19%). Preoperative determination of HCC grade by PNB has low concordance with explant pathologic grade and low sensitivity and positive predictive value in identifying poorly differentiated tumors. PNB grade did not accurately discriminate post-LT HCC recurrence and had no utility in improving prognostication compared with the MC alone. Incorporation of PNB to guide transplant candidate selection appears unjustified. Liver Transplantation 23 1123-1132 2017 AASLD. © 2017 by the American Association for the Study of Liver Diseases.

Experience With Cardiac Implantable Electrical Device Explantation After Cardiac Transplantation: A Report of 16 Cases From a Single Center in a Period of 5 Years.

PubMed

Çiftci, Orçun; Yılmaz, Kerem Can; Sezgin, Atilla; Özin, Mehmet Bülent; Müderrisoğlu, İbrahim Haldun; Haberal, Mehmet

2018-03-01

Cardiac implantable electrical devices are widely used for patients with advanced heart failure and are usually explanted during orthotopic heart transplant. However, lead fragments and the pulse generator are sometimes left after the procedure. Given the concerns of infectious and thromboembolic complications, their removal is recommended. Herein, we report our experience with cardiac implantable electrical device explantation after orthotopic heart transplant. We included recipients of heart transplants performed at Başkent University Faculty of Medicine, Department of Cardiovascular Surgery, who underwent lead and pulse generator explantation by manual traction between January 2012 and June 2017. We analyzed patient demographic, clinical, biochemical, and treatment properties. Sixteen patients (11 males, 5 females) with a median age of 45 years (range, 18-52 y) were included. Two patients (12.5%) died during follow-up but not secondary to device explantation. All patients were using immunosuppressives and 50% were receiving antiplatelet/anticoagulant agents. All pulse generators were located at the left prepectoral area, with tips of lead fragments in the superior vena cava or left subclavian vein. No procedural complications were observed. Aspirin was continued uninterrupted perioperatively, warfarin was stopped 2 days before the procedure, and low-molecular-weight heparins were skipped on the morning and evening of the procedure. One patient (6.3%) complained of postoperative pain, and another (6.3%) developed a pocket hematoma, which was treated conservatively. No patient developed fever, clinical infection, or major bleeding. Preoperative and postoperative levels of hemoglobin, white blood cells, and C-reactive protein were similar. No demographic, procedural, or biochemical variable was significantly correlated with postprocedural complications. In our cohort, explantation of lead fragments and pulse generators of cardiac implantable electrical devices was safe after heart transplant. It appears that neither antiplatelet/anticoagulant agents nor immunosuppressives seem to put patients at increased risk of postoperative complications.

Outcomes of single- vs double-cuff artificial urinary sphincter insertion in low- and high-risk profile male patients with severe stress urinary incontinence.

PubMed

Ahyai, Sascha A; Ludwig, Tim A; Dahlem, Roland; Soave, Armin; Rosenbaum, Clemens; Chun, Felix K-H; Fisch, Margit; Schmid, Marianne; Kluth, Luis A

2016-10-01

To evaluate continence and complication rates of bulbar single-cuff (SC) and distal bulbar double-cuff (DC) insertion in male patients with severe stress urinary incontinence (SUI) according to whether the men were considered low or high risk for unfavourable artificial urinary sphincter (AUS) outcomes. In all, 180 male patients who underwent AUS implantation between 2009 and 2013 were followed according to institutional standards. Patients with previous pelvic radiation therapy, open bulbar urethral or UI surgery ('high risk') underwent distal bulbar DC (123 patients) insertion, all others ('low risk') had proximal bulbar SC (57) insertion. Primary and secondary endpoints consisted of continence and complication rates. Kaplan-Meier analysis determined explantation-free survival, and Cox regression models assessed risk factors for persistent UI and explantation. The median follow-up was 24 months. Whereas there was no significant difference in pad usage/objective continence after SC vs DC insertion, superior rates of subjective/social continence and less persistent UI were reported by the patients with DC devices (all P ≤ 0.02). Overall, device explantation (erosion, infection or mechanical failure) occurred in 12.8% of patients. While early (<6 weeks) complication rates compared with SC patients were similar (P > 0.05), DC patients had a 5.7-fold higher risk of device explantation during late follow-up (P = 0.02) and significantly shorter explantation-free survival (log-rank, P = 0.003). Distal bulbar DC insertion in patients with a 'high-risk' profile (previous pelvic radiation, urethral surgery) leads to similar objective continence, but higher explantation rates when compared with patients considered 'low risk' with proximal bulbar SCs. Randomised controlled trials comparing both devices will be needed to determine whether the higher explanations rates are attributable to the DC device or to underlying risk factors. © 2016 The Authors BJU International © 2016 BJU International Published by John Wiley & Sons Ltd.

Hibiscus syriacus Extract from an Established Cell Culture Stimulates Skin Wound Healing.

PubMed

di Martino, O; Tito, A; De Lucia, A; Cimmino, A; Cicotti, F; Apone, F; Colucci, G; Calabrò, V

2017-01-01

Higher plants are the source of a wide array of bioactive compounds that support skin integrity and health. Hibiscus syriacus , family Malvaceae, is a plant of Chinese origin known for its antipyretic, anthelmintic, and antifungal properties. The aim of this study was to assess the healing and hydration properties of H. syriacus ethanolic extract (HSEE). We established a cell culture from Hibiscus syriacus leaves and obtained an ethanol soluble extract from cultured cells. The properties of the extract were tested by gene expression and functional analyses on human fibroblast, keratinocytes, and skin explants. HSEE treatment increased the healing potential of fibroblasts and keratinocytes. Specifically, HSEE significantly stimulated fibronectin and collagen synthesis by 16 and 60%, respectively, while fibroblasts contractility was enhanced by 30%. These results were confirmed on skin explants, where HSEE accelerated the wound healing activity in terms of epithelium formation and fibronectin production. Moreover, HSEE increased the expression of genes involved in skin hydration and homeostasis. Specifically, aquaporin 3 and filaggrin genes were enhanced by 20 and 58%, respectively. Our data show that HSEE contains compounds capable of stimulating expression of biomarkers relevant to skin regeneration and hydration thereby counteracting molecular pathways leading to skin damage and aging.

Hibiscus syriacus Extract from an Established Cell Culture Stimulates Skin Wound Healing

PubMed Central

di Martino, O.; Tito, A.; De Lucia, A.; Cimmino, A.; Cicotti, F.; Apone, F.; Colucci, G.

2017-01-01

Higher plants are the source of a wide array of bioactive compounds that support skin integrity and health. Hibiscus syriacus, family Malvaceae, is a plant of Chinese origin known for its antipyretic, anthelmintic, and antifungal properties. The aim of this study was to assess the healing and hydration properties of H. syriacus ethanolic extract (HSEE). We established a cell culture from Hibiscus syriacus leaves and obtained an ethanol soluble extract from cultured cells. The properties of the extract were tested by gene expression and functional analyses on human fibroblast, keratinocytes, and skin explants. HSEE treatment increased the healing potential of fibroblasts and keratinocytes. Specifically, HSEE significantly stimulated fibronectin and collagen synthesis by 16 and 60%, respectively, while fibroblasts contractility was enhanced by 30%. These results were confirmed on skin explants, where HSEE accelerated the wound healing activity in terms of epithelium formation and fibronectin production. Moreover, HSEE increased the expression of genes involved in skin hydration and homeostasis. Specifically, aquaporin 3 and filaggrin genes were enhanced by 20 and 58%, respectively. Our data show that HSEE contains compounds capable of stimulating expression of biomarkers relevant to skin regeneration and hydration thereby counteracting molecular pathways leading to skin damage and aging. PMID:29333453

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

PubMed

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

2013-02-01

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

Effects of Histone Deacetylase Inhibitor (HDACi); Trichostatin-A (TSA) on the expression of housekeeping genes.

PubMed

Mogal, Ashish; Abdulkadir, Sarki A

2006-04-01

In quantitative RT-PCR (qRT-PCR), analysis of gene expression is dependent on normalization using housekeeping genes such as 18S rRNA, GAPDH and beta actin. However, variability in their expression has been reported to be caused by factors like drug treatment, pathological states and cell-cycle phase. An emerging area of cancer research focuses on identifying the role of epigenetic alterations such as histone modifications and DNA methylation in the initiation and progression of cancer. Histone acetylation is the best studied modification so far and has been probed through the use of histone deacetylase inhibitors (HDACi). Further, modulation of histone acetylation is currently being explored as a therapeutic strategy in the treatment of cancer and HDACis have shown promise in inhibiting tumorigenesis and metastasis. Trichostatin-A (TSA) is the most widely used HDACi. Therefore, we were driven to identify a suitable internal control for RT-PCR following TSA treatment. We performed quantitative RT-PCR analysis using mouse prostate tissue explants, human prostate cancer (LNCaP) cells and human breast cancer (T-47D and ZR-75-1) cells following TSA treatment. Expression of housekeeping genes including 18S rRNA, beta actin, GAPDH and ribosomal highly-basic 23-kDa protein (rb 23-kDa, RPL13A) were compared in vehicle versus TSA treated samples. Our results showed marked variations in 18S rRNA, beta actin mRNA and GAPDH mRNA levels in mouse prostate explants and a human prostate cancer (LNCaP) cell line following TSA treatment. Furthermore, in two human breast cancer cell lines (T-47D and ZR-75-1) 18S rRNA, beta actin mRNA and GAPDH mRNA levels varied significantly. However, RPL13A mRNA levels remained constant in all the conditions tested. Therefore, we recommend use of RPL13A as a standard for normalization during TSA treatment.

Genetic and Dietary Regulation of Glyburide Efflux by the Human Placental Breast Cancer Resistance Protein Transporter.

PubMed

Bircsak, Kristin M; Gupta, Vivek; Yuen, Poi Yu Sofia; Gorczyca, Ludwik; Weinberger, Barry I; Vetrano, Anna M; Aleksunes, Lauren M

2016-04-01

Glyburide is frequently used to treat gestational diabetes owing to its low fetal accumulation resulting from placental efflux by the breast cancer resistance protein (BCRP)/ABCG2 transporter. Here we sought to determine how exposure to the dietary phytoestrogen genistein and expression of a loss-of-function polymorphism in the ABCG2 gene (C421A) impacted the transport of glyburide by BCRP using stably transfected human embryonic kidney 293 (HEK) cells, human placental choriocarcinoma BeWo cells, and human placental explants. Genistein competitively inhibited the BCRP-mediated transport of (3)H-glyburide in both wild-type (WT) and C421A-BCRP HEK-expressing cells, with greater accumulation of (3)H-glyburide in cells expressing the C421A variant. In BeWo cells, exposure to genistein for 60 minutes increased the accumulation of (3)H-glyburide 30%-70% at concentrations relevant to dietary exposure (IC50 ∼180 nM). Continuous exposure of BeWo cells to genistein for 48 hours reduced the expression of BCRP mRNA and protein by up to 40%, which impaired BCRP transport activity. Pharmacologic antagonism of the estrogen receptor attenuated the genistein-mediated downregulation of BCRP expression, suggesting that phytoestrogens may reduce BCRP levels through this hormone receptor pathway in BeWo cells. Interestingly, genistein treatment for 48 hours did not alter BCRP protein expression in explants dissected from healthy term placentas. These data suggest that whereas genistein can act as a competitive inhibitor of BCRP-mediated transport, its ability to downregulate placental BCRP expression may only occur in choriocarcinoma cells. Overall, this research provides important mechanistic data regarding how the environment (dietary genistein) and a frequent genetic variant (ABCG2, C421A) may alter the maternal-fetal disposition of glyburide. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Protocols for Callus and Somatic Embryo Initiation for Hibiscus sabdariffa L. (Malvaceae): Influence of Explant Type, Sugar, and Plant Growth Regulators

USDA-ARS?s Scientific Manuscript database

A significant work on callus induction and somatic embryogenesis was realized for Hibiscus sabdariffa. Two genotypes (Hibiscus sabdariffa and Hibiscus sabdariffa var. altissima) two sugars (sucrose and glucose) and three concentrations (1 %, 2%, 3%) of each sugar, 3 explant types (root, hypocotyl, c...

Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins

USDA-ARS?s Scientific Manuscript database

A partial-thickness epidermal explant model was colonized with GFP-expressing S. aureus and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. Oxygen concentration in explants and H2O2 in media was quantified using microelectrodes. The re...

Study of breast implant rupture: MRI versus surgical findings.

PubMed

Vestito, A; Mangieri, F F; Ancona, A; Minervini, C; Perchinunno, V; Rinaldi, S

2012-09-01

This study evaluated the role of breast magnetic resonance (MR) imaging in the selective study breast implant integrity. We retrospectively analysed the signs of breast implant rupture observed at breast MR examinations of 157 implants and determined the sensitivity and specificity of the technique in diagnosing implant rupture by comparing MR data with findings at surgical explantation. The linguine and the salad-oil signs were statistically the most significant signs for diagnosing intracapsular rupture; the presence of siliconomas/seromas outside the capsule and/or in the axillary lymph nodes calls for immediate explantation. In agreement with previous reports, we found a close correlation between imaging signs and findings at explantation. Breast MR imaging can be considered the gold standard in the study of breast implants.

Acetylcholine causes rooting in leaf explants of in vitro raised tomato (Lycopersicon esculentum Miller) seedlings.

PubMed

Bamel, Kiran; Gupta, Shrish Chandra; Gupta, Rajendra

2007-05-30

The animal neurotransmitter acetylcholine (ACh) induces rooting and promotes secondary root formation in leaf explants of tomato (Lycopersicon esculentum Miller var. Pusa Ruby), cultured in vitro on Murashige and Skoog's medium. The roots originate from the midrib of leaf explants and resemble taproot. ACh at 10(-5) M was found to be the optimum over a wide range of effective concentrations between 10(-7) and 10(-3) M. The breakdown products, choline and acetate were ineffective even at 10(-3) M concentration. ACh appears to have a natural role in tomato rhizogenesis because exogenous application of neostigmine, an inhibitor of ACh hydrolysis, could mimic the effect of ACh. Neostigmine, if applied in combination with ACh, potentiated the ACh effect.

Regeneration of Acer caudatifolium Hayata plantlets from juvenile explants.

PubMed

Durkovic, J

2003-07-01

Juvenile and fully mature Acer caudatifolium Hayata explants were assayed for their organogenic capacity. A protocol for multiple shoot culture formation and in vitro plant regeneration was developed for juvenile axillary bud cultures. Mature explants failed in shoot regeneration. Shoot multiplication was achieved by releasing apical dominance of the single elongated shoot on woody plant medium (WPM) supplemented with 0.7 mg l(-1) 6-benzylaminopurine and 0.05 mg l(-1) alpha-naphthaleneacetic acid. The highest rooting percentage was recorded on half-strength WPM containing 1.0 mg l(-1) indole-3-butyric acid. Regenerated plantlets were successfully hardened to ex vitro conditions and continued to grow after transfer to soil. No morphological aberrations were observed in the regenerates.

Differential Gene Expression in Explanted Human Retinal Pigment Epithelial Cells 24-Hours Post-Exposure to 532 nm, 3.0 ns Pulsed Laser Light and 1064 nm, 170 ps Pulsed Laser Light 12-Hours Post-Exposure: Results Compendium

DTIC Science & Technology

2004-06-01

Additionally, we offer 3 conceptual cartoons outlining our vision for the future progres of laser bioeffects research, metabonomic risk assessment...future progress of laser bioeffects research, metabonomic risk assessment modeling and knowledge building from laser bioeffects data. BACKGROUND In the...our concepts of future laser bioeffects research directions (Figure 5), a metabonomic risk assessment model of laser tissue interaction (Figure 6

A very rare venous anomaly in a living liver donor: left hepatic venous connection to the right atrium.

PubMed

Uraz, S; Duran, C; Balci, D; Akin, B; Dayangac, M; Kurt, Z; Ayanoglu, O H; Killi, R; Yuzer, Y; Tokat, Y

2007-06-01

In humans, three main hepatic veins drain the liver into the inferior vena cava below the diaphragm. This report represents the first living donor liver that had a rare anatomic variation of the left hepatic vein draining directly to the right atrium, which was detected preoperatively by routine investigations of the living donor transplantation. This type of anomaly may present potentially fatal challenges to a donor operation if not detected preoperatively, especially when the left lobe is the choice for explantation.

Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

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

Kang, Khong Bee, E-mail: dmskkb@nccs.com.sg; Zhu Congju; Wong Yinling

Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, {gamma}-H{sub 2}AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival,more » {gamma}-H{sub 2}AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G{sub 2}/M arrest and increased {gamma}-H{sub 2}AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased {gamma}-H{sub 2}AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are resistant to irradiation-induced cytotoxicity, G{sub 2}/M arrest, and DNA DSBs, compared with nonstem glioma cells. Gefitinib differentially enhances radiosensitivity of stem-like gliomaspheres by reducing EGFR-Akt activation and DNA-PKcs expression, accompanied by enhanced irradiation-induced DNA DSBs and inhibition of DSB repair.« less

Gefitinib radiosensitizes stem-like glioma cells: inhibition of epidermal growth factor receptor-Akt-DNA-PK signaling, accompanied by inhibition of DNA double-strand break repair.

PubMed

Kang, Khong Bee; Zhu, Congju; Wong, Yin Ling; Gao, Qiuhan; Ty, Albert; Wong, Meng Cheong

2012-05-01

We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, γ-H(2)AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, γ-H(2)AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G(2)/M arrest and increased γ-H(2)AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased γ-H(2)AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Stem-like gliomaspheres are resistant to irradiation-induced cytotoxicity, G(2)/M arrest, and DNA DSBs, compared with nonstem glioma cells. Gefitinib differentially enhances radiosensitivity of stem-like gliomaspheres by reducing EGFR-Akt activation and DNA-PKcs expression, accompanied by enhanced irradiation-induced DNA DSBs and inhibition of DSB repair. Copyright © 2012 Elsevier Inc. All rights reserved.

Propofol inhibits gap junctions by attenuating sevoflurane-induced cytotoxicity against rat liver cells in vitro.

PubMed

Huang, Fei; Li, Shangrong; Gan, Xiaoliang; Wang, Ren; Chen, Zhonggang

2014-04-01

Liver abnormalities are seen in a small proportion of patients following anaesthesia with sevoflurane. To investigate whether the cytotoxicity of sevoflurane against rat liver cells was mediated by gap junction intercellular communications, and the effect of propofol on sevoflurane-induced cytotoxicity. Experimental study. The study was carried out in the central laboratory of The Third Affiliated Hospital, Sun Yat-sen University. BRL-3A rat liver cells. Immortal rat liver cells BRL-3A were grown at low and high density. Colony-forming assays were performed to determine clonogenic growth of these cells. To investigate the effect of oleamide and propofol on gap junction function, we measured fluorescence transmission between cells using parachute dye-coupling assays. Immunoblotting assays were performed to determine connexin32 and connexin43 expression. Our colony formation assays revealed that, in low-density culture, sevoflurane caused no apparent inhibition of clonogenic growth of BRL-3A cells. In high-density culture, 2.2 to 4.4% sevoflurane markedly inhibited clonogenic growth of BRL-3A cells with 67.6 (0.34)% and 61.2 (0.17)% of the cells being viable, respectively (P = 0.003 vs. low-density culture), suggesting cell density dependency of sevoflurane-induced cytotoxicity. Our colony formation assays revealed that propofol markedly attenuated the suppression by sevoflurane of the clonogenic growth of BRL-3A cells (viability: propofol and sevoflurane, 91.5 (0.014)% vs. sevoflurane, 56.6 (0.019)%; P <0.01). Blocking gap junctions with 10 μmol l oleamide significantly attenuated 4.4% sevoflurane-induced suppression with a viability of 83.6 ± 0.138% (oleamide and sevoflurane vs. sevoflurane, P < 0.01). Immunoblotting assays further showed that propofol (3.2 μg ml) markedly reduced CX32 levels and significantly inhibited gap junctional intercellular communications as revealed by parachute dye-coupling assays. Values are mean (SD). This study provides the first direct evidence that sevoflurane-induced cytotoxicity, which is mediated through gap junctions, is attenuated by propofol, possibly by its action on Cx32 homomeric or heteromeric complexes.

Radiation sensitivity of Merkel cell carcinoma cell lines

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

Leonard, J.H.; Ramsay, J.R.; Birrell, G.W.

1995-07-30

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT)more » assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.« less

Mechanism of Trypanosoma cruzi Placenta Invasion and Infection: The Use of Human Chorionic Villi Explants

PubMed Central

Fretes, Ricardo E.; Kemmerling, Ulrike

2012-01-01

Congenital Chagas disease, a neglected tropical disease, endemic in Latin America, is associated with premature labor and miscarriage. During vertical transmission the parasite Trypanosoma cruzi (T. cruzi) crosses the placental barrier. However, the exact mechanism of the placental infection remains unclear. We review the congenital transmission of T. cruzi, particularly the role of possible local placental factors that contribute to the vertical transmission of the parasite. Additionally, we analyze the different methods available for studying the congenital transmission of the parasite. In that context, the ex vivo infection with T. cruzi trypomastigotes of human placental chorionic villi constitutes an excellent tool for studying parasite infection strategies as well as possible local antiparasitic mechanisms. PMID:22701129

The study of ascorbate peroxidase, catalase and peroxidase during in vitro regeneration of Argyrolobium roseum.

PubMed

Habib, Darima; Chaudhary, Muhammad Fayyaz; Zia, Muhammad

2014-01-01

Here, we demonstrate the micropropagation protocol of Argyrolobium roseum (Camb.), an endangered herb exhibiting anti-diabetic and immune-suppressant properties, and antioxidant enzymes pattern is evaluated. Maximum callogenic response (60 %) was observed from leaf explant at 1.0 mg L(-1) 1-nephthalene acetic acid (NAA) and 0.5 mg L(-1) 6-benzyl aminopurine (BA) in Murashige and Skoog (MS) medium using hypocotyl and root explants (48 % each). Addition of AgNO3 and PVP in the culture medium led to an increase in callogenic response up to 86 % from leaf explant and 72 % from hypocotyl and root explants. The best shooting response was observed in the presence of NAA, while maximum shoot length and number of shoots were achieved based on BA-supplemented MS medium. The regenerated shoots were rooted and successfully acclimatized under greenhouse conditions. Catalase and peroxidase enzymes showed ascending pattern during in vitro plant development from seed while ascorbate peroxidase showed descending pattern. Totally reverse response of these enzymes was observed during callus induction from three different explants. During shoot induction, catalase and peroxidase increased at high rate while there was a mild reduction in ascorbate peroxidase activity. Catalase and peroxidase continuously increased; on the other hand, ascorbate peroxidase activity decreased during root development and acclimatization states. The protocol described here can be employed for the mass propagation and genetic transformation of this rare herb. This study also highlights the importance and role of ascorbate peroxidase, catalase, and peroxidase in the establishment of A. roseum in vitro culture through callogenesis and organogenesis.

VEGF as a Survival Factor in Ex Vivo Models of Early Diabetic Retinopathy.

PubMed

Amato, Rosario; Biagioni, Martina; Cammalleri, Maurizio; Dal Monte, Massimo; Casini, Giovanni

2016-06-01

Growing evidence indicates neuroprotection as a therapeutic target in diabetic retinopathy (DR). We tested the hypothesis that VEGF is released and acts as a survival factor in the retina in early DR. Ex vivo mouse retinal explants were exposed to stressors similar to those characterizing DR, that is, high glucose (HG), oxidative stress (OS), or advanced glycation end-products (AGE). Neuroprotection was provided using octreotide (OCT), a somatostatin analog, and pituitary adenylate cyclase activating peptide (PACAP), two well-documented neuroprotectants. Data were obtained with real-time RT-PCR, Western blot, ELISA, and immunohistochemistry. Apoptosis was induced in the retinal explants by HG, OS, or AGE treatments. At the same time, explants also showed increased VEGF expression and release. The data revealed that VEGF is released shortly after exposure of the explants to stressors and before the level of cell death reaches its maximum. Retinal cell apoptosis was inhibited by OCT and PACAP. At the same time, OCT and PACAP also reduced VEGF expression and release. Vascular endothelial growth factor turned out to be a protective factor for the stressed retinal explants, because inhibiting VEGF with a VEGF trap further increased cell death. These data show that protecting retinal neurons from diabetic stress also reduces VEGF expression and release, while inhibiting VEGF leads to exacerbation of apoptosis. These observations suggest that the retina in early DR releases VEGF as a prosurvival factor. Neuroprotective agents may decrease the need of VEGF production by the retina, therefore limiting the risk, in the long term, of pathologic angiogenesis.

Stress urinary incontinence in female neurological patients: long-term functional outcomes after artificial urinary sphincter (AMS 800TM ) implantation.

PubMed

Phé, Véronique; Léon, Priscilla; Granger, Benjamin; Denys, Pierre; Bitker, Marc-Olivier; Mozer, Pierre; Chartier-Kastler, Emmanuel

2017-03-01

To report the long-term functional outcomes of artificial urinary sphincter (AUS) implantation in female adult neurological patients suffering from stress urinary incontinence (SUI) due to sphincter deficiency. Female patients with neurological disease suffering from SUI due to sphincter deficiency who underwent AUS (AMS 800 TM ) implantation between 1984 and 2011 were included. Continence rate defined as no need for pads and survival rates of the device without needing explantation or revision using Kaplan-Meier curves were reported. Overall, 26 patients, median age 49.2 years (IQR 28.5-59.7) were included. The median follow-up time was 7.5 years (IQR 3.9-23.8). At the end of follow-up period, 15 patients (57.7%) still had their primary AUS. The AUS was explanted in five women because of infection or erosion. Survival rates, without AUS explantation were 90%, 84%, 84%, and 74% at 5, 10, 15, 20 years, respectively. Survival rates without AUS revision were 75%, 51%, 51%, and 51% at 5, 10, 15, 20 years, respectively. 71.4% of patients with AUS were continent. When considering the 26 initial patients, including the patients in whom the AUS was explanted, the continence rate was 57.7%. For treating neurogenic sphincter deficiency in the long term, the AMS 800 TM can offer a satisfying rate of continence to female patients, with a tolerable rate of explantation and revision. Neurourol. Urodynam. 36:764-769, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Long-term functional outcomes after artificial urinary sphincter implantation in men with stress urinary incontinence.

PubMed

Léon, Priscilla; Chartier-Kastler, Emmanuel; Rouprêt, Morgan; Ambrogi, Vanina; Mozer, Pierre; Phé, Véronique

2015-06-01

To evaluate long-term functional outcomes of artificial urinary sphincters (AUSs) and to determine how many men required explantation because of stress urinary incontinence (SUI) caused by sphincter deficiency after prostate surgery. Men who had undergone placement of an AUS (American Medical Systems AMS 800®) between 1984 and 1992 to relieve SUI caused by sphincter deficiency after prostate surgery were included. Continence, defined as no need for pads, was assessed at the end of the follow-up. Kaplan-Meier survival curves estimated the survival rate of the device without needing explantation or revision. In all, 57 consecutive patients were included with a median (interquartile range, IQR) age of 69 (64-72) years. The median (IQR) duration of follow-up was 15 (8.25-19.75) years. At the end of follow-up, 25 patients (43.8%) still had their primary AUS. The AUS was explanted in nine men because of erosion (seven) and infection (two). Survival rates, without AUS explantation, were 87%, 87%, 80%, and 80% at 5, 10, 15, and 20 years, respectively. Survival rates, without AUS revision, were 59%, 28%, 15%, and 5% at 5, 10, 15, and 20 years, respectively. At the end of the follow-up, in intention-to-treat analysis, 77.2% of patients were continent. In the long term (>10 years) the AMS 800 can offer a high rate of continence to men with SUI caused by sphincter deficiency, with a tolerable rate of explantation and revision. © 2014 The Authors. BJU International © 2014 BJU International.

Electron microscopic evaluation of a gold glaucoma micro shunt after explantation.

PubMed

Berk, Thomas A; Tam, Diamond Y; Werner, Liliana; Mamalis, Nick; Ahmed, Iqbal Ike K

2015-03-01

We present a case of an explanted gold glaucoma micro shunt (GMS Plus) and the subsequent light and electron microscopic analyses. The shunt was implanted in a patient with medically refractive glaucoma. The intraocular pressure (IOP) was stable at 12 mm Hg 6 months postoperatively but spiked to 26 mm Hg 6 months later; membranous growth was visible on the implant gonioscopically. A second gold micro shunt was placed 2 years after the first. The IOP was 7 mm Hg 1 week postoperatively but increased to 23 mm Hg 3 weeks later; similar membranous growth was visible on this implant. One of the shunts was explanted, and light and scanning electron microscopic analyses revealed encapsulation around the shunt exterior and connective tissue invasion of the microstructure. This represents the first electron microscopic analysis of an explanted gold glaucoma micro shunt and the first unequivocal images of the fibrotic pseudo-capsule traversing its microchannels and fenestrations. Dr. Ahmed is a consultant to and has received research grants from Solx, Inc. No other author has a financial or proprietary interest in any material or method mentioned. Copyright © 2015 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Simple explant culture of the embryonic chicken retina with long-term preservation of photoreceptors.

PubMed

Thangaraj, Gopenath; Greif, Alexander; Layer, Paul G

2011-10-01

Structurally stable in vitro-model systems are indispensible to analyse neural development during embryogenesis, follow cellular differentiation and evaluate neurotoxicological or growth factor effects. Here we describe a three-dimensional, long-term in vitro-culture system of the embryonic chick retina which supports photoreceptor development. Retinal tissue was isolated from E6 chick eye, and cultured as explants by continuous orbital rotation to allow free floatation without any supporting materials. Young stage (E6) immature retinas were cultured for various time periods in order to follow the differentiation of cell types and plexiform layers by immunocytochemical methods. These explants could be cultured for at least 2-3 weeks with remarkable retention of retinal architecture. Interestingly, photoreceptors developed in the absence of pigment epithelium. Electron microscopic studies revealed formation of structures resembling photoreceptor outer segments, a feature not reported previously. Thus, the verification of photoreceptors, Müller cells, inner retinal cells and the inner plexiform layer described in our study establishes this explant culture as a valuable in vivo-like model system. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Comparison of somatic embryogenesis in Medicago sativa and Medicago truncatula.

PubMed

Hoori, F; Ehsanpour, A A; Mostajeran, A

2007-02-01

In this study, the regeneration through embryogenesis of two species of Medicago were studied. Seeds of Medicago sativa cv. Rehnani and M. truncatula line A17 were grown on MS medium. After 4-6 weeks, segments of leaf and stem from two species were transferred to MS medium containing 2 mg L(-1) NAA, 2,4-D and Kinetin. The results indicated that callus formation from leaf explants of M. sativa was higher than M. trancatula. In the next stage, media with different combinations of auxin, cytokinin or ethinyl estradiol were provided for regeneration. Then in two stages, explants of leaf and stem of two species were transferred on these media. Results after 3-6 weeks showed that in medium containing NAA and TDZ, stem pieces ofM. sativa produced shoots while leaf pieces on NAA and ethinyl estradiol formed roots. Leaf explants of M. truncatula in the medium containing NAA and BAP, produced somatic embryos. Also in media with auxin and ethinyl estradiol, somatic embryos were formed on calli of two species. Ethinyl estradiol and auxin together can induce somatic embryogenesis and root production on calli and stem or leaf explants.

3D tissue-like assemblies: A novel approach to investigate virus-cell interactions.

PubMed

Goodwin, Thomas J; McCarthy, Maureen; Cohrs, Randall J; Kaufer, Benedikt B

2015-11-15

Virus-host cell interactions are most commonly analyzed in cells maintained in vitro as two-dimensional tissue cultures. However, these in vitro conditions vary quite drastically from the tissues that are commonly infected in vivo. Over the years, a number of systems have been developed that allow the establishment of three-dimensional (3D) tissue structures that have properties similar to their in vivo 3D counterparts. These 3D systems have numerous applications including drug testing, maintenance of large tissue explants, monitoring migration of human lymphocytes in tissues, analysis of human organ tissue development and investigation of virus-host interactions including viral latency. Here, we describe the establishment of tissue-like assemblies for human lung and neuronal tissue that we infected with a variety of viruses including the respiratory pathogens human parainfluenza virus type 3 (PIV3), respiratory syncytial virus (RSV) and SARS corona virus (SARS-CoV) as well as the human neurotropic herpesvirus, varicella-zoster virus (VZV). Copyright © 2015 Elsevier Inc. All rights reserved.

3D Tissue-Like Assemblies: A Novel Approach to Investigate Virus-Cell Interactions

PubMed Central

Goodwin, Thomas J.; McCarthy, Maureen; Cohrs, Randall J.; Kaufer, Benedikt B.

2017-01-01

Virus-host cell interactions are most commonly analyzed in cells maintained in vitro as two-dimensional tissue cultures. However, these in vitro conditions vary quite drastically from the tissues that are commonly infected in vivo. Over the years, a number of systems have been developed that allow the establishment of three-dimensional (3D) tissue structures that have properties similar to their in vivo 3D counterparts. These 3D systems have numerous applications including drug testing, maintenance of large tissue explants, monitoring migration of human lymphocytes in tissues, analysis of human organ tissue development and investigation of virus-host interactions including viral latency. Here, we describe the establishment of tissue-like assemblies for human lung and neuronal tissue that we infected with a variety of viruses including the respiratory pathogens human parainfluenza virus type 3 (PIV3), respiratory syncytial virus (RSV) and SARS corona virus (SARS-CoV) as well as the human neurotropic herpesvirus, varicella-zoster virus (VZV) PMID:25986169

Regeneration of Cuphea wrightii (Peyr 651) and fertile C.wrightii C. tolucana hybrids from leaf explants.

PubMed

Przybecki, Z; Olejniczak, J; Adamska, E

2001-01-01

Callus was obtained from leaf explants of Cupea wrightii and Cuphea wrightii x Cuphea tolucana hybrid plants, and the plants were later regenerated. C. tolucana explants were capable of forming callus, but not of regenerating. In order to obtain callus from C. wrightii and the hybrid plants, the addition of BAP to the medium was necessary, whereas in the case of C. tolucana auxin was needed. The regeneration of the plants did not require auxin, and both forms (C. wrightii and the hybrids) regenerated in the same medium. The regeneration yield came to around 12 plants from the callus of one harvest. Some of the callus from the hybrids was subjected to colchicine treatment, which increased the number of fully fertile regenerants from 1% to almost 20%.

Hemoglobin promotes somatic embryogenesis in peanut cultures.

PubMed

Jayabalan, N; Anthony, P; Davey, M R; Power, J B; Lowe, K C

2004-02-01

Critical parameters influencing somatic embryogenesis include growth regulators and oxygen supply. Consequently, the present investigation has focused on optimization of a somatic embryogenic system for peanut (Arachis hypogaea L.) through media supplementation with the auxin, picloram. The latter at 30 mg L(-1) was optimal for inducing regeneration of somatic embryos from cultured explants of zygotic embryos. In contrast, somatic embryogenesis did not occur in the absence of this growth regulator. An assessment has also been made of the beneficial effect on somatic embryogenesis and plant regeneration of the commercial hemoglobin (Hb) solution, Erythrogen. Hemoglobin at 1:50 and 1:100 (v:v) stimulated increases in mean fresh weight (up to a maximum of 57% over control), mean number of explants producing somatic embryos (15%) and mean number of somatic embryos per explant (29%).

Evaluation of Radioresponse and Radiosensitizers in Glioblastoma Organotypic Cultures.

PubMed

Bayin, N Sumru; Ma, Lin; Placantonakis, Dimitris G; Barcellos-Hoff, Mary Helen

2018-01-01

Glioblastoma (GBM), a deadly primary brain malignancy, manifests pronounced radioresistance. Identifying agents that improve the sensitivity of tumor tissue to radiotherapy is critical for improving patient outcomes. The response to ionizing radiation is regulated by both cell-intrinsic and -extrinsic mechanisms. In particular, the tumor microenvironment is known to promote radioresistance in GBM. Therefore, model systems used to test radiosensitizing agents need to take into account the tumor microenvironment. We recently showed that GBM explant cultures represent an adaptable ex vivo platform for rapid and personalized testing of radiosensitizers. These explants preserve the cellular composition and tissue architecture of parental patient tumors and therefore capture the microenvironmental context that critically determines the response to radiotherapy. This chapter focuses on the detailed protocol for testing candidate radiosensitizing agents in GBM explants.

Genetic transformation protocols using zygotic embryos as explants: an overview.

PubMed

Tahir, Muhammad; Waraich, Ejaz A; Stasolla, Claudio

2011-01-01

Genetic transformation of plants is an innovative research tool which has practical significance for the development of new and improved genotypes or cultivars. However, stable introduction of genes of interest into nuclear genomes depends on several factors such as the choice of target tissue, the method of DNA delivery in the target tissue, and the appropriate method to select the transformed plants. Mature or immature zygotic embryos have been a popular choice as explant or target tissue for genetic transformation in both angiosperms and gymnosperms. As a result, considerable protocols have emerged in the literature which have been optimized for various plant species in terms of transformation methods and selection procedures for transformed plants. This article summarizes the recent advances in plant transformation using zygotic embryos as explants.

Atrial fibrillation driven by micro-anatomic intramural re-entry revealed by simultaneous sub-epicardial and sub-endocardial optical mapping in explanted human hearts.

PubMed

Hansen, Brian J; Zhao, Jichao; Csepe, Thomas A; Moore, Brandon T; Li, Ning; Jayne, Laura A; Kalyanasundaram, Anuradha; Lim, Praise; Bratasz, Anna; Powell, Kimerly A; Simonetti, Orlando P; Higgins, Robert S D; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul M L; Weiss, Raul; Hummel, John D; Fedorov, Vadim V

2015-09-14

The complex architecture of the human atria may create physical substrates for sustained re-entry to drive atrial fibrillation (AF). The existence of sustained, anatomically defined AF drivers in humans has been challenged partly due to the lack of simultaneous endocardial-epicardial (Endo-Epi) mapping coupled with high-resolution 3D structural imaging. Coronary-perfused human right atria from explanted diseased hearts (n = 8, 43-72 years old) were optically mapped simultaneously by three high-resolution CMOS cameras (two aligned Endo-Epi views (330 µm2 resolution) and one panoramic view). 3D gadolinium-enhanced magnetic resonance imaging (GE-MRI, 80 µm3 resolution) revealed the atrial wall structure varied in thickness (1.0 ± 0.7-6.8 ± 2.4 mm), transmural fiber angle differences, and interstitial fibrosis causing transmural activation delay from 23 ± 11 to 43 ± 22 ms at increased pacing rates. Sustained AF (>90 min) was induced by burst pacing during pinacidil (30-100 µM) perfusion. Dual-sided sub-Endo-sub-Epi optical mapping revealed that AF was driven by spatially and temporally stable intramural re-entry with 107 ± 50 ms cycle length and transmural activation delay of 67 ± 31 ms. Intramural re-entrant drivers were captured primarily by sub-Endo mapping, while sub-Epi mapping visualized re-entry or 'breakthrough' patterns. Re-entrant drivers were anchored on 3D micro-anatomic tracks (15.4 ± 2.2 × 6.0 ± 2.3 mm2, 2.9 ± 0.9 mm depth) formed by atrial musculature characterized by increased transmural fiber angle differences and interstitial fibrosis. Targeted radiofrequency ablation of the tracks verified these re-entries as drivers of AF. Integrated 3D structural-functional mapping of diseased human right atria ex vivo revealed that the complex atrial microstructure caused significant differences between Endo vs. Epi activation during pacing and sustained AF driven by intramural re-entry anchored to fibrosis-insulated atrial bundles. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

Atrial fibrillation driven by micro-anatomic intramural re-entry revealed by simultaneous sub-epicardial and sub-endocardial optical mapping in explanted human hearts

PubMed Central

Hansen, Brian J.; Zhao, Jichao; Csepe, Thomas A.; Moore, Brandon T.; Li, Ning; Jayne, Laura A.; Kalyanasundaram, Anuradha; Lim, Praise; Bratasz, Anna; Powell, Kimerly A.; Simonetti, Orlando P.; Higgins, Robert S.D.; Kilic, Ahmet; Mohler, Peter J.; Janssen, Paul M.L.; Weiss, Raul; Hummel, John D.; Fedorov, Vadim V.

2015-01-01

Aims The complex architecture of the human atria may create physical substrates for sustained re-entry to drive atrial fibrillation (AF). The existence of sustained, anatomically defined AF drivers in humans has been challenged partly due to the lack of simultaneous endocardial–epicardial (Endo–Epi) mapping coupled with high-resolution 3D structural imaging. Methods and results Coronary-perfused human right atria from explanted diseased hearts (n = 8, 43–72 years old) were optically mapped simultaneously by three high-resolution CMOS cameras (two aligned Endo–Epi views (330 µm2 resolution) and one panoramic view). 3D gadolinium-enhanced magnetic resonance imaging (GE-MRI, 80 µm3 resolution) revealed the atrial wall structure varied in thickness (1.0 ± 0.7–6.8 ± 2.4 mm), transmural fiber angle differences, and interstitial fibrosis causing transmural activation delay from 23 ± 11 to 43 ± 22 ms at increased pacing rates. Sustained AF (>90 min) was induced by burst pacing during pinacidil (30–100 µM) perfusion. Dual-sided sub-Endo–sub-Epi optical mapping revealed that AF was driven by spatially and temporally stable intramural re-entry with 107 ± 50 ms cycle length and transmural activation delay of 67 ± 31 ms. Intramural re-entrant drivers were captured primarily by sub-Endo mapping, while sub-Epi mapping visualized re-entry or ‘breakthrough’ patterns. Re-entrant drivers were anchored on 3D micro-anatomic tracks (15.4 ± 2.2 × 6.0 ± 2.3 mm2, 2.9 ± 0.9 mm depth) formed by atrial musculature characterized by increased transmural fiber angle differences and interstitial fibrosis. Targeted radiofrequency ablation of the tracks verified these re-entries as drivers of AF. Conclusions Integrated 3D structural–functional mapping of diseased human right atria ex vivo revealed that the complex atrial microstructure caused significant differences between Endo vs. Epi activation during pacing and sustained AF driven by intramural re-entry anchored to fibrosis-insulated atrial bundles. PMID:26059724

Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors.

PubMed

Anaka, Matthew; Hudson, Christopher; Lo, Pu-Han; Do, Hongdo; Caballero, Otavia L; Davis, Ian D; Dobrovic, Alexander; Cebon, Jonathan; Behren, Andreas

2013-10-11

Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma.

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells.

PubMed

Fedr, Radek; Pernicová, Zuzana; Slabáková, Eva; Straková, Nicol; Bouchal, Jan; Grepl, Michal; Kozubík, Alois; Souček, Karel

2013-05-01

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. Copyright © 2013 International Society for Advancement of Cytometry.

Regression of endometrial explants in a rat model of endometriosis treated with melatonin.

PubMed

Güney, Mehmet; Oral, Baha; Karahan, Nermin; Mungan, Tamer

2008-04-01

To determine the antioxidant, antiinflammatory, and immunomodulatory effects of melatonin on endometrial explants, the distribution of cyclooxygenase-2 (COX-2), the activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), and levels of malondialdehyde (MDA) in the rat endometriosis model. Prospective, placebo-controlled experimental study. Experimental surgery laboratory in a university department. Twenty-five rats with experimentally induced endometriosis. Endometriosis was surgically induced in 25 rats by transplanting an autologous fragment of endometrial tissue onto the inner surface of the abdominal wall. Four weeks later, three rats were killed and the remaining 22 rats given second-look laparotomies to identify and measure ectopic uterine tissue in three dimensions. After the second laparotomy, 4 weeks of vehicle and melatonin treatment were administered, then all of the rats were given a third laparotomy and killed. The volume and weight of the implants were measured. The remaining rats were randomly divided into two groups. In control group (group 1; n = 11) no medication was given. To the rats in melatonin-treated group (group 2; n = 11), 10 mg/kg a day of melatonin was administered intraperitoneally. Four weeks later, after the second laparotomy, the endometrial explants were reevaluated morphologically, and COX-2 expression was evaluated immunohistochemically and histologically. In addition, endometrial explants were analyzed for the antioxidant enzymes SOD, CAT, and MDA, a marker of lipid peroxidation. A scoring system was used to evaluate expression of COX-2 and preservation of epithelia. The pretreatment and posttreatment volumes within the control group were 135.9 +/- 31.5 and 129.4 +/- 28.7, respectively. The mean explant volume was 141.4 +/- 34.4 within the melatonin group before the treatment and 42.9 +/- 14.0 after 4 weeks of treatment. There was a statistically significant difference in spherical volumes (129.4 +/- 28.7 versus 42.9 +/- 14.0 mm(3)) of explant weights (155.8 +/- 27.1 versus 49.6 +/- 19.5 mg) and COX-2 positivity (91% versus 18.1%) between groups after the third laparotomy. In the melatonin-treated group, the endometrial explant levels of MDA statistically significantly decreased and activities of SOD and CAT significantly increased when compared with the control group. The epithelia showed statistically significantly better preservation in the control group when compared with the melatonin-treated group (2.54 +/- 0.52 versus 0.63 +/- 0.50). Melatonin causes regression and atrophy of the endometriotic lesions in rats.

Exosomes are released by bystander cells exposed to radiation-induced biophoton signals: Reconciling the mechanisms mediating the bystander effect

PubMed Central

Fernandez-Palomo, Cristian; McNeill, Fiona E.; Seymour, Colin B.; Rainbow, Andrew J.; Mothersill, Carmel E.

2017-01-01

Objective The objective of our study was to explore a possible molecular mechanism by which ultraviolet (UV) biophotons could elicit bystander responses in reporter cells and resolve the problem of seemingly mutually exclusive mechanisms of a physical UV signal & a soluble factor-mediated bystander signal. Methods The human colon carcinoma cell line, HCT116 p53 +/+, was directly irradiated with 0.5 Gy tritium beta particles to induce ultraviolet biophoton emission. Bystander cells were not directly irradiated but were exposed to the emitted UV biophotons. Medium was subsequently harvested from UV-exposed bystander cells. The exosomes extracted from this medium were incubated with reporter cell populations. These reporter cells were then assayed for clonogenic survival and mitochondrial membrane potential with and without prior treatment of the exosomes with RNase. Results Clonogenic cell survival was significantly reduced in reporter cells incubated with exosomes extracted from cells exposed to secondarily-emitted UV. These exosomes also induced significant mitochondrial membrane depolarization in receiving reporter cells. Conversely, exosomes extracted from non-UV-exposed cells did not produce bystander effects in reporter cells. The treatment of exosomes with RNase prior to their incubation with reporter cells effectively abolished bystander effects in reporter cells and this suggests a role for RNA in mediating the bystander response elicited by UV biophotons and their produced exosomes. Conclusion This study supports a role for exosomes released from UV biophoton-exposed bystander cells in eliciting bystander responses and also indicates a reconciliation between the UV-mediated bystander effect and the bystander effect which has been suggested in the literature to be mediated by soluble factors. PMID:28278290

Concomitant administration of radiation with eribulin improves the survival of mice harboring intracerebral glioblastoma.

PubMed

Miki, Shunichiro; Imamichi, Shoji; Fujimori, Hiroaki; Tomiyama, Arata; Fujimoto, Kenji; Satomi, Kaishi; Matsushita, Yuko; Matsuzaki, Sanae; Takahashi, Masamichi; Ishikawa, Eiichi; Yamamoto, Tetsuya; Matsumura, Akira; Mukasa, Akitake; Nishikawa, Ryo; Masutomi, Kenkichi; Narita, Yoshitaka; Masutani, Mitsuko; Ichimura, Koichi

2018-05-14

Glioblastoma is the most common and devastating type of malignant brain tumor. We recently found that eribulin suppresses glioma growth in vitro and in vivo and that eribulin is efficiently transferred into mouse brain tumors at a high concentration. Eribulin is a non-taxane microtubule inhibitor approved for breast cancer and liposarcoma. Cells arrested in M-phase by chemotherapeutic agents such as microtubule inhibitors are highly sensitive to radiation-induced DNA damage. Several recent case reports demonstrated the clinical benefits of eribulin combined with radiation therapy for metastatic brain tumors. In this study, we investigated the efficacy of a combined eribulin and radiation treatment on human glioblastoma cells. The glioblastoma cell lines U87MG, U251MG, U118MG, and SJ28 cells, a patient-derived sphere culture cell line, were used to determine the radiosensitizing effect of eribulin using western blotting, flow cytometry, and clonogenic assay. Subcutaneous and intracerebral glioma xenografts were generated in mice to assess the efficacy of the combined treatment. The combination of eribulin and radiation enhanced DNA damage in vitro. The clonogenic assay of U87MG demonstrated the radiosensitizing effect of eribulin. The concomitant eribulin and radiation treatment significantly prolonged the survival of mice harboring intracerebral glioma xenografts compared with eribulin or radiation alone (p<0.0001). In addition, maintenance administration of eribulin after the concomitant treatment further controlled brain tumor growth. Aberrant microvasculature was decreased in these tumors. Concomitant treatment with eribulin and radiation followed by maintenance administration of eribulin may serve as a novel therapeutic strategy for glioblastomas. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Protective Role of Hsp27 Protein Against Gamma Radiation-Induced Apoptosis and Radiosensitization Effects of Hsp27 Gene Silencing in Different Human Tumor Cells

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

Aloy, Marie-Therese; Hospices Civils de Lyon, Service de Radiotherapie, Centre Hospitalier Lyon-Sud, Pierre-Benite; Hadchity, Elie

Purpose: The ability of heat shock protein 27 (Hsp27) to protect cells from stressful stimuli and its increased levels in tumors resistant to anticancer therapeutics suggest that it may represent a target for sensitization to radiotherapy. In this study, we investigate the protective role of Hsp27 against radiation-induced apoptosis and the effect of its attenuation in highly expressing radioresistant cancer cell lines. Methods and Materials: We examined clonogenic death and the kinetics of apoptotic events in different tumor cell lines overexpressing or underexpressing Hsp27 protein irradiated with photons. The radiosensitive Jurkat cell line, which does not express Hsp27 constitutively ormore » in response to {gamma}-rays, was stably transfected with Hsp27 complementary DNA. Attenuation of Hsp27 expression was accomplished by antisense or RNAi (interfering RNA) strategies in SQ20B head-and-neck squamous carcinoma, PC3 prostate cancer, and U87 glioblastoma radioresistant cells. Results: We measured concentration-dependent protection against the cytotoxic effects of radiation in Jurkat-Hsp27 cells, which led to a 50% decrease in apoptotic cells at 48 hours in the highest expressing cells. Underlying mechanisms leading to radiation resistance involved a significant increase in glutathione levels associated with detoxification of reactive oxygen species, a delay in mitochondrial collapse, and caspase activation. Conversely, attenuation of Hsp27 in SQ20B cells, characterized by their resistance to apoptosis, sensitizes cells to irradiation. This was emphasized by increased apoptosis, decreased glutathione basal level, and clonogenic cell death. Sensitization to irradiation was confirmed in PC3 and U87 radioresistant cells. Conclusion: Hsp27 gene therapy offers a potential adjuvant to radiation-based therapy of resistant tumors.« less

MMPs-Mediated ECM Remodeling

PubMed

Mali, Aniket V; Joshi, Asavari A; Hegde, Mahabaleshwar V; Kadam, Shivajirao S

2017-04-01

Background: To enhance their own survival, tumor cells can manipulate their microenvironment through remodeling of the extra cellular matrix (ECM). The urokinase-type plasminogen activator (uPA) system catalyzes plasmin production which further mediates activation of matrix metalloproteinases (MMPs) and plays an important role in breast cancer invasion and metastasis through ECM remodeling. This provides a potential target for therapeutic intervention of breast cancer treatment. Enterolactone (EL) is derived from dietary flax lignans in the human body and is known to have anti-breast cancer activity. We here investigated molecular and cellular mechanisms of EL action on the uPA-plasmin- MMPs system. Methods: MTT and trypan blue dye exclusion assays, anchorage-dependent clonogenic assays and wound healing assays were carried out to study effects on cell proliferation and viability, clonogenicity and migration capacity, respectively. Real-time PCR was employed to study gene expression and gelatin zymography was used to assess MMP-2 and MMP-9 activities. All data were statistically analysed and presented as mean ± SEM values. Results: All the findings collectively demonstrated anticancer and antimetastatic potential of EL with antiproliferative, antimigratory and anticlonogenic cellular mechanisms. EL was found to exhibit multiple control of plasmin activation by down-regulating uPA expression and also up-regulating its natural inhibitor, PAI-1, at the mRNA level. Further, EL was found to down-regulate expression of MMP-2 and MMP-9 genes, and up-regulate TIMP-1 and TIMP-2; natural inhibitors of MMP-2 and MMP-9, respectively. This may be as a consequence of inhibition of plasmin activation, resulting in robust control over migration and invasion of breast cancer cells during metastasis. Conclusions: EL suppresses proliferation, migration and metastasis of MDA-MB-231 breast cancer cells by inhibiting induced ECM remodeling by the ‘uPA-plasmin-MMPs system’. Creative Commons Attribution License

Cryopreservation of cord blood CD34+ cells before or after thrombopoietin expansion differentially affects early platelet recovery in NOD SCID mice.

PubMed

van Hensbergen, Yvette; van der Garde, Mark; Brand, Anneke; Slot, Manon C; de Graaf-Dijkstra, Alice; Watt, Suzanne; Zwaginga, Jaap Jan

2015-07-01

Expansion of human cord blood (CB) CD34+ cells with thrombopoietin (TPO) can accelerate delayed platelet (PLT) recovery after transplantation into immunodeficient mice. Clinical implementation, however, will depend on practical and effective protocols. The best timing of TPO expansion in relation to cryopreservation in this respect is unknown. In this study, we evaluated whether the order of cryopreservation and TPO expansion affected the expansion rate and numbers of clonogenic hematopoietic progenitor cells in vitro or PLT and longer-term hematopoietic repopulation in NOD SCID mice in vivo. Our results demonstrate higher expansion rates and the generation of higher numbers of multilineage and megakaryocytic progenitors (granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units and megakaryocyte colony-forming units) in vitro when freshly isolated CB CD34+ cells are first cultured with TPO and then cryopreserved and thawed as compared to TPO expansion after CD34+ cell cryopreservation. In contrast, the cells produced with the latter strategy showed higher expression of CD62L and a superior stromal cell-derived factor-1α-mediated migration. This might play a role in an also observed superior early PLT recovery after transplantation of these cells into NOD SCID mice. The hematopoietic engraftment in the marrow 6 weeks after transplantation was not different between the two strategies. Although TPO expansion before cryopreservation would yield higher nucleated cell and clonogenic myeloid and megakaryocyte cell numbers and enable earlier availability, CB TPO expansion after cryopreservation is likely to be clinically more effective, despite the lower number of cells obtained after expansion. Moreover, the latter strategy is logistically more feasible. © 2015 AABB.

Adamantanyl-Histone Deacetylase Inhibitor H6CAHA Exhibits Favorable Pharmacokinetics and Augments Prostate Cancer Radiation Sensitivity

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

Konsoula, Zacharoula; Cao Hong; Velena, Alfredo

2011-04-01

Purpose: To evaluate pharmacological properties of H6CAHA, an adamantyl-hydroxamate histone deacetylase inhibitor, and to investigate its effect on prostate cancer cells following exposure to {gamma}-radiation in vitro and in vivo. Methods and Materials: H6CAHA was assessed for in vitro solubility, lipophilicity and growth inhibition, and in vivo plasma pharmacokinetics. The effect of H6CAHA on radiation clonogenic survival and DNA damage repair was evaluated in human prostate cancer (PC3, DU145, LNCaP) and nonmalignant control epithelial (RWPE1 and 267B1) cell lines. The effect of this agent on the growth of prostate cancer xenografts was also assessed in mice. Results: H6CAHA demonstrated goodmore » solubility and permeability profiles and preferentially inhibited the growth of prostate cancer cells over nonmalignant cells. Plasma pharmacokinetics revealed that the area under the curve of H6CAHA was 8.08 {+-} 0.91 {mu}M x h, and its half-life was 11.17 {+-} 0.87 h. Radiation clonogenic assays revealed that H6CAHA decreased the survival of prostate cancer cells at the dose that exerted limited effect on normal cells. Concomitantly, delayed DNA damage repair following combination treatment was evident in cancer cells, indicated by the prolonged appearance of {gamma}H2AX and Rad51 foci and suppression of DNA damage repair genes (ATM, BRCA1, and BRCA2). Combined modality of H6CAHA (daily intraperitoneal injections for 10 days) with {gamma}-radiation (10 x 2 Gy) completely blocked the growth of PC3 tumor xenografts (p < 0.001) over 60 days. Conclusion: These results support the potential therapeutic value of H6CAHA in combination with radiation and support the rationale for further clinical investigation.« less

A new strategy for umbilical cord blood collection developed at the first Colombian public cord blood bank increases total nucleated cell content.

PubMed

Vanegas, Diana; Triviño, Lady; Galindo, Cristian; Franco, Leidy; Salguero, Gustavo; Camacho, Bernardo; Perdomo-Arciniegas, Ana-María

2017-09-01

The total nucleated cell dosage of umbilical cord blood (UCB) is an important factor in determining successful allogeneic hematopoietic stem cell transplantation after a minimum human leukocyte antigen donor-recipient match. The northern South American population is in need of a new-generation cord blood bank that cryopreserves only units with high total nucleated cell content, thereby increasing the likelihood of use. Colombia set up a public cord blood bank in 2014; and, as a result of its research for improving high total nucleated cell content, a new strategy for UCB collection was developed. Data from 2933 collected and 759 cryopreserved cord blood units between 2014 and 2015 were analyzed. The correlation of donor and collection variables with cellularity was evaluated. Moreover, blood volume, cell content, CD34+ count, clonogenic capacity, and microbial contamination were assessed comparing the new method, which combines in utero and ex utero techniques, with the conventional strategies. Multivariate analysis confirmed a correlation between neonatal birth weight and cell content. The new collection method increased total nucleated cell content in approximately 26% and did not alter pre-cryopreservation and post-thaw cell recovery, viability, or clonogenic ability. Furthermore, it showed a remarkably low microbial contamination rate (1.2%). The strategy for UCB collection developed at the first Colombian public cord blood bank increases total nucleated cell content and does not affect unit quality. The existence of this bank is a remarkable breakthrough for Latin-American patients in need of this kind of transplantation. © 2017 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

Silibinin inhibits hypoxia-induced HIF-1α-mediated signaling, angiogenesis and lipogenesis in prostate cancer cells: In vitro evidence and in vivo functional imaging and metabolomics

PubMed Central

Deep, Gagan; Kumar, Rahul; Nambiar, Dhanya K.; Jain, Anil K.; Ramteke, Anand M.; Serkova, Natalie J.; Agarwal, Chapla; Agarwal, Rajesh

2017-01-01

Hypoxia is associated with aggressive phenotype and poor prognosis in prostate cancer (PCa) patients suggesting that PCa growth and progression could be controlled via targeting hypoxia-induced signaling and biological effects. Here, we analyzed silibinin (a natural flavonoid) efficacy to target cell growth, angiogenesis and metabolic changes in human PCa, LNCaP and 22Rv1 cells under hypoxic condition. Silibinin treatment inhibited the proliferation, clonogenicity and endothelial cells tube formation by hypoxic (1% O2) PCa cells. Interestingly, hypoxia promoted a lipogenic phenotype in PCa cells via activating acetyl-Co A carboxylase (ACC) and fatty acid synthase (FASN) that was inhibited by silibinin treatment. Importantly, silibinin treatment strongly decreased hypoxia-induced HIF-1α expression in PCa cells together with a strong reduction in hypoxia-induced NADPH oxidase (NOX) activity. HIF-1α overexpression in LNCaP cells significantly increased the lipid accumulation and NOX activity; however, silibinin treatment reduced HIF-1α expression, lipid levels, clonogenicity and NOX activity even in HIF-1α overexpressing LNCaP cells. In vivo, silibinin feeding (200 mg/kg body weight) to male nude mice with 22Rv1 tumors, specifically inhibited tumor vascularity (measured by dynamic contrast-enhanced MRI) resulting in tumor growth inhibition without directly inducing necrosis (as revealed by diffusion-weighted MRI). Silibinin feeding did not significantly affect tumor glucose uptake measured by FDG-PET; however, reduced the lipid synthesis measured by quantitative 1H-NMR metabolomics. IHC analyses of tumor tissues confirmed that silibinin feeding decreased proliferation and angiogenesis as well as reduced HIF-1α, FASN and ACC levels. Together, these findings further support silibinin usefulness against PCa through inhibiting hypoxia-induced signaling. PMID:27533043

Evaluation of preventive and therapeutic activity of novel non-steroidal anti-inflammatory drug, CG100649, in colon cancer: Increased expression of TNF-related apoptosis-inducing ligand receptors enhance the apoptotic response to combination treatment with TRAIL.

PubMed

Woo, Jong Kyu; Kang, Ju-Hee; Jang, Yeong-Su; Ro, Seonggu; Cho, Joong Myung; Kim, Hwan-Mook; Lee, Sang-Jin; Oh, Seung Hyun

2015-04-01

Non-steroidal anti-inflammatory drugs (NSAIDs) have been suggested as the potential new class of preventive or therapeutic antitumor agents. The aim of the present study was to evaluate the antitumor activity of the novel NSAID, CG100649. CG100649 is a novel NSAID dual inhibitor for COX-2 and carbonic anhydrase (CA)-I/-II. In the present study, we investigated the alternative mechanism by which CG100649 mediated suppression of the colon cancer growth and development. The anchorage‑dependent and -independent clonogenic assay showed that CG100649 inhibited the clonogenicity of human colon cancer cells. The flow cytometric analysis showed that CG100649 induced the G2/M cell cycle arrest in colon cancer cells. Animal studies showed that CG100649 inhibited the tumor growth in colon cancer xenograft in nude mice. Furthermore, quantitative PCR and FACS analysis demonstrated that CG100649 upregulated the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors (DR4 and DR5) but decreased the expression of decoy receptors (DcR1 and DcR2) in colon cancer cells. The results showed that CG100649 treatment sensitized TRAIL‑mediated growth suppression and apoptotic cell death. The combination treatment resulted in significant repression of the intestinal polyp formation in APCmin/+ mice. Our data clearly demonstrated that CG100649 contains preventive and therapeutic activity for colon cancer. The present study may be useful for identification of the potential benefit of the NSAID CG100649, for the achievement of a better treatment response in colon cancer.

A Mitochondria-Targeted Nitroxide/Hemigramicidin S Conjugate Protects Mouse Embryonic Cells Against Gamma Irradiation

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

Jiang Jianfei; Belikova, Natalia A.; Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA

2008-03-01

Purpose: To evaluate the in vitro radioprotective effect of the mitochondria-targeted hemigramicidin S-conjugated 4-amino-2,2,6,6-tetramethyl-piperidine-N-oxyl (hemi-GS-TEMPO) 5-125 in {gamma}-irradiated mouse embryonic cells and adenovirus-12 SV40 hybrid virus transformed human bronchial epithelial cells BEAS-2B and explore the mechanisms involved in its radioprotective effect. Methods and Materials: Cells were incubated with 5-125 before (10 minutes) or after (1 hour) {gamma}-irradiation. Superoxide generation was determined by using dihydroethidium assay, and lipid oxidation was quantitated by using a fluorescence high-performance liquid chromatography-based Amplex Red assay. Apoptosis was characterized by evaluating the accumulation of cytochrome c in the cytosol and externalization of phosphatidylserine on the cellmore » surface. Cell survival was measured by means of a clonogenic assay. Results: Treatment (before and after irradiation) of cells with 5-125 at low concentrations (5, 10, and 20 {mu}M) effectively suppressed {gamma}-irradiation-induced superoxide generation, cardiolipin oxidation, and delayed irradiation-induced apoptosis, evaluated by using cytochrome c release and phosphatidylserine externalization. Importantly, treatment with 5-125 increased the clonogenic survival rate of {gamma}-irradiated cells. In addition, 5-125 enhanced and prolonged {gamma}-irradiation-induced G{sub 2}/M phase arrest. Conclusions: Radioprotection/mitigation by hemi-GS-TEMPO likely is caused by its ability to act as an electron scavenger and prevent superoxide generation, attenuate cardiolipin oxidation in mitochondria, and hence prevent the release of proapoptotic factors from mitochondria. Other mechanisms, including cell-cycle arrest at the G{sub 2}/M phase, may contribute to the protection.« less

Tumourigenicity and radiation resistance of mesenchymal stem cells.

PubMed

D'Andrea, Filippo P; Horsman, Michael R; Kassem, Moustapha; Overgaard, Jens; Safwat, Akmal

2012-05-01

Cancer stem cells are believed to be more radiation resistant than differentiated tumour cells of the same origin. It is not known, however, whether normal nontransformed adult stem cells share the same radioresistance as their cancerous counterpart. Nontumourigenic (TERT4) and tumourigenic (TRET20) cell lines, from an immortalised mesenchymal stem cell line, were grown in culture prior to irradiation and gene expression analysis. Radiation resistance was measured using a clonogenic assay. Differences in gene expression between the two cell lines, both under nontreated and irradiated conditions, were assessed with microarrays (Affymetrix Human Exon 1.0 ST array). The cellular functions affected by the altered gene expressions were assessed through gene pathway mapping (Ingenuity Pathway Analysis). Based on the clonogenic assay the nontumourigenic cell line was found to be more sensitive to radiation than the tumourigenic cell line. Using the exon chips, 297 genes were found altered between untreated samples of the cell lines whereas only 16 genes responded to radiation treatment. Among the genes with altered expression between the untreated samples were PLAU, PLAUR, TIMP3, MMP1 and LOX. The pathway analysis based on the alteration between the untreated samples indicated cancer and connective tissue disorders. This study has shown possible common genetic events linking tumourigenicity and radiation response. The PLAU and PLAUR genes are involved in apoptosis evasion while the genes TIMP3, MMP1 and LOX are involved in regulation of the surrounding matrix. The first group may contribute to the difference in radiation resistance observed and the latter could be a major contributor to the tumourigenic capabilities by degrading the intercellular matrix. These results also indicate that cancer stem cells are more radiation resistant than stem cells of the same origin.

A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses

PubMed Central

Stevenson, Katrina; Gilmour, Lesley; Hamilton, Graham; Chalmers, Anthony J

2017-01-01

Abstract Background. Glioblastoma (GBM) is the most common primary brain tumor, with dismal prognosis. The failure of drug–radiation combinations with promising preclinical data to translate into effective clinical treatments may relate to the use of simplified 2-dimensional in vitro GBM cultures. Methods. We developed a customized 3D GBM culture system based on a polystyrene scaffold (Alvetex) that recapitulates key histological features of GBM and compared it with conventional 2D cultures with respect to their response to radiation and to molecular targeted agents for which clinical data are available. Results. In 3 patient-derived GBM lines, no difference in radiation sensitivity was observed between 2D and 3D cultures, as measured by clonogenic survival. Three different molecular targeted agents, for which robust clinical data are available were evaluated in 2D and 3D conditions: (i) temozolomide, which improves overall survival and is standard of care for GBM, exhibited statistically significant effects on clonogenic survival in both patient-derived cell lines when evaluated in the 3D model compared with only one cell line in 2D cells; (ii) bevacizumab, which has been shown to increase progression-free survival when added to standard chemoradiation in phase III clinical trials, exhibited marked radiosensitizing activity in our 3D model but had no effect on 2D cells; and (iii) erlotinib, which had no efficacy in clinical trials, displayed no activity in our 3D GBM model, but radiosensitized 2D cells. Conclusions. Our 3D model reliably predicted clinical efficacy, strongly supporting its clinical relevance and potential value in preclinical evaluation of drug–radiation combinations for GBM. PMID:27576873

Exosomes are released by bystander cells exposed to radiation-induced biophoton signals: Reconciling the mechanisms mediating the bystander effect.

PubMed

Le, Michelle; Fernandez-Palomo, Cristian; McNeill, Fiona E; Seymour, Colin B; Rainbow, Andrew J; Mothersill, Carmel E

2017-01-01

The objective of our study was to explore a possible molecular mechanism by which ultraviolet (UV) biophotons could elicit bystander responses in reporter cells and resolve the problem of seemingly mutually exclusive mechanisms of a physical UV signal & a soluble factor-mediated bystander signal. The human colon carcinoma cell line, HCT116 p53 +/+, was directly irradiated with 0.5 Gy tritium beta particles to induce ultraviolet biophoton emission. Bystander cells were not directly irradiated but were exposed to the emitted UV biophotons. Medium was subsequently harvested from UV-exposed bystander cells. The exosomes extracted from this medium were incubated with reporter cell populations. These reporter cells were then assayed for clonogenic survival and mitochondrial membrane potential with and without prior treatment of the exosomes with RNase. Clonogenic cell survival was significantly reduced in reporter cells incubated with exosomes extracted from cells exposed to secondarily-emitted UV. These exosomes also induced significant mitochondrial membrane depolarization in receiving reporter cells. Conversely, exosomes extracted from non-UV-exposed cells did not produce bystander effects in reporter cells. The treatment of exosomes with RNase prior to their incubation with reporter cells effectively abolished bystander effects in reporter cells and this suggests a role for RNA in mediating the bystander response elicited by UV biophotons and their produced exosomes. This study supports a role for exosomes released from UV biophoton-exposed bystander cells in eliciting bystander responses and also indicates a reconciliation between the UV-mediated bystander effect and the bystander effect which has been suggested in the literature to be mediated by soluble factors.

Glioma Cell Death Induced by Irradiation or Alkylating Agent Chemotherapy Is Independent of the Intrinsic Ceramide Pathway

PubMed Central

Gramatzki, Dorothee; Herrmann, Caroline; Happold, Caroline; Becker, Katrin Anne; Gulbins, Erich; Weller, Michael; Tabatabai, Ghazaleh

2013-01-01

Background/Aims Resistance to genotoxic therapy is a characteristic feature of glioma cells. Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and glucosylceramide synthase (GCS) catalyzes ceramide metabolism. Increased ceramide levels have been suggested to enhance chemotherapy-induced death of cancer cells. Methods Microarray and clinical data for ASM and GCS in astrocytomas WHO grade II–IV were acquired from the Rembrandt database. Moreover, the glioblastoma database of the Cancer Genome Atlas network (TCGA) was used for survival data of glioblastoma patients. For in vitro studies, increases in ceramide levels were achieved either by ASM overexpression or by the GCS inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) in human glioma cell lines. Combinations of alkylating chemotherapy or irradiation and ASM overexpression, PPMP or exogenous ceramide were applied in parental cells. The anti-glioma effects were investigated by assessing proliferation, metabolic activity, viability and clonogenicity. Finally, viability and clonogenicity were assessed in temozolomide (TMZ)-resistant cells upon treatment with PPMP, exogenous ceramide, alkylating chemotherapy, irradiation or their combinations. Results Interrogations from the Rembrandt and TCGA database showed a better survival of glioblastoma patients with low expression of ASM or GCS. ASM overexpression or PPMP treatment alone led to ceramide accumulation but did not enhance the anti-glioma activity of alkylating chemotherapy or irradiation. PPMP or exogenous ceramide induced acute cytotoxicity in glioblastoma cells. Combined treatments with chemotherapy or irradiation led to additive, but not synergistic effects. Finally, no synergy was found when TMZ-resistant cells were treated with exogenous ceramide or PPMP alone or in combination with TMZ or irradiation. Conclusion Modulation of intrinsic glioma cell ceramide levels by ASM overexpression or GCS inhibition does not enhance the anti-glioma activity of alkylating chemotherapy or irradiation. PMID:23667632

Mocetinostat combined with gemcitabine for the treatment of leiomyosarcoma: Preclinical correlates

PubMed Central

Braggio, Danielle; Zewdu, Abeba; Casadei, Lucia; Batte, Kara; Bid, Hemant Kumar; Koller, David; Yu, Peter; Iwenofu, Obiajulu Hans; Strohecker, Anne; Choy, Edwin; Lev, Dina; Pollock, Raphael

2017-01-01

Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastatic disease. Chemotherapeutic intervention has demonstrated to have modest clinical efficacy with no curative potential in LMS patients. Previously, we demonstrated pan-HDAC inhibition to have a superior effect in various complex karyotypic sarcomas. In this study, our goal is to evaluate the therapeutic efficacy of mocetinostat alone and in combination with gemcitabine in LMS. Human leiomyosarcoma (LMS) cell lines were used for in vitro and in vivo studies. Compounds tested included the class I HDAC inhibitor, mocetinostat, and nucleoside analog, gemcitabine. MTS and clonogenic assays were used to evaluate the effect of mocetinostat on LMS cell growth. Cleaved caspase 3/7 analysis was used to determine the effects of mocetinostat on apoptosis. Compusyn software was used to determine in vitro synergy studies for the combination of mocetinostat plus gemcitabine. A LMS xenograft model in SCID mice was used to test the impact of mocetinostat alone, gemcitabine alone and the combination of mocetinostat plus gemcitabine. Mocetinostat abrogated LMS cell growth and clonogenic potential, and enhanced apoptosis in LMS cell lines. The combination of mocetinostat plus gemcitabine exhibited a synergistic effect in LMS cells in vitro. Similarly, mocetinostat combined with gemcitabine resulted in superior anti-LMS effects in vivo. Mocetinostat reduced the expression of gemcitabine-resistance markers RRM1, RRM2, and increased the expression of gemcitabine-sensitivity marker, hENT1, in LMS cells. LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of mocetinostat combined with gemcitabine for the treatment of LMS. PMID:29186204

Heat shock protein 90 (HSP90) is overexpressed in p16-negative oropharyngeal squamous cell carcinoma, and its inhibition in vitro potentiates the effects of chemoradiation.

PubMed

Patel, Kirtesh; Wen, Jing; Magliocca, Kelly; Muller, Susan; Liu, Yuan; Chen, Zhuo Georgia; Saba, Nabil; Diaz, Roberto

2014-11-01

Cisplatin and radiation therapy remain the current standard for treating locally advanced SCCHN. Novel treatment approaches are needed, especially in patients with human papilloma virus (HPV)-negative disease who have worse outcomes despite multimodality therapy. Using our institutional review board approved database, we obtained twenty oropharyngeal squamous cell carcinoma (SCC) tissue samples: ten p16 positive, ten p16-negative. Because p16 expression is strongly associated with HPV positivity in oropharyngeal SCC, p16 status was used as a marker of HPV. We subsequently analyzed, via immunohistochemistry, heat shock protein 90 (HSP90) protein levels. Using HPV-positive and HPV-negative SCC cell lines, we compared baseline HSP90 expression levels and the effect of the HSP90 inhibitor ganetespib on viability and apoptosis. Clonogenic survival of HPV-negative cells treated with ganetespib, radiation therapy, and/or cisplatin was then investigated. We characterize the effects of ganetespib on proteins that are thought to drive DNA damage resistance in HPV-negative cells. HSP90 expression was significantly higher in p16-negative compared with p16-positive samples (p = 0.016) and in HPV-negative cell lines compared with positive cells. Ganetespib increased cytotoxicity and induced apoptosis in HPV-negative more than positive cells. Adding ganetespib to cisplatin and/or radiation therapy in HPV-negative cells further decreased clonogenic survival. Finally, ganetespib downregulated expressions of EGFR, ERK, AKT, p53, and HIF-1α. Ganetespib inhibited HPV-negative SCCHN viability and potentiated cell kill when combined with cisplatin or radiation therapy in vitro. With HSP90 expression higher in HPV-negative cells and in p16-negative patients, further exploration of the clinical activity of HSP90 inhibitors in SCCHN is warranted.

Left Ventricular Assist Device as a Bridge to Recovery for Patients With Advanced Heart Failure.

PubMed

Jakovljevic, Djordje G; Yacoub, Magdi H; Schueler, Stephan; MacGowan, Guy A; Velicki, Lazar; Seferovic, Petar M; Hothi, Sandeep; Tzeng, Bing-Hsiean; Brodie, David A; Birks, Emma; Tan, Lip-Bun

2017-04-18

Left ventricular assist devices (LVADs) have been used as an effective therapeutic option in patients with advanced heart failure, either as a bridge to transplantation, as destination therapy, or in some patients, as a bridge to recovery. This study evaluated whether patients undergoing an LVAD bridge-to-recovery protocol can achieve cardiac and physical functional capacities equivalent to those of healthy controls. Fifty-eight male patients-18 implanted with a continuous-flow LVAD, 16 patients with LVAD explanted (recovered patients), and 24 heart transplant candidates (HTx)-and 97 healthy controls performed a maximal graded cardiopulmonary exercise test with continuous measurements of respiratory gas exchange and noninvasive (rebreathing) hemodynamic data. Cardiac function was represented by peak exercise cardiac power output (mean arterial blood pressure × cardiac output) and functional capacity by peak exercise O 2 consumption. All patients demonstrated a significant exertional effort as demonstrated with the mean peak exercise respiratory exchange ratio >1.10. Peak exercise cardiac power output was significantly higher in healthy controls and explanted LVAD patients compared with other patients (healthy 5.35 ± 0.95 W; explanted 3.45 ± 0.72 W; LVAD implanted 2.37 ± 0.68 W; and HTx 1.31 ± 0.31 W; p < 0.05), as was peak O 2 consumption (healthy 36.4 ± 10.3 ml/kg/min; explanted 29.8 ± 5.9 ml/kg/min; implanted 20.5 ± 4.3 ml/kg/min; and HTx 12.0 ± 2.2 ml/kg/min; p < 0.05). In the LVAD explanted group, 38% of the patients achieved peak cardiac power output and 69% achieved peak O 2 consumption within the ranges of healthy controls. The authors have shown that a substantial number of patients who recovered sufficiently to allow explantation of their LVAD can even achieve cardiac and physical functional capacities nearly equivalent to those of healthy controls. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Effect of postmortem time interval on in vitro culture potential of goat skin tissues stored at room temperature.

PubMed

Singh, Mahipal; Ma, Xiaoling; Sharma, Anil

2012-09-01

Animal cloning using somatic cell nuclear transfer technology has renewed the interest in postmortem tissue storage, since these tissues can be used to reintroduce the lost genes back into the breeding pool in animal agriculture, preserve the genetic diversity, and revive the endangered species. However, for successful cloning of animals, integrity of nuclear DNA is essential. Cell viability and their potential to in vitro culture ensure nuclear integrity. The aim of this study was to determine the limits of postmortem time interval within which live cells can be recovered from goat skin tissues. To test the postmortem tissue storage limits, we cultured 2-3 mm(2) skin pieces (n = 70) from the ears of three breeds of goats (n = 7) after 0, 2, 4, and 6 days of postmortem storage at 24°C. After 10 days of culture, outgrowth of fibroblast-like cells (>50 cells) around the explants was scored. All the explants irrespective of breed displayed outgrowth of cells on the dish containing fresh tissues (i.e., day 0 of storage). However, the number of explants exhibiting outgrowth reduced with increasing time interval. Only 53.85 % explants displayed outgrowth after 2 days of tissue storage. The number of explants displaying outgrowth was much smaller after 4 (16.67 %) and 6 days (13.3 %) of storage. In general, the number of outgrowing cells per explant, on a given day, also decreased with increasing postmortem storage time interval. To test the differences between cell cultures, we established secondary cultures from one of the goats exhibiting outgrowth of cells after 6 days of tissue storage and compared them to similar cells from fresh tissues. Comparison of both the cell lines revealed similar cell morphology and growth curves and had doubling times of 23.04 and 22.56 h, respectively. These results suggest that live cells can be recovered from goat (and perhaps other animal) tissues stored at room temperature even after 6 days of their death with comparable growth profiles and, thus, can be used for tissue banking for preservation of superior genetics, genetic diversity, and cloning of animals.

Normal fates and states of specification of different regions in the axolotl gastrula.

PubMed

Cleine, J H; Slack, J M

1985-04-01

A fate map was constructed for four regions of the early gastrula of Ambystoma mexicanum using orthotopic grafts from donors labelled with FLDx (fluoresceinated-lysinated-dextran). The region around the animal pole gave rise to epidermis only and did not include prospective neural plate. The dorsal marginal zone contributed to cephalic endoderm and to the whole length of the axial mesoderm (notochord and somites), the lateral marginal zone to lateroventral and somitic mesoderm, and the ventral marginal zone to lateroventral mesoderm. It was found that the dorsal marginal zone contributed relatively more to the anterior regions of the mesodermal mantle and the ventral marginal zone more to its posterior parts. The same regions of the gastrula and also vegetal yolky tissue were cultured as explants and labelled with tritiated mannose. Their glycoprotein synthesis pattern was compared to those of the neurula tissues to which they contribute in vivo. Animal pole explants synthesized large amounts of the epidermis-specific marker epimucin. Dorsal marginal zone explants did not synthesize epimucin but did make amounts of S2 and S6 indicative of mesoderm, as well as the notochord-specific markers S2.2 and S3.2. Lateral marginal zone explants showed the same pattern as the dorsal marginal zone including the two notochord-specific markers, although they do not contribute to notochord in vivo. Ventral marginal zone explants were more variable in their behaviour. Yolky tissue from the vegetal hemisphere of the gastrula or the archenteron floor of the neurula synthesized mainly polydisperse material of high molecular weight rather than discrete glycoproteins. The results indicate that at the early gastrula stage states of specification exist which correspond to the three germ layers, ecto-, meso- and endoderm. The ectodermal specification of animal pole explants is quite robust and cannot easily be changed by variation of the culture conditions. However treatment with a concentrated pellet of vegetalizing factor does induce a change to mesodermal specification, which is clearly detectable in the pattern of glycoprotein synthesis. Similar inductive interactions between different regions of the early embryo are thought to occur during normal development.

Calcification of different designs of silicone intraocular lenses in eyes with asteroid hyalosis.

PubMed

Stringham, Jack; Werner, Liliana; Monson, Bryan; Theodosis, Raymond; Mamalis, Nick

2010-08-01

To describe the association between calcification of older and newer designs of silicone intraocular lenses (IOLs) and asteroid hyalosis. Case series with clinicopathologic correlation. Sixteen silicone IOLs explanted because of decreased visual acuity associated with opacifying deposits on the posterior optic surface. All 16 lenses underwent gross and light microscopic analyses. Selected lenses underwent alizarin red staining or scanning electron microscopy coupled with energy dispersive x-ray spectroscopy for elemental composition. Clinical data in each case were obtained by a questionnaire sent to the explanting surgeons. Clinical data in relation to 111 hydrophilic acrylic lenses explanted because of calcification also were assessed for comparison. Deposit morphologic features and location were evaluated under gross and light microscopy. The calcified nature of the deposits was assessed by histochemical staining and surface analyses. Clinical data obtained included age at IOL implantation, gender, implantation and explantation dates, as well as history of neodymium:yttrium-aluminum-garnet laser treatment. The presence of asteroid hyalosis in the affected eye was investigated for the explanted silicone and hydrophilic acrylic lenses. The 16 lenses were of 8 designs manufactured from different silicone materials, which were explanted 9.21+/-3.66 years after implantation. Neodymium:yttrium-aluminum-garnet laser applications performed in 12 cases partially removed deposits from the lens, followed by a gradual increase in their density after the procedures. The presence of asteroid hyalosis was confirmed in 13 cases; no notes regarding this condition were found in patient charts in the other 3 cases. The deposits were only on the posterior optic surface of the silicone lenses and were composed of calcium and phosphate. A history of asteroid hyalosis was not found in relation to any of the 111 cases of postoperative calcification of hydrophilic acrylic lenses. Including this current series, there are 22 cases of calcification of silicone lenses involving 8 designs manufactured from different silicone materials described in the literature. The presence of asteroid hyalosis was confirmed in 86.4% of cases. These findings may be added to the list of pros and cons surgeons consider when selecting or recommending an IOL. Copyright 2010 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

E2f1 mediates high glucose-induced neuronal death in cultured mouse retinal explants.

PubMed

Wang, Yujiao; Zhou, Yi; Xiao, Lirong; Zheng, Shijie; Yan, Naihong; Chen, Danian

2017-10-02

Diabetic retinopathy (DR) is the most common complication of diabetes and remains one of the major causes of blindness in the world; infants born to diabetic mothers have higher risk of developing retinopathy of prematurity (ROP). While hyperglycemia is a major risk factor, the molecular and cellular mechanisms underlying DR and diabetic ROP are poorly understood. To explore the consequences of retinal cells under high glucose, we cultured wild type or E2f1 -/- mouse retinal explants from postnatal day 8 with normal glucose, high osmotic or high glucose media. Explants were also incubated with cobalt chloride (CoCl 2 ) to mimic the hypoxic condition. We showed that, at 7 days post exposure to high glucose, retinal explants displayed elevated cell death, ectopic cell division and intact retinal vascular plexus. Cell death mainly occurred in excitatory neurons, such as ganglion and bipolar cells, which were also ectopically dividing. Many Müller glial cells reentered the cell cycle; some had irregular morphology or migrated to other layers. High glucose inhibited the hyperoxia-induced blood vessel regression of retinal explants. Moreover, inactivation of E2f1 rescued high glucose-induced ectopic division and cell death of retinal neurons, but not ectopic cell division of Müller glial cells and vascular phenotypes. This suggests that high glucose has direct but distinct effects on retinal neurons, glial cells and blood vessels, and that E2f1 mediates its effects on retinal neurons. These findings shed new light onto mechanisms of DR and the fetal retinal abnormalities associated with maternal diabetes, and suggest possible new therapeutic strategies.

A rapid and efficient protocol for in vitro multiplication of genetically uniform Stevia rebaudiana (Bertoni).

PubMed

Khan, A; Jayanthi, M; Gantasala, Nagavara Prasad; Bhooshan, N; Rao, Uma

2016-07-01

Stevia rebaudiana (Bertoni), commonly called candy leaf or sweet leaf, endemic to South America, is an important medicinal plant. As a source of low calorie natural sweetener 'stevoside', it is used in obesity, diabetes, treatment of heartburn and tooth decay, and also serves as a food supplement. Large scale commercial propagation of S. rebaudiana demands a suitable protocol. Here, we propose an improved protocol for in vitro multiplication of S. rebaudiana from nodal explants. In this protocol, the effect of laboratory grade urea on multiple shoot induction from nodal explants was studied. The nodal explants were initially cultured on Murashige and Skoog (MS) basal media for 2 weeks which facilitated the axillary bud break. Further, culturing of these explants on MS medium fortified with 6 benzyl amninopurine (BAP) (2 mg/L) and Naphthalene acetic acid (NAA) (1 mg/L) with and .without urea (5 mg/L) for a period of 40 days revealed maximum shoot production of 44.56 from a single nodal explant in media supplemented with urea as compared to 22.44 without urea. The differences in the number of shoots produced were significant and these shoots readily rooted in MS media with NAA (4 mg/L). Primary and secondary hardening was successful in these plants. There were no visible morphological abnormalities observed in the micropropagated plantlets. Genetic analysis from random samples also revealed that these plants are genetically uniform. The advantage of the present protocol is that the complete process of multiple shoot induction, rooting and hardening could be completed within a period of 6 months as compared to the existing protocols.

Cardiomyocyte Circadian Oscillations Are Cell-Autonomous, Amplified by β-Adrenergic Signaling, and Synchronized in Cardiac Ventricle Tissue

PubMed Central

Welsh, David K.

2016-01-01

Circadian clocks impact vital cardiac parameters such as blood pressure and heart rate, and adverse cardiac events such as myocardial infarction and sudden cardiac death. In mammals, the central circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus, synchronizes cellular circadian clocks in the heart and many other tissues throughout the body. Cardiac ventricle explants maintain autonomous contractions and robust circadian oscillations of clock gene expression in culture. In the present study, we examined the relationship between intrinsic myocardial function and circadian rhythms in cultures from mouse heart. We cultured ventricular explants or dispersed cardiomyocytes from neonatal mice expressing a PER2::LUC bioluminescent reporter of circadian clock gene expression. We found that isoproterenol, a β-adrenoceptor agonist known to increase heart rate and contractility, also amplifies PER2 circadian rhythms in ventricular explants. We found robust, cell-autonomous PER2 circadian rhythms in dispersed cardiomyocytes. Single-cell rhythms were initially synchronized in ventricular explants but desynchronized in dispersed cells. In addition, we developed a method for long-term, simultaneous monitoring of clock gene expression, contraction rate, and basal intracellular Ca2+ level in cardiomyocytes using PER2::LUC in combination with GCaMP3, a genetically encoded fluorescent Ca2+ reporter. In contrast to robust PER2 circadian rhythms in cardiomyocytes, we detected no rhythms in contraction rate and only weak rhythms in basal Ca2+ level. In summary, we found that PER2 circadian rhythms of cardiomyocytes are cell-autonomous, amplified by adrenergic signaling, and synchronized by intercellular communication in ventricle explants, but we detected no robust circadian rhythms in contraction rate or basal Ca2+. PMID:27459195

Cross-linked xenogenic collagen implantation in the sheep model for vaginal surgery.

PubMed

Endo, Masayuki; Urbankova, Iva; Vlacil, Jaromir; Sengupta, Siddarth; Deprest, Thomas; Klosterhalfen, Bernd; Feola, Andrew; Deprest, Jan

The properties of meshes used in reconstructive surgery affect the host response and biomechanical characteristics of the grafted tissue. Whereas durable synthetics induce a chronic inflammation, biological grafts are usually considered as more biocompatible. The location of implantation is another determinant of the host response: the vagina is a different environment with specific function and anatomy. Herein, we evaluated a cross-linked acellular collagen matrix (ACM), pretreated by the anti-calcification procedure ADAPT® in a sheep model for vaginal surgery. Ten sheep were implanted with a cross-linked ACM, and six controls were implanted with a polypropylene (PP; 56 g/m 2 ) control. One implant was inserted in the lower rectovaginal septum, and one was used for abdominal wall defect reconstruction. Grafts were removed after 180 days; all graft-related complications were recorded, and explants underwent bi-axial tensiometry and contractility testing. Half of ACM-implanted animals had palpable induration in the vaginal implantation area, two of these also on the abdominal implant. One animal had a vaginal exposure. Vaginal ACMs were 63 % less stiff compared to abdominal ACM explants ( p  = 0.01) but comparable to vaginal PP explants. Seven anterior vaginal ACM explants showed areas of graft degradation on histology. There was no overall difference in vaginal contractility. Considering histologic degradation in the anterior vaginal implant as representative for the host, posterior ACM explants of animals with degradation had a 60 % reduced contractility as compared to PP ( p  = 0.048). Three abdominal implants showed histologic degradation; those were more compliant than non-degraded implants. Vaginal implantation with ACM was associated with graft-related complications (GRCs) and biomechanical properties comparable to PP. Partially degraded ACM had a decreased vaginal contractility.

Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

NASA Technical Reports Server (NTRS)

Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

1998-01-01

Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

Exogenous transforming growth factor-β1 enhances smooth muscle differentiation in embryonic mouse jejunal explants.

PubMed

Coletta, Riccardo; Roberts, Neil A; Randles, Michael J; Morabito, Antonino; Woolf, Adrian S

2017-01-13

An ex vivo experimental strategy that replicates in vivo intestinal development would in theory provide an accessible setting with which to study normal and dysmorphic gut biology. The current authors recently described a system in which mouse embryonic jejunal segments were explanted onto semipermeable platforms and fed with chemically defined serum-free media. Over 3 days in organ culture, explants formed villi and they began to undergo spontaneous peristalsis. As defined in the current study, the wall of the explanted gut failed to form a robust longitudinal smooth muscle (SM) layer as it would do in vivo over the same time period. Given the role of transforming growth factor β1 (TGFβ1) in SM differentiation in other organs, it was hypothesized that exogenous TGFβ1 would enhance SM differentiation in these explants. In vivo, TGFβ receptors I and II were both detected in embryonic longitudinal jejunal SM cells and, in organ culture, exogenous TGFβ1 induced robust differentiation of longitudinal SM. Microarray profiling showed that TGFβ1 increased SM specific transcripts in a dose dependent manner. TGFβ1 proteins were detected in amniotic fluid at a time when the intestine was physiologically herniated. By analogy with the requirement for exogenous TGFβ1 for SM differentiation in organ culture, the TGFβ1 protein that was demonstrated to be present in the amniotic fluid may enhance intestinal development when it is physiologically herniated in early gestation. Future studies of embryonic intestinal cultures should include TGFβ1 in the defined media to produce a more faithful model of in vivo muscle differentiation. Copyright © 2017 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd. Copyright © 2017 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.

Effect of medium composition and light on root and rhinacanthin formation in Rhinacanthus nasutus cultures.

PubMed

Panichayupakaranant, P; Meerungrueang, W

2010-11-01

Rhinacanthus nasutus (L.) Kurz (Acanthaceae) has long been used in Thai traditional medicine for treatment of tinea versicolor, ringworm, pruritic rash, and abscess. The active constituents are known as a group of naphthoquinone esters, rhinacanthins. This work focused on establishment of R. nasutus root cultures and determination of rhinacanthin production. Induction of R. nasutus root formation was accomplished on solid Gamborg's B5 (B5) medium, supplied with 0.1 mg/L indole-3-butyric acid (IBA) and 20 g/L sucrose. The effects of explants (whole leaf explants and four-side excised leaf explants), light and medium composition on root and rhinacanthin formation were investigated. The root formation from the whole leaf explants was 10 times higher than that from the four-side excised leaf explants. In addition, light possessed an inhibitory effect on the root and rhinacanthin formation of R. nasutus. Medium manipulation found that Murashige and Skoog (MS) medium supplied with 3 mg/L IBA and 30 g/L sucrose was the most suitable for induction of the root formation. Unfortunately, the obtained root cultures produced only rhinacanthin-C in very low amount, 0.026 mg/g dry weight (DW), when they were transferred into the same MS liquid medium. With semisolid medium (4 g/L agar) of the same MS composition, however, the root cultures appeared to produce higher content of rhinacanthin-C, -D and -N (3.45, 0.07 and 0.07 mg/g DW, respectively). Our finding suggests that culturing in semisolid medium is capable of improving of rhinacanthin production in R. nasutus root cultures.

Treatment with the non-ionic surfactant poloxamer P188 reduces DNA fragmentation in cells from bovine chondral explants exposed to injurious unconfined compression.

PubMed

Baars, D C; Rundell, S A; Haut, R C

2006-06-01

Excessive mechanical loading to a joint has been linked with the development of post-traumatic osteoarthritis (OA). Among the suspected links between impact trauma to a joint and associated degeneration of articular cartilage is an acute reduction in chondrocyte viability. Recently, the non-ionic surfactant poloxamer 188 (P188) has been shown to reduce by approximately 50% the percentage of non-viable chondrocytes 24 h post-injury in chondral explants exposed to 25 MPa of unconfined compression. There is a question whether these acutely 'saved' chondrocytes will continue to degrade over time, as P188 is only thought to act by acute repair of damaged cell membranes. In order to investigate the degradation of traumatized chondrocytes in the longer term, the current study utilized TUNEL staining to document the percentage of cells suffering DNA fragmentation with and without an immediate 24 h period of exposure of the explants to P188 surfactant. In the current study, as in the previous study by this laboratory, chondral explants were excised from bovine metacarpophalangeal joints and subjected to 25 MPa of unconfined compression. TUNEL staining was performed at 1 h, 4 days, and 7 days post-impact. The current study found that P188 was effective in reducing the percentage of cells with DNA fragmentation in impacted explants by approximately 45% at 4 and 7 days post-impact. These data suggest that early P188 intervention was effective in preventing DNA fragmentation of injured chondrocytes. The current hypothesis is that this process was mitigated by the acute repair of damaged plasma membranes by the non-ionic surfactant P188, and that most repaired cells did not continue to degrade as measured by the fragmentation of their DNA.

Microbiology of Explanted Suture Segments from Infected and Noninfected Surgical Patients

PubMed Central

Krepel, Candace J.; Marks, Richard M.; Rossi, Peter J.; Sanger, James; Goldblatt, Matthew; Graham, Mary Beth; Rothenburger, Stephen; Collier, John; Seabrook, Gary R.

2013-01-01

Sutures under selective host/environmental factors can potentiate postoperative surgical site infection (SSI). The present investigation characterized microbial recovery and biofilm formation from explanted absorbable (AB) and nonabsorbable (NAB) sutures from infected and noninfected sites. AB and NAB sutures were harvested from noninfected (70.9%) and infected (29.1%) sites in 158 patients. At explantation, devices were sonicated and processed for qualitative/quantitative bacteriology; selective sutures were processed for scanning electron microscopy (SEM). Bacteria were recovered from 85 (53.8%) explanted sites; 39 sites were noninfected, and 46 were infected. Suture recovery ranged from 11.1 to 574.6 days postinsertion. A significant difference in mean microbial recovery between noninfected (1.2 isolates) and infected (2.7 isolates) devices (P < 0.05) was noted. Staphylococcus epidermidis, Staphylococcus aureus, coagulase-negative staphylococci (CNS), Peptostreptococcus spp., Bacteroides fragilis, Escherichia coli, Enterococcus spp., Pseudomonas aeruginosa, and Serratia spp. were recovered from infected devices, while commensal skin flora was recovered from noninfected devices. No significant difference in quantitative microbial recovery between infected monofilament and multifilament sutures was noted. Biofilm was present in 100% and 66.6% of infected and noninfected devices, respectively (P < 0.042). We conclude that both monofilament and braided sutures provide a hospitable surface for microbial adherence: (i) a significant difference in microbial recovery from infected and noninfected sutures was noted, (ii) infected sutures harbored a mixed flora, including multidrug-resistant health care-associated pathogens, and (iii) a significant difference in the presence or absence of a biofilm in infected versus noninfected explanted devices was noted. Further studies to document the benefit of focused risk reduction strategies to minimize suture contamination and biofilm formation postimplantation are warranted. PMID:23175247

Myogenic specification in somites: induction by axial structures.

PubMed

Buffinger, N; Stockdale, F E

1994-06-01

Specification of the myogenic phenotype in somites was examined in the early chick embryo using organotypic explant cultures stained with monoclonal antibodies to myosin heavy chain. It was found that myogenic specification (formation of muscle fibers in explants of somites or segmental plates cultured alone) does not occur until Hamburger and Hamilton stage 11 (12-14 somites). At this stage, only the somites in the rostral half of the embryo are myogenically specified. By Hamburger and Hamilton stage 12 (15-17 somites), the three most caudal somites were not specified to be myogenic while most or all of the more rostral somites are specified to myogenesis. Somites from older embryos (stage 13-15, 18-26 somites) showed the same pattern of myogenic specification--all but the three most caudal somites were specified. We investigated the effects of the axial structures, the notochord and neural tube, on myogenic specification. Both the notochord and neural tube were able to induce myogenesis in unspecified somites. In contrast, the neural tube, but not the notochord, was able to induce myogenesis in explants of segmental plate, a structure which is not myogenic when cultured alone. When explants of specified somites were stained with antibodies to slow or fast MyHC, it was found that myofiber diversity (fast and fast slow fibers) was established very early in development (as early as Hamburger and Hamilton stage 11). We also found fiber diversity in explants of unspecified somites (the three most caudal somites from stage 11 to 15) when they were recombined with notochord or neural tube. We conclude that myogenic specification in the embryo results in diverse fiber types and is an inductive process which is mediated by factors produced by the neural tube and notochord.