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Sample records for basic ex-vivo investigation

  1. Prion structure investigated in situ, ex vivo, and in vitro by FTIR spectroscopy

    Science.gov (United States)

    Kneipp, Janina; Miller, Lisa M.; Spassov, Sashko; Sokolowski, Fabian; Lasch, Peter; Beekes, Michael; Naumann, Dieter

    2004-07-01

    Syrian hamster nervous tissue was investigated by FTIR microspectroscopy with conventional and synchrotron infrared light sources. Various tissue structures from the cerebellum and medulla oblongata of scrapie-infected and control hamsters were investigated at a spatial resolution of 50 μm. Single neurons in dorsal root ganglia of scrapie-infected hamsters were analyzed by raster scan mapping at 6 μm spatial resolution. These measurements enabled us to (i) scrutinize structural differences between infected and non-infected tissue and (ii) analyze for the first time the distribution of different protein structures in situ within single nerve cells. Single nerve cells exhibited areas of increased β-sheet content, which co-localized consistently with accumulations of the pathological prion protein (PrPSc). Spectral data were also obtained from purified, partly proteinase K digested PrPSc isolated from scrapie-infected nervous tissue of hamsters to elucidate similarities/dissimilarities between prion structure in situ and ex vivo. A further comparison is drawn to the recombinant Syrian hamster prion protein SHaPrP90-232, whose in vitro transition from the predominantly a-helical isoform to β-sheet rich oligomeric structures was also investigated by FTIR spectroscopy.

  2. Development of an ex vivo model for investigating the bacterial association to the gut epithelium of pigs

    DEFF Research Database (Denmark)

    Sugiharto, Sugiharto; Jensen, Bent Borg; Lauridsen, Charlotte

    2012-01-01

    To study enterotoxigenic Escherichia coli (ETEC) association to the gut of pigs, a simple and reproducible experimental model would be helpful. The aim of this experiment was to establish a model for studying the association of ETEC to the gut epithelium of pigs. Intestinal segments were prepared...... and they associated more (P = 0.10) at distal than mid SI. The E. coli did not differ (P > 0.05) between genotypes in F4-inoculated segment. In conclusion, the ex vivo model may be feasible to investigate the ETEC association to the gut epithelium of pigs....

  3. Ex vivo lung perfusion.

    Science.gov (United States)

    Reeb, Jeremie; Cypel, Marcelo

    2016-03-01

    Lung transplantation is an established life-saving therapy for patients with end-stage lung disease. Unfortunately, greater success in lung transplantation is hindered by a shortage of lung donors and the relatively poor early-, mid-, and long-term outcomes associated with severe primary graft dysfunction. Ex vivo lung perfusion has emerged as a modern preservation technique that allows for a more accurate lung assessment and improvement in lung quality. This review outlines the: (i) rationale behind the method; (ii) techniques and protocols; (iii) Toronto ex vivo lung perfusion method; (iv) devices available; and (v) clinical experience worldwide. We also highlight the potential of ex vivo lung perfusion in leading a new era of lung preservation. PMID:26700566

  4. Investigation of the effect of hydration on dermal collagen in ex vivo human skin tissue using second harmonic generation microscopy

    Science.gov (United States)

    Samatham, Ravikant; Wang, Nicholas K.; Jacques, Steven L.

    2016-02-01

    Effect of hydration on the dermal collagen structure in human skin was investigated using second harmonic generation microscopy. Dog ears from the Mohs micrographic surgery department were procured for the study. Skin samples with subject aged between 58-90 years old were used in the study. Three dimensional Multiphoton (Two-photon and backward SHG) control data was acquired from the skin samples. After the control measurement, the skin tissue was either soaked in deionized water for 2 hours (Hydration) or kept at room temperature for 2 hours (Desiccation), and SHG data was acquired. The data was normalized for changes in laser power and detector gain. The collagen signal per unit volume from the dermis was calculated. The desiccated skin tissue gave higher backward SHG compared to respective control tissue, while hydration sample gave a lower backward SHG. The collagen signal decreased with increase in hydration of the dermal collagen. Hydration affected the packing of the collagen fibrils causing a change in the backward SHG signal. In this study, the use of multiphoton microscopy to study the effect of hydration on dermal structure was demonstrated in ex vivo tissue.

  5. Ex vivo lung perfusion

    OpenAIRE

    Machuca, Tiago N; Cypel, Marcelo

    2014-01-01

    Lung transplantation (LTx) is an established treatment option for eligible patients with end-stage lung disease. Nevertheless, the imbalance between suitable donor lungs available and the increasing number of patients considered for LTx reflects in considerable waitlist mortality. Among potential alternatives to address this issue, ex vivo lung perfusion (EVLP) has emerged as a modern preservation technique that allows for more accurate lung assessment and also improvement of lung function. I...

  6. Computer-assisted ex vivo, normothermic small bowel perfusion

    OpenAIRE

    Stangl, M.J.; Krapp, J.; Theodorou, D; Eder, M.; Hammer, C; Land, W.; Schildberg, Friedrich Wilhelm

    2000-01-01

    Background: In the present study, a technique for computer-assisted, normothermic, oxygenated, ex vivo, recirculating small bowel perfusion was established as a tool to investigate organ pretreatment protocols and ischemia/reperfusion phenomena. A prerequisite for the desired setup was an organ chamber for ex vivo perfusion and the use of syngeneic whole blood as perfusate. Methods: The entire small bowel was harvested from Lewis rats and perfused in an organ chamber ex vivo for at least 2 h....

  7. A method to investigate radial glia cell behavior using two-photon time-lapse microscopy in an ex vivo model of spinal cord development.

    Directory of Open Access Journals (Sweden)

    Janelle M.P. Pakan

    2014-04-01

    Full Text Available The mammalian central nervous system (CNS develops from multipotent progenitor cells, which proliferate and differentiate into the various cell types of the brain and spinal cord. Despite the wealth of knowledge from progenitor cell culture studies, there is a significant lack of understanding regarding dynamic progenitor cell behavior over the course of development. This is in part due to shortcomings in the techniques available to study these processes in living tissues as they are occurring. In order to investigate cell behavior under physiologically relevant conditions we established an ex vivo model of the developing rat spinal cord. This method allows us to directly observe specific populations of cells ex vivo in real time and over extended developmental periods as they undergo proliferation, migration and differentiation in the CNS. Previous investigations of progenitor cell behavior have been limited in either spatial or temporal resolution (or both due to the necessity of preserving tissue viability and avoiding phototoxic effects of fluorescent imaging. The method described here overcomes these obstacles. Using two-photon and confocal microscopy and transfected organotypic spinal cord slice cultures we have undertaken detailed imaging of a unique population of neural progenitors, radial glial cells. This method uniquely enables analysis of large populations as well as individual cells; ultimately resulting in a 4D dataset of progenitor cell behavior for up to seven days during embryonic development. This approach can be adapted to study a variety of cell populations at different stages of development using appropriate promoter driven fluorescent protein expression. The ability to control the tissue micro-environment makes this ex vivo method a powerful tool to elucidate the underlying molecular mechanisms regulating cell behavior during embryonic development.

  8. The pathogenesis of Randall's plaque: a papilla cartography of Ca compounds through an ex vivo investigation based on XANES spectroscopy.

    Science.gov (United States)

    Carpentier, Xavier; Bazin, Dominique; Jungers, Paul; Reguer, Solenn; Thiaudière, Dominique; Daudon, Michel

    2010-05-01

    At the surface of attached kidney stones, a particular deposit termed Randall's plaque (RP) serves as a nucleus. This structural particularity as well as other major public health problems such as diabetes type-2 may explain the dramatic increase in urolithiasis now affecting up to 20% of the population in the industrialized countries. Regarding the chemical composition, even if other phosphate phases such as whitlockite or brushite can be found as minor components (less than 5%), calcium phosphate apatite as well as amorphous carbonated calcium phosphate (ACCP) are the major components of most RPs. Through X-ray absorption spectroscopy performed at the Ca K-absorption edge, a technique specific to synchrotron radiation, the presence and crystallinity of the Ca phosphate phases present in RP were determined ex vivo. The sensitivity of the technique was used as well as the fact that the measurements can be performed directly on the papilla. The sample was stored in formol. Moreover, a first mapping of the chemical phase from the top of the papilla to the deep medulla is obtained. Direct structural evidence of the presence of ACCP as a major constituent is given for the first time. This set of data, coherent with previous studies, shows that this chemical phase can be considered as one precursor in the genesis of RP.

  9. Investigating the cubosomal ability for transnasal brain targeting: In vitro optimization, ex vivo permeation and in vivo biodistribution.

    Science.gov (United States)

    Abdelrahman, Fatma Elzahraa; Elsayed, Ibrahim; Gad, Mary Kamal; Badr, Ahmed; Mohamed, Magdi Ibrahim

    2015-07-25

    The aim of this study was to enhance the risperidone delivery to the brain through the transnasal route via optimization of cubosomal gel. Cubosomes were prepared using glycerol mono-oleate (GMO), Pluronic F127 (PF127) and Tween 80 (T80). The prepared formulae were characterized by testing their particle size, polydispersity index, zeta potential, entrapment efficiency, in vitro drug release and transmission electron microscopy. Central composite design was planned for the formulae optimization and the selected formula (containing PF127 with concentration 15 mg/g GMO and T80 with concentration of 20mg/L) was re-prepared in presence of gelling polymer (gellan gum or polyox). The optimal cubosomal gel (containing 0.4% w/v polyox) had been subjected to ex-vivo permeation, histopathological evaluation and in vivo biodistribution studies. It showed significantly higher transnasal permeation and better distribution to the brain, when compared to the used control (drug solution and/or suspension). Finally, the cubosomal gel could be considered as a promising carrier for brain targeting of CNS acting drugs through the transnasal route.

  10. Ex-vivo lung perfusion.

    Science.gov (United States)

    Van Raemdonck, Dirk; Neyrinck, Arne; Cypel, Marcelo; Keshavjee, Shaf

    2015-06-01

    This review outlines the new and promising technique of ex vivo lung perfusion and its clinical potential to increase the number of transplantable lungs and to improve the early and late outcome after transplantation. The rationale, the experimental background, the technique and protocols, and available devices for ex vivo lung perfusion are discussed. The current clinical experience worldwide and ongoing clinical trials are reviewed. PMID:24629039

  11. Ex vivo lung graft perfusion.

    Science.gov (United States)

    Briot, Raphaël; Gennai, Stéphane; Maignan, Maxime; Souilamas, Redha; Pison, Christophe

    2016-04-01

    This review proposes an update of the state of the art and the ongoing clinical trials of ex vivo lung perfusion for lung transplantation in patients. Ex vivo lung perfusion techniques (EVLP) can be used to evaluate a lung graft outside of the body. The goal of EVLP is to study the functional status of lung grafts that were first rejected for transplantation because they did not match all criteria for a conventional transplantation. After an EVLP evaluation, some of these lungs may be requalified for a possible transplantation in patients. This article proposes an overview of the developments of EVLP techniques. During EVLP, the perfusion and ventilation of the isolated lung preparation are very progressive in order to avoid oedema due to ischaemia-reperfusion injuries. Lung evaluation is mainly based on gasometric (PaO2/FiO2) and rheological criteria (low pulmonary arterial resistance). Several series of patients transplanted with EVLP evaluated lungs have been recently published with promising results. EVLP preparations also allow a better understanding of the physiopathology and treatments of ischaemia-reperfusion injuries. Organ procurements from "non-heart-beating" donors will probably require a wider application of these ex vivo techniques. The development of semi-automated systems might facilitate the clinical use of EVLP techniques. PMID:26746565

  12. Porcine Ex Vivo intestinal segment model

    OpenAIRE

    Ripken, D.; Hendriks, H.F.J.

    2015-01-01

    This chapter describes the use of the porcine ex vivo intestinal segment model. This includes the advantages and disadvantages of the segment model and a detailed description of the isolation and culture as well as the applications of the porcine ex vivo intestinal segment model in practice. Compared to the Ussing chamber (Chap. 24) the porcine ex vivo small intestinal segment model is a relatively simple to use intestinal tissue model. The main difference being that the tissue segment is not...

  13. Porcine Ex Vivo intestinal segment model

    NARCIS (Netherlands)

    Ripken, D.; Hendriks, H. F J

    2015-01-01

    This chapter describes the use of the porcine ex vivo intestinal segment model. This includes the advantages and disadvantages of the segment model and a detailed description of the isolation and culture as well as the applications of the porcine ex vivo intestinal segment model in practice. Compare

  14. Porcine Ex Vivo intestinal segment model

    NARCIS (Netherlands)

    Ripken, D.; Hendriks, H.F.J.

    2015-01-01

    This chapter describes the use of the porcine ex vivo intestinal segment model. This includes the advantages and disadvantages of the segment model and a detailed description of the isolation and culture as well as the applications of the porcine ex vivo intestinal segment model in practice. Comp

  15. Influence of water dilution on percutaneous absorption of N-vinyl-2-pyrrolidone in vivo and ex vivo in rats and ex vivo in humans.

    Science.gov (United States)

    Marquet, Fabrice; Payan, Jean-Paul; Beydon, Dominique; Wathier, Ludivine; Ferrari, Elisabeth; Grandclaude, Marie-Christine

    2015-11-01

    N-vinyl-2-pyrrolidone (NVP) is mainly used as a monomer in the production of polyvinylpyrrolidone or copolymers. Percutaneous absorption is an important source of exposure in the work environment. However, few studies have investigated this route of absorption. In this study, percutaneous absorption of neat or aqueous NVP solutions was measured in vivo and ex vivo in rats, and ex vivo in humans. Penetration and absorption fluxes were very similar following in vivo exposure to neat NVP (0.54 and 0.43 mg/cm(2)/h, respectively). Exposing rats to a 50% aqueous solution of NVP increased both fluxes threefold (to 1.48 and 1.55 mg/cm(2)/h, respectively). Ex vivo, the absorption flux increased with solutions from 10 to 25% of NVP, reached a plateau (between 25 and 50% in rat, 25 and 75% in human) and then decreased with neat NVP. In vivo and ex vivo absorption fluxes measured using rat skin were similar, supporting the hypothesis that the ex vivo measurements were a good representation of what was observed in vivo. Thus, for humans, the ex vivo measurements are likely the same as would be determined in vivo. PMID:25160662

  16. Ebola Virus Persistence in Semen Ex Vivo.

    Science.gov (United States)

    Fischer, Robert J; Judson, Seth; Miazgowicz, Kerri; Bushmaker, Trent; Munster, Vincent J

    2016-02-01

    On March 20, 2015, a case of Ebola virus disease was identified in Liberia that most likely was transmitted through sexual contact. We assessed the efficiency of detecting Ebola virus in semen samples by molecular diagnostics and the stability of Ebola virus in ex vivo semen under simulated tropical conditions.

  17. Ebola Virus Persistence in Semen Ex Vivo

    OpenAIRE

    Fischer, Robert J.; Judson, Seth; Miazgowicz, Kerri; Bushmaker, Trent; Munster, Vincent J.

    2016-01-01

    On March 20, 2015, a case of Ebola virus disease was identified in Liberia that most likely was transmitted through sexual contact. We assessed the efficiency of detecting Ebola virus in semen samples by molecular diagnostics and the stability of Ebola virus in ex vivo semen under simulated tropical conditions.

  18. Ex vivo laser confocal microscopy findings of cultured Acanthamoeba trophozoites

    Directory of Open Access Journals (Sweden)

    Yamazaki N

    2012-08-01

    Full Text Available Natsuko Yamazaki,1 Akira Kobayashi,1 Hideaki Yokogawa,1 Yasuhisa Ishibashi,2 Yosaburo Oikawa,3 Masaharu Tokoro,4 Kazuhisa Sugiyama11Department of Ophthalmology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; 2Department of Ophthalmology, East Washinomiya Hospital, Kuki, Japan; 3Department of Medical Zoology, Kanazawa Medical University, Kahoku, Japan; 4Department of Parasitology, Kanazawa University Graduate School of Medical Science, Kanazawa, JapanPurpose: The purpose of the current study was to investigate ex vivo laser confocal microscopic findings of cultured Acanthamoeba trophozoites obtained from Acanthamoeba keratitis patients.Methods: Eight cultured samples of Acanthamoeba trophozoites from eight eyes of seven patients (mean age, 26.9 years; age range, 18–52 years were used. Seven samples were from corneal scrapings of Acanthamoeba keratitis patients and one sample was from the solution in a soft contact lens case. Ex vivo laser confocal microscopy was performed to qualitatively evaluate the shape and degree of light reflection of the living Acanthamoeba trophozoites.Results: Ex vivo laser confocal microscopy demonstrated highly reflective, high-contrast Acanthamoeba trophozoites with no walls (mean size, 25.4 µm; range, 17.1–58.5 µm. The shapes of the trophozoites were highly pleomorphic, and some showed characteristic acanthopodia by laser confocal microscopy.Conclusion: Ex vivo laser confocal microscopy was effective in demonstrating cultured Acanthamoeba trophozoites of various shapes and sizes. The observations of the current study may be helpful when similar structures are identified under in vivo conditions.Keywords: Acanthamoeba, trophozoite, laser confocal microscopy

  19. Ex vivo lung perfusion and reconditioning.

    Science.gov (United States)

    Yeung, Jonathan C; Cypel, Marcelo; Massad, Ehab; Keshavjee, Shaf

    2011-01-01

    Normothermic ex vivo lung perfusion can act as a platform for the evaluation and repair of donor lungs. An acellular hyperosmolar solution is perfused anterograde through the donor lungs at 40% of the estimated cardiac output. Following oxygenation of the perfusate by the lung, it passes through a hollow fiber oxygenator supplied with a hypoxic gas mixture to remove oxygen and to maintain physiological carbon dioxide levels. Flow through a heat exchanger to maintain normothermia and a leukocyte filter to remove demarginated leukocytes completes the circuit. Lung function can be measured by the difference in PO2 between the perfusate postlung and postmembrane and by physiological parameters. Utilization of this method of ex vivo donor lung evaluation should reduce concerns of primary graft dysfunction and increase utilization rates of donor lungs. PMID:24412979

  20. Investigation of ex vivo stability of fesoterodine in human plasma and its simultaneous determination together with its active metabolite 5-HMT by LC-ESI-MS/MS: Application to a bioequivalence study.

    Science.gov (United States)

    Parekh, Jignesh M; Sanyal, Mallika; Yadav, Manish; Shrivastav, Pranav S

    2013-01-15

    Fesoterodine is a non-selective muscarinic-receptor antagonist, used in the treatment of overactive bladder syndrome. A highly sensitive, selective and rapid method has been developed for the simultaneous determination of fesoterodine and its active metabolite, 5-hydroxymethyl tolterodine (5-HMT) in human plasma by liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS). Due to rapid conversion of parent drug to 5-HMT, ex vivo stability of fesoterodine in human plasma was extensively studied to optimize the extraction protocol. The analytes and their deuterated analogs were quantitatively extracted from 100μL human plasma by liquid-liquid extraction in methyl tert-butyl ether: n-hexane. The chromatographic separation of analytes was achieved on a Kromasil C18 (100mm×4.6mm, 5μm) column under isocratic conditions. The method was validated over a dynamic concentration range of 0.01-10ng/mL for both the analytes. Ion-suppression effects were investigated by post-column infusion of analytes. The precision (% CV) values for the calculated slopes of calibration curves, which would reflect the relative matrix effect, were less than 1.5% for both the analytes. The intra-batch and inter-batch precision (% CV) across quality control levels varied from 1.82 to 3.73% and the mean extraction recovery was >96% for both the analytes. The method was successfully applied to a bioequivalence study of 8mg fesoterodine tablet formulation (test and reference) in 12 healthy Indian subjects under fasted and fed condition. The assay reproducibility estimated by reanalysis of incurred samples showed a change of ±12.0%. PMID:23266359

  1. Evaluation of the influence of cardiac motion on the accuracy and reproducibility of longitudinal measurements and the corresponding image quality in optical frequency domain imaging: an ex vivo investigation of the optimal pullback speed.

    Science.gov (United States)

    Koyama, Kohei; Yoneyama, Kihei; Mitarai, Takanobu; Kuwata, Shingo; Kongoji, Ken; Harada, Tomoo; Akashi, Yoshihiro J

    2015-08-01

    Longitudinal measurement using intravascular ultrasound is limited because the motorized pullback device assumes no cardiac motion. A newly developed intracoronary imaging modality, optical frequency domain imaging (OFDI), has higher resolution and an increased auto-pullback speed with presumably lesser susceptibility to cardiac motion artifacts during pullback for longitudinal measurement; however, it has not been fully investigated. We aimed to clarify the influence of cardiac motion on the accuracy and reproducibility of longitudinal measurements obtained using OFDI and to determine the optimal pullback speed. This ex vivo study included 31 stents deployed in the mid left anterior descending artery under phantom heartbeat and coronary flow simulation. Longitudinal stent lengths were measured twice using OFDI at three pullback speeds. Differences in stent lengths between OFDI and microscopy and between two repetitive pullbacks were assessed to determine accuracy and reproducibility. Furthermore, three-dimensional (3D) reconstruction was used for evaluating image quality. With regard to differences in stent length between OFDI and microscopy, the intraclass correlation coefficient values were 0.985, 0.994, and 0.995 at 10, 20, and 40 mm/s, respectively. With regard to reproducibility, the values were 0.995, 0.996, and 0.996 at 10, 20, and 40 mm/s, respectively. 3D reconstruction showed a superior image quality at 10 and 20 mm/s compared with that at 40 mm/s. OFDI demonstrated high accuracy and reproducibility for longitudinal stent measurements. Moreover, its accuracy and reproducibility were remarkable at a higher pullback speed. A 20-mm/s pullback speed may be optimal for clinical and research purposes.

  2. Ex vivo culture of human fetal gonads

    DEFF Research Database (Denmark)

    Jørgensen, A; Nielsen, J.E.; Perlman, S;

    2015-01-01

    '-deoxyuridine (BrdU) incorporation assay. MAIN RESULTS AND THE ROLE OF CHANCE: A novel ex vivo 'hanging-drop' culture model for human fetal gonads was successfully established. Continued proliferation of cells without signs of increased apoptosis was observed after 2 weeks of culture. In cultured fetal ovaries......STUDY QUESTION: What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads? SUMMARY ANSWER: RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal...... phenotype in fetal testis cultures. WHAT IS KNOWN ALREADY: One of the first manifestations of sex differentiation is the initiation of meiosis in fetal ovaries. In contrast, meiotic entry is actively prevented in the fetal testis at this developmental time-point. It has previously been shown that RA...

  3. Avaliação e recondicionamento pulmonar ex vivo Ex vivo lung evaluation and reconditioning

    Directory of Open Access Journals (Sweden)

    Paulo Manuel Pêgo-Fernandes

    2010-12-01

    Full Text Available OBJETIVO: Apenas 15% dos pulmões doados são aproveitados para transplante. Um novo método de Perfusão Pulmonar Ex Vivo (PPEV foi desenvolvido e pode ser usado para avaliação e recondicionamento de pulmões "marginais" e rejeitados para o transplante. Esse trabalho relata nossa experiência com a avaliação funcional da PPEV. MÉTODOS: Foram estudados pulmões de 12 doadores considerados inapropriados para transplante pulmonar. Após a captação, os pulmões são perfundidos ex vivo com Steen Solution, uma solução de composição eletrolítica extracelular com alta pressão coloidosmótica. Um oxigenador de membrana ligado ao circuito recebe uma mistura gasosa (nitrogênio e dióxido de carbono e "desoxigena" o perfusato, mantendo uma concentração de gases semelhante a do sangue venoso. Os pulmões são gradualmente aquecidos, perfundidos e ventilados. A avaliação dos órgãos é feita por gasometrias e medidas como a resistência vascular pulmonar (RVP e complacência pulmonar (CP. RESULTADOS: A PaO2 (FiO2 100% passou de um valor médio de 193,3 mmHg no doador para 495,3 mmHg durante a PPEV. Após uma hora de PPEV, a RVP média era de 737,3 dinas/seg/ cm5 e a CP era de 42,2 ml/cmH2O. CONCLUSÕES: O modelo de avaliação pulmonar ex vivo pode melhorar a capacidade de oxigenação de pulmões "marginais" inicialmente rejeitados para transplante. Isso denota um grande potencial do método para aumentar a disponibilidade de pulmões para transplante e, possivelmente, reduzir o tempo de espera nas filas.OBJECTIVE: Only about 15% of the potential candidates for lung donation are considered suitable for transplantation. A new method for ex vivo lung perfusion (EVLP has been developed and can be used for evaluation and reconditioning of "marginal" and unacceptable lungs. This is a report of functional evaluation experience with ex vivo perfusion of twelve donor lungs deemed unacceptable in São Paulo, Brazil. METHODS: After harvesting, the

  4. Ex Vivo and In Vivo Regulation of Lipocalin-2, a Novel Adipokine, by Insulin

    OpenAIRE

    Tan, Bee K.; Adya, Raghu; Shan, Xiaoye; Syed, Farhatullah; Lewandowski, Krzysztof C.; O'Hare, John P.; Randeva, Harpal S

    2009-01-01

    OBJECTIVE—Lipocalin-2, a novel adipokine, has been shown to be elevated in obese, insulin-resistant, and diabetic subjects. We therefore sought to study the ex vivo and in vivo effects of insulin on lipocalin-2 levels in humans. RESEARCH DESIGN AND METHODS—We investigated the in vivo effects of insulin (hyperinsulinemia) on circulating lipocalin-2 levels by enzyme-linked immunosorbent assay via a prolonged insulin-glucose infusion. The ex vivo effect of insulin on adipose tissue lipocalin-2 p...

  5. The Role of Hibiscus sabdariffa L. (Roselle) in Maintenance of Ex Vivo Murine Bone Marrow-Derived Hematopoietic Stem Cells

    OpenAIRE

    Zariyantey Abdul Hamid; Winnie Hii Lin Lin; Basma Jibril Abdalla; Ong Bee Yuen; Elda Surhaida Latif; Jamaludin Mohamed; Nor Fadilah Rajab; Chow Paik Wah; Muhd Khairul Akmal Wak Harto; Siti Balkis Budin

    2014-01-01

    Hematopoietic stem cells- (HSCs-) based therapy requires ex vivo expansion of HSCs prior to therapeutic use. However, ex vivo culture was reported to promote excessive production of reactive oxygen species (ROS), exposing HSCs to oxidative damage. Efforts to overcome this limitation include the use of antioxidants. In this study, the role of Hibiscus sabdariffa L. (Roselle) in maintenance of cultured murine bone marrow-derived HSCs was investigated. Aqueous extract of Roselle was added at var...

  6. Respiratory symptoms and ex vivo cytokine release are associated in workers processing herring

    DEFF Research Database (Denmark)

    Bønløkke, Jakob Hjort; Thomassen, Mads; Viskum, Sven;

    2004-01-01

    OBJECTIVE: To determine the prevalence of respiratory symptoms among workers processing herring and assess ex vivo cytokine release in response to agents at their workplace. METHODS: We applied a questionnaire, and performed skin prick testing and pulmonary investigations in 36 workers at two her...

  7. The Impact of Food Bioactives on Health: In Vitro and Ex Vivo Models

    NARCIS (Netherlands)

    Verhoeckx, Kitty; Cotter, Paul; López-Expósito, Iván; Kleiveland, Charlotte; Lea, Tor; Mackie, Alan; Requena, Teresa; Swiatecka, Dominika; Wichers, Harry

    2015-01-01

    This book describes in vitro and ex vivo models that can be employed to investigate effects of digested food products on the GIT, or specific components thereof. Many such models exist and include, for example, those used to study digestion and fermentation in the small and large intestine, to inves

  8. Increasing time interval and decreasing allergen dose interval improves ex vivo desensitization of human blood basophils

    DEFF Research Database (Denmark)

    Witting Christensen, Sara K; Krohn, Inge Kortekaas; Thuraiaiyah, Jani;

    2016-01-01

    BACKGROUND: Desensitization is a method for inducing temporary tolerance to allergen. The mechanism underlying desensitization is yet to be established. METHODS: Basophil granulocytes in whole blood from grass pollen allergic subjects were desensitized ex vivo by sequential addition of increasing...... for investigation of the mechanism of desensitization. This article is protected by copyright. All rights reserved....

  9. Ex vivo lung perfusion in Brazil

    Directory of Open Access Journals (Sweden)

    Luis Gustavo Abdalla

    2016-04-01

    Full Text Available Objective: To evaluate the use of ex vivo lung perfusion (EVLP clinically to prepare donor lungs for transplantation. Methods: A prospective study involving EVLP for the reconditioning of extended-criteria donor lungs, the criteria for which include aspects such as a PaO2/FiO2 ratio < 300 mmHg. Between February of 2013 and February of 2014, the lungs of five donors were submitted to EVLP for up to 4 h each. During EVLP, respiratory mechanics were continuously evaluated. Once every hour during the procedure, samples of the perfusate were collected and the function of the lungs was evaluated. Results: The mean PaO2 of the recovered lungs was 262.9 ± 119.7 mmHg at baseline, compared with 357.0 ± 108.5 mmHg after 3 h of EVLP. The mean oxygenation capacity of the lungs improved slightly over the first 3 h of EVLP-246.1 ± 35.1, 257.9 ± 48.9, and 288.8 ± 120.5 mmHg after 1, 2, and 3 h, respectively-without significant differences among the time points (p = 0.508. The mean static compliance was 63.0 ± 18.7 mmHg, 75.6 ± 25.4 mmHg, and 70.4 ± 28.0 mmHg after 1, 2, and 3 h, respectively, with a significant improvement from hour 1 to hour 2 (p = 0.029 but not from hour 2 to hour 3 (p = 0.059. Pulmonary vascular resistance remained stable during EVLP, with no differences among time points (p = 0.284. Conclusions: Although the lungs evaluated remained under physiological conditions, the EVLP protocol did not effectively improve lung function, thus precluding transplantation.

  10. Novel Sensor-Enabled Ex Vivo Bioreactor: A New Approach towards Physiological Parameters and Porcine Artery Viability

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    Raghavendra Mundargi

    2015-01-01

    Full Text Available The aim of the present work is to design and construct an ex vivo bioreactor system to assess the real time viability of vascular tissue. Porcine carotid artery as a model tissue was used in the ex vivo bioreactor setup to monitor its viability under physiological conditions such as oxygen, pressure, temperature, and flow. The real time tissue viability was evaluated by monitoring tissue metabolism through a fluorescent indicator “resorufin.” Our ex vivo bioreactor allows real time monitoring of tissue responses along with physiological conditions. These ex vivo parameters were vital in determining the tissue viability in sensor-enabled bioreactor and our initial investigations suggest that, porcine tissue viability is considerably affected by high shear forces and low oxygen levels. Histological evaluations with hematoxylin and eosin and Masson’s trichrome staining show intact endothelium with fresh porcine tissue whereas tissues after incubation in ex vivo bioreactor studies indicate denuded endothelium supporting the viability results from real time measurements. Hence, this novel viability sensor-enabled ex vivo bioreactor acts as model to mimic in vivo system and record vascular responses to biopharmaceutical molecules and biomedical devices.

  11. Otospheres derived from neonatal mouse cochleae retain the progenitor cell phenotype after ex vivo expansions.

    Science.gov (United States)

    Lou, Xiang-Xin; Nakagawa, Takayuki; Ohnishi, Hiroe; Nishimura, Koji; Ito, Juichi

    2013-02-01

    Because of their limited regenerative potential, cochlear hair cell loss is one of the major causes of permanent hearing loss in mammals. However, recent studies have shown that postnatal cochlear epithelia retain the progenitor cells that form otospheres. Otospheres are capable of self-renewing and differentiating into inner ear cell lineages, thereby suggesting a promising source for hair cell regeneration. We investigated retention of the progenitor cell phenotype in otospheres after ex vivo expansion, which is crucial for transplantation approaches. Reverse transcriptase-polymerase chain reaction and immunocytochemical analyses showed that otospheres derived from neonatal mice retained expression of stem and cochlear cell markers. After in vitro differentiation, otosphere-consisting cells differentiated into hair cell phenotypes after ex vivo expansion. However, the capacity of otospheres for self-renewal weakened with subsequent generations of ex vivo expansion. Our results indicate that ex vivo expanded-otospheres are useful experimental tools for studying hair cell regeneration in transplantation approaches and that the mechanisms for retention of the progenitor cell phenotype in otospheres should be investigated. PMID:23238450

  12. Polydimethylsiloxane embedded mouse aorta ex vivo perfusion model: proof-of-concept study focusing on atherosclerosis.

    Science.gov (United States)

    Wang, Xueya; Wolf, Marc P; Keel, Rahel Bänziger; Lehner, Roman; Hunziker, Patrick R

    2012-07-01

    Existing mouse artery ex vivo perfusion models have utilized arteries such as carotid, uterine, and mesenteric arteries, but not the aorta. However, the aorta is the principal vessel analyzed for atherosclerosis studies in vivo. We have devised a mouse aorta ex vivo perfusion model that can bridge this gap. Aortas from apoE((-/-)) mice are embedded in a transparent, gas-permeable, and elastic polymer matrix [polydimethylsiloxane (PDMS)] and artificially perfused with cell culture medium under cell culture conditions. After 24 h of artificial ex vivo perfusion, no evidence of cellular apoptosis is detected. Utilizing a standard confocal microscope, it is possible to image specific receptor targeting of cells in atherosclerotic plaques during 24 h. Imaging motion artifacts are minimal due to the polymer matrix embedding. Re-embedding of the aorta enables tissue sectioning and immuno-histochemical analysis. The ex vivo data are validated by comparison with in vivo experiments. This model can save animal lives via production of multiple endpoints in a single experiment, is easy to apply, and enables straightforward comparability with pre-existing atherosclerosis in vivo data. It is suited to investigate atherosclerotic disease in particular and vascular biology in general.

  13. Ex vivo tools for the clonal analysis of zebrafish hematopoiesis.

    Science.gov (United States)

    Svoboda, Ondrej; Stachura, David L; Machonova, Olga; Zon, Leonard I; Traver, David; Bartunek, Petr

    2016-05-01

    This protocol describes the ex vivo characterization of zebrafish hematopoietic progenitors. We show how to isolate zebrafish hematopoietic cells for cultivation and differentiation in colony assays in semi-solid media. We also describe procedures for the generation of recombinant zebrafish cytokines and for the isolation of carp serum, which are essential components of the medium required to grow zebrafish hematopoietic cells ex vivo. The outcome of these clonal assays can easily be evaluated using standard microscopy techniques after 3-10 d in culture. In addition, we describe how to isolate individual colonies for further imaging and gene expression profiling. In other vertebrate model organisms, ex vivo assays have been crucial for elucidating the relationships among hematopoietic stem cells (HSCs), progenitor cells and their mature progeny. The present protocol should facilitate such studies on cells derived from zebrafish. PMID:27123951

  14. In vitro and ex vivo antiangiogenic activity of Salvia officinalis.

    Science.gov (United States)

    Keshavarz, Maryam; Mostafaie, Ali; Mansouri, Kamran; Bidmeshkipour, Ali; Motlagh, Hamid Reza Mohammadi; Parvaneh, Shahram

    2010-10-01

    Angiogenesis is a key process in the promotion of cancer and its metastasis. Herein, the antiangiogenic activity of Salvia officinalis extract and its fractions was investigated. S. officinalis aerial parts were extracted with ethanol and its successive hexane, ethyl acetate, n-butanol and aqueous fractions were evaluated for their antiangiogenic activities using human umbilical vein endothelial cells (HUVEC) capillary tube formation and rat aorta models in a three-dimensional collagen matrix. Furthermore, antimigrative effects of the fractions were assessed using a wound healing model. The ethanol extract of S. officinalis (ESO) potently inhibited capillary tube formation in HUVEC and rat aorta models of angiogenesis, and its hexane fraction (HSO) exerted the highest inhibitory effect. In addition, the ethanol extract of S. officinalis and its hexane fraction showed a dose-dependent inhibitory activity on the migration of the endothelial cells in the wound healing model. Furthermore, ESO inhibited endothelial cell proliferation at 50-200 μg/mL in a dose-dependent manner. These findings indicated some new pharmacological activities of S. officinalis such as antiangiogenic in vitro and ex vivo, and antimigrative activity in vitro. Therefore, S. officinalis could be a candidate as a useful herb with therapeutic or preventive activity against angiogenesis related disorders.

  15. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting: Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4{sup +} T cells compared

    Energy Technology Data Exchange (ETDEWEB)

    Ford, A.L.; Goodsall, A.L.; Sedgwick, J.D. [Centenary Institute of Cancer Medicine and Cell Biology, Sydney (Australia)] [and others

    1995-05-01

    Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4{sup +} cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45{sup low}CD11b/c{sup +} is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45{sup high}CD11b/c{sup +}) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45{sup high}CD11 b/c{sup +} transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4{sup +} myelin basic protein (MBP)-reactive T cells. CD45{sup high}CD11b/c{sup +} CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4{sup +} T cells to proliferate or secrete IL-2. 60 refs., 6 figs., 1 tab.

  16. In vitro and ex vivo strategies for intracellular delivery

    Science.gov (United States)

    Stewart, Martin P.; Sharei, Armon; Ding, Xiaoyun; Sahay, Gaurav; Langer, Robert; Jensen, Klavs F.

    2016-10-01

    Intracellular delivery of materials has become a critical component of genome-editing approaches, ex vivo cell-based therapies, and a diversity of fundamental research applications. Limitations of current technologies motivate development of next-generation systems that can deliver a broad variety of cargo to diverse cell types. Here we review in vitro and ex vivo intracellular delivery approaches with a focus on mechanisms, challenges and opportunities. In particular, we emphasize membrane-disruption-based delivery methods and the transformative role of nanotechnology, microfluidics and laboratory-on-chip technology in advancing the field.

  17. Pathogenic Effects of Human Herpesvirus 6 in Human Lymphoid Tissue Ex Vivo

    OpenAIRE

    Grivel, Jean-Charles; Santoro, Fabio; Chen, Silvia; Fagá, Giovanni; Malnati, Mauro S.; Ito, Yoshinori; Margolis, Leonid; Lusso, Paolo

    2003-01-01

    Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive agent that has been suggested to act as a cofactor in the progression of human immunodeficiency virus disease. However, the lack of suitable experimental models has hampered the elucidation of the mechanisms of HHV-6-mediated immune suppression. Here, we used ex vivo lymphoid tissue to investigate the cellular tropism and pathogenic mechanisms of HHV-6. Viral strains belonging to both HHV-6 subgroups (A and B) were able to product...

  18. Modeling placental transport: correlation of in vitro BeWo cell permeability and ex vivo human placental perfusion

    DEFF Research Database (Denmark)

    Poulsen, Marie Sønnegaard; Rytting, Erik; Mose, Tina;

    2009-01-01

    The placental passage of three compounds with different physicochemical properties was recently investigated in ex vivo human placental perfusion experiments (caffeine, benzoic acid, and glyphosate) [Mose, T., Kjaerstad, M.B., Mathiesen, L., Nielsen, J.B., Edelfors, S., Knudsen, L.E., 2008....... Placental passage of benzoic acid, caffeine, and glyphosate in an ex vivo human perfusion system. J. Toxicol. Environ. Health, Part A 71, 984-991]. In this work, the transport of these same three compounds, plus the reference compound antipyrine, was investigated using BeWo (b30) cell monolayers. Transport...

  19. Depleted uranium disturbs immune parameters in zebrafish, Danio rerio: an ex vivo/in vivo experiment.

    Science.gov (United States)

    Gagnaire, Béatrice; Bado-Nilles, Anne; Sanchez, Wilfried

    2014-10-01

    In this study, we investigated the effects of depleted uranium (DU), the byproduct of nuclear enrichment of uranium, on several parameters related to defence system in the zebrafish, Danio rerio, using flow cytometry. Several immune cellular parameters were followed on kidney leucocytes: cell proportion, cell mortality, phagocytosis activity and associated oxidative burst and lysosomal membrane integrity (LMI). Effects of DU were tested ex vivo after 17 h of contact between DU and freshly isolated leucocytes from 0 to 500 µg DU/L. Moreover, adult zebrafish were exposed in vivo during 3 days at 20 and 250 µg DU/L. Oxidative burst results showed that DU increased reactive oxygen species (ROS) basal level and therefore reduced ROS stimulation index in both ex vivo and in vivo experiments. ROS PMA-stimulated level was also increased at 250 µg DU/L in vivo only. Furthermore, a decrease of LMI was detected after in vivo experiments. Cell mortality was also decreased at 20 µg DU/L in ex vivo experiment. However, phagocytosis activity was not modified in both ex vivo and in vivo experiments. A reduction of immune-related parameters was demonstrated in zebrafish exposed to DU. DU could therefore decrease the ability of fish to stimulate its own immune system which could, in turn, enhance the susceptibility of fish to infection. These results encourage the development and the use of innate immune analysis by flow cytometry in order to understand the effects of DU and more generally radionuclides on fish immune system and response to infectious diseases. PMID:24723161

  20. 5-Azacytidine and Rapamycin: different and synergistic effect on ex vivo expansion of natural human T Regulatory cells

    OpenAIRE

    Conteduca, Giuseppina

    2015-01-01

    BACKGROUND: Natural T regulatory cells (Treg) are challenging to expand ex vivo, and this has severely hindered in vivo evaluation of their therapeutic potential. 5-Azacytidine (5-azaC) and Rapamycin (RAPA) are immunosuppressive drugs that promote selectively the expansion of CD4+CD25highFoxp3+ regulatory T cells OBJECTIVE: We investigated whether 5-azaC and RAPA could be used together to promote the ex vivo expansion of Tregs purified from adult human peripheral blood. METHODS: CD4+C...

  1. Hematopoietic stem cells: ex-vivo expansion and therapeutic potential for myocardial ischemia

    Directory of Open Access Journals (Sweden)

    Jingwei Lu

    2010-03-01

    Full Text Available Jingwei Lu, Vincent J Pompili, Hiranmoy DasCardiovascular Stem Cell Research Laboratory, The Dorothy M Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, OH 43210, USAAbstract: Despite recent advances in cardiovascular medicine, ischemic heart disease remains the major cause of death in the United States and abroad. Cell-based therapy for degenerative diseases like myocardial ischemia using stem cells is currently under serious investigation. Various types of stem cells are being considered to be candidates for cell transplantation in cell-based therapy. Hematopoietic stem cells are one of the most promising cell types as several studies demonstrated their ability to improve ischemic cardiac functions by enhancing neovascularization and by reducing the total size of scar tissue. However, in order to procure sufficient numbers of functional stem cells, ex-vivo expansion technology became critically important. In this review, we focus on the state-of-the-art ex-vivo technology for the expansion of hematopoietic stem cells, and the underlying mechanisms regulating stem cell self-renewal as well as differentiation.Keywords: ischemic heart disease, ex-vivo expansion, hematopoietic stem cells, cytokines, nanofibers

  2. Current status of ex vivo gene therapy for hematological disorders: a review of clinical trials in Japan around the world.

    Science.gov (United States)

    Tani, Kenzaburo

    2016-07-01

    Gene therapies are classified into two major categories, namely, in vivo and ex vivo. Clinical trials of human gene therapy began with the ex vivo techniques. Based on the initial successes of gene-therapy clinical trials, these approaches have spread worldwide. The number of gene therapy trials approved worldwide increased gradually starting in 1989, reaching 116 protocols per year in 1999, and a total of 2210 protocols had been approved by 2015. Accumulating clinical evidence has demonstrated the safety and benefits of several types of gene therapy, with the exception of serious adverse events in several clinical trials. These painful experiences were translated backward to basic science, resulting in the development of several new technologies that have influenced the recent development of ex vivo gene therapy in this field. To date, six gene therapies have been approved in a limited number of countries worldwide. In Japan, clinical trials of gene therapy have developed under the strong influence of trials in the US and Europe. Since the initial stages, 50 clinical trials have been approved by the Japanese government. In this review, the history and current status of clinical trials of ex vivo gene therapy for hematological disorders are introduced and discussed. PMID:27289360

  3. Ex vivo fracture resistance of direct resin composite complete crowns with and without posts on maxillary premolars.

    NARCIS (Netherlands)

    Fokkinga, W.A.; Bell, A.M. Le; Kreulen, C.M.; Lassila, L.V.; Vallittu, P.K.; Creugers, N.H.J.

    2005-01-01

    AIM: To investigate ex vivo the fracture resistance and failure mode of direct resin composite complete crowns with and without various root canal posts made on maxillary premolars. METHODOLOGY: The clinical crowns of 40 human extracted single-rooted maxillary premolars were sectioned at the cemento

  4. Rat precision-cut intestinal slices to study P-gp activity and the potency of its inhibitors ex vivo

    NARCIS (Netherlands)

    Li, Ming; de Graaf, Inge A M; de Jager, Marina H; Groothuis, Geny M M

    2015-01-01

    Rat Precision-Cut Intestinal Slices (PCIS) were evaluated as ex vivo model to study the regional gradient of P-gp activity, and to investigate whether the rank order of inhibitory potency of P-gp inhibitors can be correctly reproduced in this model with more accurate IC50 values than with current in

  5. Precision cut intestinal slices are an appropriate ex vivo model to study NSAID-induced intestinal toxicity in rats

    NARCIS (Netherlands)

    Niu, Xiaoyu; de Graaf, Inge A. M.; van der Bij, Hendrik A.; Groothuis, Geny M. M.

    2014-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used therapeutic agents, however, they are associated with a high prevalence of intestinal side effects. In this investigation, rat precision cut intestinal slices (PCIS) were evaluated as an ex vivo model to study NSAID-induced intestinal to

  6. High-wavenumber FT-Raman spectroscopy for in vivo and ex vivo measurements of breast cancer

    DEFF Research Database (Denmark)

    Garcia-Flores, A. F.; Raniero, L.; Canevari, R. A.;

    2011-01-01

    The identification of normal and cancer breast tissue of rats was investigated using high-frequency (HF) FT-Raman spectroscopy with a near-infrared excitation source on in vivo and ex vivo measurements. Significant differences in the Raman intensities of prominent Raman bands of lipids and protei...

  7. Mechanical Properties of Healthy and ex vivo Onychomycosis Nails and the Influence of a Porphyrin-propylene Glycol Antifungal Formulation

    NARCIS (Netherlands)

    A. Hosseinzoi (Amu); F. Galli (Federica); L. Incrocci (Luca); T. Smijs (Threes)

    2016-01-01

    textabstractAims: To investigate nail penetration enhancing effectiveness of a novel drug formulation and ingredients, 40% propylene glycol (PG) and 40 μM multifunctional photosensitizer (MFPS). Proposed formulation was proven effective in photodynamic treatment (PDT) of ex vivo fungal infections of

  8. Ex vivo culture of patient tissue & examination of gene delivery.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

  9. An organotypic slice model for ex vivo study of neural, immune, and microbial interactions of mouse intestine.

    Science.gov (United States)

    Schwerdtfeger, Luke A; Ryan, Elizabeth P; Tobet, Stuart A

    2016-02-15

    Organotypic tissue slices provide seminatural, three-dimensional microenvironments for use in ex vivo study of specific organs and have advanced investigative capabilities compared with isolated cell cultures. Several characteristics of the gastrointestinal tract have made in vitro models for studying the intestine challenging, such as maintaining the intricate structure of microvilli, the intrinsic enteric nervous system, Peyer's patches, the microbiome, and the active contraction of gut muscles. In the present study, an organotypic intestinal slice model was developed that allows for functional investigation across regions of the intestine. Intestinal tissue slices were maintained ex vivo for several days in a physiologically relevant environment that preserved normal enterocyte structure, intact and proliferating crypt cells, submucosal organization, and muscle wall composure. Cell death was measured by a membrane-impermeable DNA binding indicator, ethidium homodimer, and less than 5% of cells were labeled in all regions of the villi and crypt epithelia at 24 h ex vivo. This tissue slice model demonstrated intact myenteric and submucosal neuronal plexuses and functional interstitial cells of Cajal to the extent that nonstimulated, segmental contractions occurred for up to 48 h ex vivo. To detect changes in physiological responses, slices were also assessed for segmental contractions in the presence and absence of antibiotic treatment, which resulted in slices with lesser or greater amounts of commensal bacteria, respectively. Segmental contractions were significantly greater in slices without antibiotics and increased native microbiota. This model renders mechanisms of neuroimmune-microbiome interactions in a complex gut environment available to direct observation and controlled perturbation.

  10. Ex-vivo response to blood products and haemostatic agents after paediatric cardiac surgery

    DEFF Research Database (Denmark)

    Hvas, Anne-Mette; Andreasen, Jo B; Christiansen, Kirsten;

    2013-01-01

    cardiac surgery. The haemostatic potential of various factor concentrates (fibrinogen concentrate, recombinant factor VIIa and factor XIII), fresh frozen plasma (FFP), pooled platelets and tranexamic acid was investigated. After surgery, the coagulation profiles revealed significantly prolonged clotting...... of fibrinogen concentrate, FFP or tranexamic acid improved clot stability significantly. Whole blood coagulation was significantly impaired after cardiac surgery in children. Ex-vivo studies showed a total reversal of the coagulopathy after addition of pooled platelets and significantly improved clot stability...... after addition of fibrinogen concentrate, FFP and tranexamic acid, respectively....

  11. PASSIVE CAVITATION DETECTION DURING PULSED HIFU EXPOSURES OF EX VIVO TISSUES AND IN VIVO MOUSE PANCREATIC TUMORS

    Science.gov (United States)

    Li, Tong; Chen, Hong; Khokhlova, Tatiana; Wang, Yak-Nam; Kreider, Wayne; He, Xuemei; Hwang, Joo Ha

    2014-01-01

    Pulsed high-intensity focused ultrasound (pHIFU) has been demonstrated to enhance vascular permeability, disrupt tumor barriers and enhance drug penetration into tumor tissue through acoustic cavitation. Monitoring of cavitation activity during pHIFU treatments and knowing the ultrasound pressure levels sufficient to reliably induce cavitation in a given tissue are therefore very important. Here, three metrics of cavitation activity induced by pHIFU and evaluated by confocal passive cavitation detection were introduced: cavitation probability, cavitation persistence and the level of the broadband acoustic emissions. These metrics were used to characterize cavitation activity in several ex vivo tissue types (bovine tongue and liver and porcine adipose tissue and kidney) and gel phantoms (polyacrylamide and agarose) at varying peak-rarefactional focal pressures (1–12 MPa) during the following pHIFU protocol: frequency 1.1 MHz, pulse duration 1 ms, pulse repetition frequency 1 Hz. To evaluate the relevance of the measurements in ex vivo tissue, cavitation metrics were also investigated and compared in the ex vivo and in vivo murine pancreatic tumors that develop spontaneously in transgenic KPC mice and closely recapitulate human disease in their morphology. The cavitation threshold, defined at 50 % cavitation probability, was found to vary broadly among the investigated tissues (within 2.5–10 MPa), depending mostly on the water-lipid ratio that characterizes the tissue composition. Cavitation persistence and the intensity of broadband emissions depended both on tissue structure and lipid concentration. Both the cavitation threshold and broadband noise emission level were similar between ex vivo and in vivo pancreatic tumor tissue. The largest difference between in vivo and ex vivo settings was found in the pattern of cavitation occurrence throughout pHIFU exposure: it was sporadic in vivo, but ex vivo it decreased rapidly and stopped over the first few pulses

  12. Ex vivo infection of human embryonic spinal cord neurons prior to transplantation into adult mouse cord

    Directory of Open Access Journals (Sweden)

    Dénes Ádám

    2010-05-01

    Full Text Available Abstract Background Genetically modified pseudorabies virus (Prv proved suitable for the delivery of foreign genes to rodent embryonic neurons ex vivo and maintaining foreign gene expression after transplantation into spinal cord in our earlier study. The question arose of whether human embryonic neurons, which are known to be more resistant to Prv, could also be infected with a mutant Prv. Specifically, we investigated whether a mutant Prv with deleted ribonucleotide reductase and early protein 0 genes has the potential to deliver marker genes (gfp and β-gal into human embryonic spinal cord neurons and whether the infected neurons maintain expression after transplantation into adult mouse cord. Results The results revealed that the mutant Prv effectively infected human embryonic spinal cord neurons ex vivo and the grafted cells exhibited reporter gene expression for several weeks. Grafting of infected human embryonic cells into the spinal cord of immunodeficient (rnu-/rnu- mice resulted in the infection of some of the host neurons. Discussion These results suggest that Prv is suitable for the delivery of foreign genes into transplantable human cells. This delivery method may offer a new approach to use genetically modified cells for grafting in animal models where spinal cord neuronal loss or axon degeneration occurs.

  13. Linarin Inhibits the Acetylcholinesterase Activity In-vitro and Ex-vivo.

    Science.gov (United States)

    Feng, Xinchi; Wang, Xin; Liu, Youping; Di, Xin

    2015-01-01

    Linarin is a flavone glycoside in the plants Flos chrysanthemi indici, Buddleja officinalis, Cirsium setosum, Mentha arvensis and Buddleja davidii, and has been reported to possess analgesic, antipyretic, anti-inflammatory and neuroprotective activities. In this paper, linarin was investigated for its AChE inhibitory potential both in-vitro and ex-vivo. Ellman's colorimetric method was used for the determination of AChE inhibitory activity in mouse brain. In-vitro assays revealed that linarin inhibited AChE activity with an IC50 of 3.801 ± 1.149 μM. Ex-vivo study showed that the AChE activity was significantly reduced in both the cortex and hippocampus of mice treated intraperitoneally with various doses of linarin (35, 70 and 140 mg/Kg). The inhibition effects produced by high dose of linarin were the same as that obtained after huperzine A treatment (0.5 mg/Kg). Molecular docking study revealed that both 4'-methoxyl group and 7-O-sugar moiety of linarin played important roles in ligand-receptor binding and thus they are mainly responsible for AChE inhibitory activity. In view of its potent AChE inhibitory activity, linarin may be a promising therapeutic agent for the treatment of some diseases associated with AChE, such as glaucoma, myasthenia gravis, gastric motility and Alzheimer's disease.

  14. Angiogenic Potency of Bone Marrow Stromal Cells Improved by ex Vivo Hypoxia Prestimulation

    Institute of Scientific and Technical Information of China (English)

    毛晓波; 曾秋棠; 王祥; 曹林生; 白智峰

    2004-01-01

    To study the angiogenic potency of hypoxia-prestimulated bone marrow stromal cells (BMSCs) when transplanted into acute myocardial infarction models of rats. BMSCs were cultured under hypoxia condition for 24 h. Their expression of VEGF was investigated. The rat acute myocardial infarction models were made by coronary artery ligation and divided into 3 groups at random.In normoxia group, twice-passaged BMSCs were labeled with Bromodeoxyuridine (BrdU) and then implanted into the infarction regions and ischemic border of the recipients in 4 weeks. The rats in hypoxia group were implanted with hypoxia-prestimulated BMSCs. In control group, the model rats received only DMEM medium injection. Six-weeks after AMI, the infarction regions were examined to identify the angiogenesis and the expression of the VEGF. Our results showed that viable cells labeled with BrdU could be identified in the host hearts. The infarction regions in normoxia and hypoxia groups had a greater capillary density and increased VEGF expression than the regions in control group. The capillary density and VEGF expression in hypoxia group were higher than in normoxia group. It is concluded that the enhanced expression of VEGF in BMSCs could be induced by ex vivo hypoxia stimulation. BMSCs implantation promoted the angiogenesis in myocardial infarction tissue via supplying exogenic VEGF. Angiogenic potency of bone marrow stromal cells was improved by ex vivo hypoxia prestimulation though the enhanced VEGF expression.

  15. In vitro and ex vivo effect of hyaluronic acid on erythrocyte flow properties

    Directory of Open Access Journals (Sweden)

    Palatnik S

    2010-02-01

    Full Text Available Abstract Background Hyaluronic acid (HA is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA patients, a serum HA-increasing pathology. Methods Erythrocyte deformability (by filtration through nucleopore membranes and erythrocyte aggregability (EA were tested on blood from healthy donors additioned with purified HA. EA was measured by transmitted light and analyzed with a mathematical model yielding two parameters, the aggregation rate and the size of the aggregates. Conformational changes of cytoskeleton proteins were estimated by electron paramagnetic resonance spectroscopy (EPR. Results In vitro, erythrocytes treated with HA showed increased rigidity index (RI and reduced aggregability, situation strongly related to the rigidization of the membrane cytoskeleton triggered by HA, as shown by EPR results. Also, a significant correlation (r: 0.77, p Conclusions Our results lead us to postulate the hypothesis that HA interacts with the erythrocyte surface leading to modifications in erythrocyte rheological and flow properties, both ex vivo and in vitro.

  16. Ex vivo expansion of circulating CD34(+) cells enhances the regenerative effect on rat liver cirrhosis.

    Science.gov (United States)

    Nakamura, Toru; Koga, Hironori; Iwamoto, Hideki; Tsutsumi, Victor; Imamura, Yasuko; Naitou, Masako; Masuda, Atsutaka; Ikezono, Yu; Abe, Mitsuhiko; Wada, Fumitaka; Sakaue, Takahiko; Ueno, Takato; Ii, Masaaki; Alev, Cantas; Kawamoto, Atsuhiko; Asahara, Takayuki; Torimura, Takuji

    2016-01-01

    Ex vivo expansion of autologous cells is indispensable for cell transplantation therapy of patients with liver cirrhosis. The aim of this study was to investigate the efficacy of human ex vivo-expanded CD34(+) cells for treatment of cirrhotic rat liver. Recipient rats were intraperitoneally injected with CCl4 twice weekly for 3 weeks before administration of CD34(+) cells. CCl4 was then re-administered twice weekly for 3 more weeks, and the rats were sacrificed. Saline, nonexpanded or expanded CD34(+) cells were injected via the spleen. After 7 days, CD34(+) cells were effectively expanded in a serum-free culture medium. Expanded CD34(+) cells were also increasingly positive for cell surface markers of VE-cadherin, VEGF receptor-2, and Tie-2. The expression of proangiogenic growth factors and adhesion molecules in expanded CD34(+) cells increased compared with nonexpanded CD34(+) cells. Expanded CD34(+) cell transplantation reduced liver fibrosis, with a decrease of αSMA(+) cells. Assessments of hepatocyte and sinusoidal endothelial cell proliferative activity indicated the superior potency of expanded CD34(+) cells over non-expanded CD34(+) cells. The inhibition of integrin αvβ3 and αvβ5 disturbed the engraftment of transplanted CD34(+) cells and aggravated liver fibrosis. These findings suggest that expanded CD34(+) cells enhanced the preventive efficacy of cell transplantation in a cirrhotic model. PMID:27162932

  17. Consequences of Mrp2 deficiency for diclofenac toxicity in the rat intestine ex vivo.

    Science.gov (United States)

    Niu, Xiaoyu; de Graaf, Inge A M; van de Vegte, Dennis; Langelaar-Makkinje, Miriam; Sekine, Shuichi; Groothuis, Geny M M

    2015-02-01

    The non-steroidal anti-inflammatory drug diclofenac (DCF) has a high prevalence of intestinal side effects in humans and rats. It has been reported that Mrp2 transporter deficient rats (Mrp2) are more resistant to DCF induced intestinal toxicity. This was explained in vivo by impaired Mrp2-dependent biliary transport of DCF-acylglucuronide (DAG), leading to decreased intestinal exposure to DAG and DCF. However, it is not known to what extent adaptive changes in the Mrp2 intestine itself influence its sensitivity to DCF toxicity without the influence of liver metabolites. To investigate this, DCF toxicity and disposition were studied ex vivo by precision-cut intestinal slices and Ussing chamber using intestines from wild type(WT) and Mrp2 rats. The results show that adaptive changes due to Mrp2 deficiency concerning Mrp2, Mrp3 and BCRP gene expression, GSH content and DAG formation were different between liver and intestine. Furthermore, Mrp2 intestine was intrinsically more resistant to DCF toxicity than its WT counterpart ex vivo. This can at least partly be explained by a reduced DCF uptake by the Mrp2 intestine, but isnot related to the other adaptive changes in the intestine. The extrapolation of this data to humans with MRP2 deficiency is uncertain due to species differences in activity and regulation of transporters.

  18. Polyethylene glycol diffusion in ex vivo skin tissue

    Science.gov (United States)

    Genin, V. D.; Tuchina, D. K.; Bashkatov, A. N.; Genina, E. A.; Tuchin, V. V.

    2015-11-01

    Optical clearing of the rat skin under the action of polyethylene glycol (PEG) with molecular weight 300 and 400 Dalton was studied ex vivo. The collimated transmittance was measured at the wavelength range 500-900 nm. It was found that collimated transmittance of skin samples increased, whereas weight, thickness and area of the samples decreased during PEG penetration in skin tissue. A mechanism of the optical clearing under the action of PEG is discussed. Taking into account the kinetics of volume and thickness of the skin samples, diffusion coefficient of PEGs in skin tissue has been estimated as (1.83±2.22)×10-6 cm2/s and (1.70±1.47)×10-6 cm2/s for PEG-300 and PEG-400, respectively. The presented results can be useful for enhancement of many methods of laser therapy and optical diagnostics of skin diseases and localization of subcutaneous neoplasms.

  19. The clinical potential of ex vivo lung perfusion.

    Science.gov (United States)

    Cypel, Marcelo; Keshavjee, Shaf

    2012-02-01

    The number of patients listed for lung transplantation largely exceeds the number of available transplantable organs because of both a shortage of organ donors and a low utilization rate of donor lungs. Normothermic ex vivo lung perfusion (EVLP) is a method that maintains the organ in physiologically protective conditions outside the body during preservation, and shows great promise to increase utilization of donor lungs by allowing more accurate evaluation, as well as treatment and repair, of damaged donor lungs prior to transplantation. This article will cover the rationale, technical details and results of experimental and clinical studies with EVLP. The significant potential applications of EVLP in lung transplantation, lung regeneration and oncology are discussed. PMID:22283576

  20. Assessment of donor heart viability during ex vivo heart perfusion.

    Science.gov (United States)

    White, Christopher W; Ambrose, Emma; Müller, Alison; Li, Yun; Le, Hoa; Hiebert, Brett; Arora, Rakesh; Lee, Trevor W; Dixon, Ian; Tian, Ganghong; Nagendran, Jayan; Hryshko, Larry; Freed, Darren

    2015-10-01

    Ex vivo heart perfusion (EVHP) may facilitate resuscitation of discarded donor hearts and expand the donor pool; however, a reliable means of demonstrating organ viability prior to transplantation is required. Therefore, we sought to identify metabolic and functional parameters that predict myocardial performance during EVHP. To evaluate the parameters over a broad spectrum of organ function, we obtained hearts from 9 normal pigs and 37 donation after circulatory death pigs and perfused them ex vivo. Functional parameters obtained from a left ventricular conductance catheter, oxygen consumption, coronary vascular resistance, and lactate concentration were measured, and linear regression analyses were performed to identify which parameters best correlated with myocardial performance (cardiac index: mL·min(-1)·g(-1)). Functional parameters exhibited excellent correlation with myocardial performance and demonstrated high sensitivity and specificity for identifying hearts at risk of poor post-transplant function (ejection fraction: R(2) = 0.80, sensitivity = 1.00, specificity = 0.85; stroke work: R(2) = 0.76, sensitivity = 1.00, specificity = 0.77; minimum dP/dt: R(2) = 0.74, sensitivity = 1.00, specificity = 0.54; tau: R(2) = 0.51, sensitivity = 1.00, specificity = 0.92), whereas metabolic parameters were limited in their ability to predict myocardial performance (oxygen consumption: R(2) = 0.28; coronary vascular resistance: R(2) = 0.20; lactate concentration: R(2) = 0.02). We concluded that evaluation of functional parameters provides the best assessment of myocardial performance during EVHP, which highlights the need for an EVHP device capable of assessing the donor heart in a physiologic working mode.

  1. Optimized isolation enables Ex vivo analysis of microglia from various central nervous system regions

    NARCIS (Netherlands)

    De Haas, Alexander H.; Boddeke, Hendricus W. G. M.; Brouwer, Nieske; Biber, Knut

    2007-01-01

    Ex vivo analysis is an accurate and convenient way to study in vivo microglia phenotype and function. However, current microglia isolation protocols for ex vivo analysis show many differences in isolation steps (perfusion, removal of meninges and blood vessels, mechanical dissociation, enzymatic dis

  2. Precision cut intestinal slices are an appropriate ex vivo model to study NSAID-induced intestinal toxicity in rats.

    Science.gov (United States)

    Niu, Xiaoyu; de Graaf, Inge A M; van der Bij, Hendrik A; Groothuis, Geny M M

    2014-10-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used therapeutic agents, however, they are associated with a high prevalence of intestinal side effects. In this investigation, rat precision cut intestinal slices (PCIS) were evaluated as an ex vivo model to study NSAID-induced intestinal toxicity. Firstly, PCIS were incubated with 0-200 μM diclofenac (DCF), one of the most intensively studied NSAIDs, to investigate whether they could correctly reflect the toxic mechanisms. DCF induced intestinal toxicity in PCIS was shown by morphological damage and ATP depletion. DCF induced endoplasmic-reticulum (ER) stress, mitochondrial injury and oxidative stress were reflected by up-regulated HSP-70 (heat shock protein 70) and BiP (binding immunoglobulin protein) gene expression, caspase 9 activation, GSH (glutathione) depletion and HO-1 (heme oxygenase 1) gene up-regulation respectively. Furthermore, DCF intestinal metabolites, which gave rise to protein adduct but not toxicity, were detected in PCIS. Secondly, PCIS were incubated with various concentrations of five NSAIDs. Typical NSAID-induced morphological changes were observed in PCIS. The ex vivo toxicity ranking (diflunisal> diclofenac = indomethacin > naproxen ≫ aspirin) showed good correlation with published in vitro and in vivo data, with diflunisal being the only exception. In conclusion, PCIS correctly reflect the various mechanisms of DCF-induced intestinal toxicity, and can serve as an ex vivo model for the prediction of NSAID-induced intestinal toxicity. PMID:25014874

  3. ELITE--the ex vivo training unit for NOTES: development and validation.

    Science.gov (United States)

    Fiolka, Adam; Gillen, Sonja; Meining, Alexander; Feussner, Hubertus

    2010-10-01

    Skill training is an essential part of surgical education. Every physician has to get familiar with the various operation techniques and needs to handle the different instruments. However, mechanical and computer-based VR-simulators offer only one specific procedure, either laparoscopic or endoscopic. We designed the universal training system ELITE (endoscopic-laparoscopic interdisciplinary training entity) which is a new full synthetic ex vivo surgical training model for laparoscopic surgery, combined endoluminal/endocavitary procedures ("hybrid surgery") and NOTES. The aim of the current investigation was to integrate respiration and electro dissection into the model, and the evaluation of both innovations. The ELITE is a full-size replica of a human female torso including a gas-tight abdominal wall and offering various accesses to the abdomen. A complete organ package including liver, gallbladder, spleen, gastrointestinal tract, including the mesentery and omentum is available for this system. Cholecystectomy and appendectomy can be simulated realistically with this new training system. For more realistic conditions during operations breathing-induced organ motion could be integrated into this system. Two latex balloons were inserted into the system to imitate the function of the diaphragm. They are inflated and deflated according to the respiration cycle and move the artificial organs in a natural way. Physicians, including endoscopic/laparoscopic novices and experts, were asked to train different NOTES procedures on the model. Performance of their training and subjective appraisal of the model itself were evaluated. The opportunity of electrodissection of the gallbladder and appendix and simulation of breath excursion of the diaphragm could successfully be implemented into the training system. One recently published study showed that ELITE is a suitable tool to train different surgical procedures. All subjects (novices and endoscopic/laparoscopic experts

  4. Expression of Plasmodium vivax crt-o Is Related to Parasite Stage but Not Ex Vivo Chloroquine Susceptibility.

    Science.gov (United States)

    Pava, Zuleima; Handayuni, Irene; Wirjanata, Grennady; To, Sheren; Trianty, Leily; Noviyanti, Rintis; Poespoprodjo, Jeanne Rini; Auburn, Sarah; Price, Ric N; Marfurt, Jutta

    2015-11-02

    Chloroquine (CQ)-resistant Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivax crt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax β-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5; P < 0.001). Twenty-nine isolates fulfilled the criteria for ex vivo drug susceptibility testing and showed high variability in CQ responses (median, 107.9 [range, 6.5 to 345.7] nM). After controlling for the parasite stage, we found that pvcrt-o expression levels did not correlate with the ex vivo response to CQ or with that to any of the other antimalarials tested. Our results highlight the importance of development-stage composition for measuring pvcrt-o expression and suggest that pvcrt-o transcription is not a primary determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance.

  5. Imaging the pancreas: from ex vivo to non-invasive technology

    DEFF Research Database (Denmark)

    Holmberg, D; Ahlgren, U

    2008-01-01

    While many recently published reviews have covered non-invasive nuclear imaging techniques, the aim of this review is to focus on current developments in optical imaging technologies for investigating the pancreas. Several of these modalities are being developed into non-invasive, real-time monit......While many recently published reviews have covered non-invasive nuclear imaging techniques, the aim of this review is to focus on current developments in optical imaging technologies for investigating the pancreas. Several of these modalities are being developed into non-invasive, real......-time monitoring routines for pancreatic diseases. However, they also provide pre-clinical ex vivo and/or intravital tools for three-dimensional quantitative assessments of cellular and molecular events, with levels of specificity and resolution difficult to achieve with other currently available modalities....

  6. Human Ex-Vivo Liver Model for Acetaminophen-induced Liver Damage

    Science.gov (United States)

    Schreiter, Thomas; Sowa, Jan-Peter; Schlattjan, Martin; Treckmann, Jürgen; Paul, Andreas; Strucksberg, Karl-Heinz; Baba, Hideo A.; Odenthal, Margarete; Gieseler, Robert K.; Gerken, Guido; Arteel, Gavin E.; Canbay, Ali

    2016-01-01

    Reliable test systems to identify hepatotoxicity are essential to predict unexpected drug-related liver injury. Here we present a human ex-vivo liver model to investigate acetaminophen-induced liver injury. Human liver tissue was perfused over a 30 hour period with hourly sampling from the perfusate for measurement of general metabolism and clinical parameters. Liver function was assessed by clearance of indocyanine green (ICG) at 4, 20 and 28 hours. Six pieces of untreated human liver specimen maintained stable liver function over the entire perfusion period. Three liver sections incubated with low-dose acetaminophen revealed strong damage, with ICG half-lives significantly higher than in non-treated livers. In addition, the release of microRNA-122 was significantly higher in acetaminophen-treated than in non-treated livers. Thus, this model allows for investigation of hepatotoxicity in human liver tissue upon applying drug concentrations relevant in patients. PMID:27550092

  7. Comparative study on measured variables and sensitivity to bone microstructural changes induced by weightlessness between in vivo and ex vivo micro-CT scans.

    Science.gov (United States)

    Sun, Lian Wen; Wang, Chao; Pu, Fang; Li, De Yu; Niu, Hai Jun; Fan, Yu Bo

    2011-01-01

    Depending on the experimental design, micro-CT can be used to examine bones either in vivo or ex vivo (excised fresh or formalin-fixed). In this study we investigated if differences exist in the variables measured by micro-CT between in vivo and ex vivo scans and which kind of scan is more sensitive to the changes of bone microstructure induced by simulated weightlessness. Rat tail suspension was used to simulate the weightless condition. The same bone from either normal or tail-suspended rats was scanned by micro-CT both in vivo and ex vivo (fresh and fixed by formalin). Then, bone mineral density (BMD) and microstructural characteristics were analyzed. The results showed that no significant differences existed in the microstructural parameters of trabecular bone among in vivo, fresh, and formalin-fixed bone scans from both femurs and tibias, although BMD exhibited differences. On the other hand, most parameters of the tail-suspended rats measured by micro-CT deteriorated compared with controls. Ex vivo scanning appeared to be more sensitive to bone microstructural changes induced by tail suspension than in vivo scanning. In general, the results indicate that values obtained in vivo and ex vivo (fresh and fixed) are comparable, thus allowing for meaningful comparison of experimental results from different studies irrespective of the type of scans. In addition, this study suggests that it is better to use ex vivo scanning when evaluating bone microstructure under weightlessness. However, researchers can select any type of scan depending upon the objective and the demands of the experiment.

  8. In vitro and ex vivo evaluations on transdermal delivery of the HIV inhibitor IQP-0410.

    Directory of Open Access Journals (Sweden)

    Anthony S Ham

    Full Text Available The aim of this study was to investigate the physicochemical and in vitro/ex vivo characteristics of the pyrmidinedione IQP-0410 formulated into transdermal films. IQP-0410 is a potent therapeutic anti-HIV nonnucleoside reverse transcriptase inhibitor that would be subjected to extensive first pass metabolism, through conventional oral administration. Therefore, IQP-0410 was formulated into ethyl cellulose/HPMC-based transdermal films via solvent casting. In mano evaluations were performed to evaluate gross physical characteristics. In vitro release studies were performed in both Franz cells and USP-4 dissolution vessels. Ex vivo release and permeability assays were performed on human epidermal tissue models, and the permeated IQP-0410 was collected for in vitro HIV-1 efficacy assays in CEM-SS cells and PBMCs. Film formulation D3 resulted in pliable, strong transdermal films that were loaded with 2% (w/w IQP-0410. Composed of 60% (w/w ethyl cellulose and 20% (w/w HPMC, the films contained < 1.2% (w/w of water and were hygroscopic resulting in significant swelling under humid conditions. The water permeable nature of the film resulted in complete in vitro dissolution and drug release in 26 hours. When applied to ex vivo epidermal tissues, the films were non-toxic to the tissue and also were non-toxic to HIV target cells used in the in vitro efficacy assays. Over a 3 day application, the films delivered IQP-0410 through the skin tissue at a zero-order rate of 0.94 ± 0.06 µg/cm(2/hr with 134 ± 14.7 µM collected in the basal media. The delivered IQP-0410 resulted in in vitro EC50 values against HIV-1 of 2.56 ± 0.40 nM (CEM-SS and 0.58 ± 0.03 nM (PBMC. The film formulation demonstrated no significant deviation from target values when packaged in foil pouches under standard and accelerated environmental conditions. It was concluded that the transdermal film formulation was a potentially viable method of administering IQP-0410 that warrants

  9. Ex-vivo detection of neural events using THz BioMEMS

    CERN Document Server

    Abbas, Abdennour; Croix, Dominique; Salzet, Michel; Bocquet, Bertrand

    2009-01-01

    Background: Electromagnetic frequencies up to a few terahertz (THz) can yield real-time and noninvasive measurements on biological matter. Unfortunately, strong absorption in aqueous solutions and low spatial resolution return difficult free-space investigations. A new approach based on integrated THz circuits was used. The authors designed and fabricated a BioMEMS (Biological MicroElectro-Mechanical System) compatible with microfluidic circulation and electromagnetic propagation. It is dedicated to the ex vivo detection of nitric oxide synthase (NOS) activity, which is involved in neurodegenerative phenomena. Material/Methods: The biological model was a leech's central nervous system. After its injury, the production of NO was observed and measured in the far-THz spectral domain. The nerve cord was put inside a BioMEMS realized in polydimethylsiloxane (PDMS) sealed on a glass wafer. Glass is a good material for supporting high-frequency integrated waveguides such as coplanar waveguides (CPWs). Measurements w...

  10. CD80 antigen expression as a predictor of ex vivo chemosensitivity in chronic lymphocytic leukemia.

    Science.gov (United States)

    Kivekäs, Ilkka; Hulkkonen, Janne; Hurme, Mikko; Vilpo, Leena; Vilpo, Juhani

    2002-05-01

    We investigated the correlation between expression of 31 surface membrane antigens and chemosensitivity of peripheral blood mononuclear cells from 36 patients with CLL. The sensitivity of CLL cells to nine drugs (2'-chlorodeoxyadenosine, cisplatin, chlorambucil, cyclosporin A, doxorubicin, fludarabine, prednisolone, verapamil and vincristine) and two types of irradiation (gamma and UV-irradiation) was determined from dose-response curves of 4-day cultures ex vivo. The results indicated that the CLL cases responding to purine analogs (2'-chlorodeoxyadenosine and fludarabine) can be identified according to CD80 expression: all resistant cases had low or negative CD80 expression. No other correlations were revealed. CD80 may be a surrogate chemosensitivity marker for purine analogs. PMID:11916516

  11. Ex vivo estimation of photoacoustic imaging for detecting thyroid microcalcifications.

    Directory of Open Access Journals (Sweden)

    Jeeun Kang

    Full Text Available BACKGROUND: The aim of this study was to evaluate the diagnostic utility of PAI at detecting thyroid microcalcifications at 700 nm laser wavelengths. METHODS: This study included 36 resected samples in 18 patients. To evaluate the PA manifestation of microcalcifications in PAI, gray level histogram and co-occurrence matrix (COM texture parameters were extracted from the 3 fixed ROI US and PA images, respectively, per sample. We compared the textural parameters obtained from specimen PAIs between samples with punctate microcalcifications on specimen radiography and those without microcalcifications. RESULTS: On specimen US, the mean value (2748.4 ± 862.5 of samples with microcalcifications on specimen radiography was higher than that (1961.9 ± 780.2 of those without microcalcifications (P = 0.007. However, there were no significant differences in textural parameters obtained from specimen PAIs between samples with punctate microcalcifications on specimen radiography and those without when applying both the mean value of the three slices of thyroid specimens and the value of the thyroid specimen slice which had the highest value of the mean values in specimen US. CONCLUSION: PAI did not show significant PA contrast on thyroid microcalcifications indicating that the experimental setup and protocols should be enhanced, e.g., method of complete blood rejection from ex vivo specimens, the multi-wavelength spectroscopic PA imaging method which can solely extract the PA signal from microcalcifications even with high spectral interferences, or PA imaging with narrower slice thickness using 2-dimensional array transducer, etc.

  12. Ex Vivo Perfusion Treatment of Infection in Human Donor Lungs.

    Science.gov (United States)

    Nakajima, D; Cypel, M; Bonato, R; Machuca, T N; Iskender, I; Hashimoto, K; Linacre, V; Chen, M; Coutinho, R; Azad, S; Martinu, T; Waddell, T K; Hwang, D M; Husain, S; Liu, M; Keshavjee, S

    2016-04-01

    Ex vivo lung perfusion (EVLP) is a platform to treat infected donor lungs with antibiotic therapy before lung transplantation. Human donor lungs that were rejected for transplantation because of clinical concern regarding infection were randomly assigned to two groups. In the antibiotic group (n = 8), lungs underwent EVLP for 12 h with high-dose antibiotics (ciprofloxacin 400 mg or azithromycin 500 mg, vancomycin 15 mg/kg, and meropenem 2 g). In the control group (n = 7), lungs underwent EVLP for 12 h without antibiotics. A quantitative decrease in bacterial counts in bronchoalveolar lavage (BAL) was found in all antibiotic-treated cases but in only two control cases. Perfusate endotoxin levels at 12 h were significantly lower in the antibiotic group compared with the control group. EVLP with broad-spectrum antibiotic therapy significantly improved pulmonary oxygenation and compliance and reduced pulmonary vascular resistance. Perfusate endotoxin levels at 12 h were strongly correlated with levels of perfusates tumor necrosis factor α, IL-1β and macrophage inflammatory proteins 1α and 1β at 12 h. In conclusion, EVLP treatment of infected donor lungs with broad-spectrum antibiotics significantly reduced BAL bacterial counts and endotoxin levels and improved donor lung function. PMID:26730551

  13. Ex vivo laser lipolysis assisted with radially diffusing optical applicator

    Science.gov (United States)

    Hwang, Jieun; Hau, Nguyen Trung; Park, Sung Yeon; Rhee, Yun-Hee; Ahn, Jin-Chul; Kang, Hyun Wook

    2016-05-01

    Laser-assisted lipolysis has been implemented to reduce body fat in light of thermal interactions with adipose tissue. However, using a flat fiber with high irradiance often needs rapid cannula movements and even undesirable thermal injury due to direct tissue contact. The aim of the current study was to explore the feasibility of a radially diffusing optical applicator to liquefy the adipose tissue for effective laser lipolysis. The proposed diffuser was evaluated with a flat fiber in terms of temperature elevation and tissue liquefaction after laser lipolysis with a 980-nm wavelength. Given the same power (20 W), the diffusing applicator generated a 30% slower temperature increase with a 25% lower maximum temperature (84±3.2°C in 1 min ptissue, compared with the flat fiber. Under the equivalent temperature development, the diffuser induced up to fivefold larger area of the adipose liquefaction due to radial light emission than the flat fiber. Ex vivo tissue tests for 5-min irradiation demonstrated that the diffuser (1.24±0.15 g) liquefied 66% more adipose tissue than the flat fiber (0.75±0.05 g). The proposed diffusing applicator can be a feasible therapeutic device for laser lipolysis due to low temperature development and wide coverage of thermal treatment.

  14. Development of domperidone bilayered matrix type transdermal patches: physicochemical, in vitro and ex vivo characterization

    Directory of Open Access Journals (Sweden)

    S.K Madishetti

    2010-09-01

    Full Text Available "nBackground and the purpose of the study: Domperidone (DOM is a dopamine- receptor (D2 antagonist, which is widely used in the treatment of motion-sickness. The pharmacokinetic parameters make DOM a suitable candidate for transdermal delivery. The purpose of the present investigation was to develop transdermal delivery systems for DOM and to evaluate their physicochemical characteristics, in vitro release an ex vivo permeation through rat abdominal skin and their mechanical properties. "nMethods: Bilayered matrix type transdermal drug delivery systems (TDDS of DOM were prepared by film casting technique using hydroxypropyl methyl cellulose as primary and Eudragit RL 100 as secondary layers. Brij-35 was incorporated as a solubilizer, d-limonene and propylene glycol were employed as permeation enhancer and plasticizer respectively. The prepared TDDS were extensively evaluated for in vitro release, moisture absorption, moisture content, water vapor transmission, ex vivo permeation through rat abdominal skin, mechanical properties and stability studies. The physicochemical interaction between DOM and polymers were investigated by Differential Scanning Calorimetry (DSC and Fourier Transform Infrared Spectroscopy (FTIR. "nResults: All the formulations exhibited satisfactory physicochemical and mechanical characteristics. The optimized formulation F6 showed maximum cumulative percentage of drug release (90.7%, permeation (6806.64 μg in 24 hrs, flux (86.02 μg /hr/cm2 and permeation coefficient of 0.86x10-2 cm/hr. Values of tensile strength (4.34 kg/mm2 and elastic modulus (5.89 kg/cm2 revealed that formulation F6 was strong but not brittle. DSC and FTIR studies showed no evidence of interaction between the drug and polymers. A shelf life of 2 years is predicted for the TDDS. Conclusions: Domperidone bilayered matrix type transdermal therapeutic systems could be prepared with the required flux and suitable mechanical properties.

  15. Ex vivo T2 relaxation: associations with age-related neuropathology and cognition.

    Science.gov (United States)

    Dawe, Robert J; Bennett, David A; Schneider, Julie A; Leurgans, Sue E; Kotrotsou, Aikaterini; Boyle, Patricia A; Arfanakis, Konstantinos

    2014-07-01

    The transverse relaxation time constant, T(2), is sensitive to brain tissue's free water content and the presence of paramagnetic materials such as iron. In this study, ex vivo magnetic resonance imaging was used to investigate alterations in T(2) related to Alzheimer's disease (AD) pathology and other types of neuropathology common in old age, as well as the relationship between T(2) alterations and cognition. Cerebral hemispheres were obtained from 371 deceased older adults. Using fast spin-echo imaging with multiple echo times, T(2) maps were produced and warped to a study-specific template. Hemispheres underwent neuropathologic examination for identification of AD pathology and other common age-related neuropathologies. Voxelwise linear regression was carried out to detect regions of pathology-related T(2) alterations and, in separate analyses, regions in which T(2) alterations were linked to antemortem cognitive performance. AD pathology was associated with T(2) prolongation in white matter of all lobes and T(2) shortening in the basal ganglia and insula. Gross infarcts were associated with T(2) prolongation in white matter of all lobes, and in the thalamus and basal ganglia. Hippocampal sclerosis was associated with T(2) prolongation in the hippocampus and white matter of the temporal lobe. After controlling for neuropathology, T(2) prolongation in the frontal lobe white matter was associated with lower performance in the episodic, semantic, and working memory domains. In addition, voxelwise analysis of in vivo and ex vivo T(2) values indicated a positive relationship between the two, though further investigation is necessary to accurately translate findings of the present study to the in vivo case.

  16. Ex vivo testing of immune responses in precision-cut lung slices

    International Nuclear Information System (INIS)

    The aim of this study was the establishment of precision-cut lung slices (PCLS) as a suitable ex vivo alternative approach to animal experiments for investigation of immunomodulatory effects. For this purpose we characterized the changes of cytokine production and the expression of cell surface markers after incubation of PCLS with immunoactive substances lipopolysaccharide (LPS), macrophage-activating lipopeptide-2 (MALP-2), interferon γ (IFNγ), and dexamethasone. Viability of PCLS from wild-type and CD11c-enhanced yellow fluorescent protein (CD11-EYFP)-transgenic mice was controlled by measurement of lactate dehydrogenase (LDH) enzyme activity and live/dead fluorescence staining using confocal microscopy. Cytokines and chemokines were detected with Luminex technology and ELISA. Antigen presenting cell (APC) markers were investigated in living mouse PCLS in situ using confocal microscopy. LPS triggered profound pro-inflammatory effects in PCLS. Dexamethasone prevented LPS-induced production of cytokines/chemokines such as interleukin (IL)-5, IL-1α, TNFα, IL-12(p40), and RANTES in PCLS. Surface expression of MHC class II, CD40, and CD11c, but not CD86 was present in APCs of naive PCLS. Incubation with LPS enhanced specifically the expression of MHC class II on diverse cells. MALP-2 only failed to alter cytokine or chemokine levels, but was highly effective in combination with IFNγ resulting in increased levels of TNFα, IL-12(p40), RANTES, and IL-1α. PCLS showed characteristic responses to typical pro-inflammatory stimuli and may thus provide a suitable ex vivo technique to predict the immunomodulatory potency of inhaled substances

  17. An elegant technique for ex vivo imaging in experimental research—Optical coherence tomography (OCT)

    DEFF Research Database (Denmark)

    Tschernig, T.; Thrane, Lars; Jørgensen, Thomas Martini;

    2013-01-01

    Optical coherence tomography (OCT) is an elegant technology for imaging of tissues and organs and has been established for clinical use for around a decade. Thus, it is used in vivo but can also serve as a valuable ex vivo imaging tool in experimental research. Here, a brief overview is given with...... a focus on an ex vivo application of OCT. Image and video examples of freshly obtained murine lungs are included. The main advantage of OCT for ex vivo analysis is the non-contact, non-invasive, and non-destructive fast acquisition of a three-dimensional data set with micrometer-resolution....

  18. Ex vivo bioluminescence detection of alcelaphine herpesvirus 1 infection during malignant catarrhal fever.

    Science.gov (United States)

    Dewals, Benjamin; Myster, Françoise; Palmeira, Leonor; Gillet, Laurent; Ackermann, Mathias; Vanderplasschen, Alain

    2011-07-01

    Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest asymptomatically, causes malignant catarrhal fever (WD-MCF) when cross-species transmitted to a variety of susceptible species of the Artiodactyla order. Experimentally, WD-MCF can be reproduced in rabbits. WD-MCF is described as a combination of lymphoproliferation and degenerative lesions in virtually all organs and is caused by unknown mechanisms. Recently, we demonstrated that WD-MCF is associated with the proliferation of CD8(+) cells supporting a latent type of infection in lymphoid tissues. Here, we investigated the macroscopic distribution of AlHV-1 infection using ex vivo bioluminescence imaging in rabbit to determine whether it correlates with the distribution of lesions in lymphoid and nonlymphoid organs. To reach that goal, a recombinant AlHV-1 strain was produced by insertion of a luciferase expression cassette (luc) in an intergenic region. In vitro, the reconstituted AlHV-1 luc(+) strain replicated comparably to the parental strain, and luciferase activity was detected by bioluminescence imaging. In vivo, rabbits infected with the AlHV-1 luc(+) strain developed WD-MCF comparably to rabbits infected with the parental wild-type strain, with hyperthermia and increases of both CD8(+) T cell frequencies and viral genomic charge over time in peripheral blood mononuclear cells and in lymph nodes at time of euthanasia. Bioluminescent imaging revealed that AlHV-1 infection could be detected ex vivo in lymphoid organs but also in lung, liver, and kidney during WD-MCF, demonstrating that AlHV-1 infection is prevalent in tissue lesions. Finally, we show that the infiltrating mononuclear leukocytes in nonlymphoid organs are mainly CD8(+) T cells and that latency is predominant during WD-MCF.

  19. Micro CT imaging assessment for spatial distribution of magnetic nanoparticles in an ex vivo thrombolysis model

    Science.gov (United States)

    Wang, Fu-Sheng; Chao, Tsi-Chian; Tu, Shu-Ju

    2012-03-01

    In recent nanotechnology development, iron-based magnetic nanoparticles (MNPs) have been used in several investigations on biomedical research for small animal experiments. Their important applications include targeted drug delivery for therapeutic purpose, contrast agent for magnetic resonance imaging, and hyperthermia treatment for tumors. These MNPs can be guided by an external magnetic field due to their physical characteristics of superparamagnetism. In a recent report, authors indicated that covalently bound recombinant tissue plasminogen activator (rtPA) to MNP (MNPrtPA) with preserved enzyme activity may be guided by a bar magnet and induce target thrombolysis in an embolic model in rats. Delivery of rtPA by binding the thrombolytic drug to MNPs will improve the possibility of the drug to be delivered under magnetic guidance and retained in a local targeted area in the circulation system. In this work, an ex vivo intravascular thrombolysis model was developed to study the impact of external magnetic field on the penetration of MNP-rtPA in the blood clot samples. The samples were then scanned by a micro CT system for quantification. Images of MNPs show strong contrast with their surrounding blood clot materials. The optimum drug loading was found when 0.5 mg/ml rtPA is conjugated with 10 mg SiO2-MNP where 98% drug was attached to the carrier with full retention of its thrombolytic activity. Effective thrombolysis with tPA bound to SiO2-MNP under magnetic guidance was demonstrated in our ex vivo model where substantial reduction in time for blood clot lysis was observed compared with control groups without magnetic field application.

  20. DNA damage in Human Limbal Epithelial Cells expanded ex vivo.

    Directory of Open Access Journals (Sweden)

    Yolanda Lorenzo Corrales

    2015-04-01

    Full Text Available Limbal stem cell deficiency, secondary to insults and diseases, may be treated by transplantation of ex vivo engineered epithelial grafts. We here present preliminary data on levels of cellular DNA damage in grafts produced in two different types of culture medium. Cultures were initiated using corneo-limbal donor tissue after removal of the central area for transplant purposes. Explants (approx. 2x2 mm were positioned epithelial side down on tissue culture treated polyester membranes and expanded for four weeks in Dulbecco’s Modified Eagle Medium F12 Nutrient Mixture (Ham [DMEM/F12 (1:1] with either (1 H. medium; 10% human serum or (2 COM; 5% fetal bovine serum (FBS, Epidermal Growth Factor (EGF, insulin-transferrin-sodiumselenzine (ITS , cholera toxin-A, dimethyl sulfoxide (DMSO and hydrocortisone. Cells were dissociated using Trypsin-EDTA (0.05% for 30 min., the enzyme activity was inhibited by medium and serum. The cell suspension was transferred to tubes on ice and processed using the Comet Assay. Duplicate samples from each culture were analyzed in each assay by visual scoring. Using a fluorescence microscope, 100 comets (50 from each gel were classified into five categories, 0-4, representing increasing relative tail intensities. Summing the scores (0-4 of 100 comets therefore gives an overall score of between 0 and 400 arbitrary units. Preliminary data show some levels of DNA damage in cells dissociated from the grafts regardless of the type of culture medium used. Anyway more experiments with other donors have to be done to have some conclusions. Recent studies have shown that medium with human serum equally support production of grafts containing differentiated as well as undifferentiated cells suitable for clinical transplantation. Preliminary data from our experiments indicate that levels of molecular damage to the DNA do not increase in cells cultured in H. medium despite its lacks of complexity.

  1. Stratum corneum damage and ex vivo porcine skin water absorption - a pilot study

    DEFF Research Database (Denmark)

    Duch Lynggaard, C; Bang Knudsen, D; Jemec, G B E

    2009-01-01

    A simple ex vivo screening technique would be of interest for mass screening of substances for potential barrier disruptive qualities. Ex vivo water absorption as a marker of skin barrier integrity was studied on pig ear skin. Skin water absorption was quantified by weighing and weight changes were...... found to reflect prehydration barrier damage. It is suggested that this simple model may be elaborated to provide a rapid, economical screening tool for potential skin irritants....

  2. The effect of radiofrequency ablation on different organs: Ex vivo and in vivo comparative studies

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yoo Na [Department of Radiology and Center for Imaging Science, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Kangnam-Ku, Seoul 135-710 (Korea, Republic of); Rhim, Hyunchul, E-mail: rhimhc@skku.edu [Department of Radiology and Center for Imaging Science, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Kangnam-Ku, Seoul 135-710 (Korea, Republic of); Choi, Dongil; Kim, Young-sun; Lee, Min Woo; Chang, Ilsoo; Lee, Won Jae; Lim, Hyo K. [Department of Radiology and Center for Imaging Science, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Kangnam-Ku, Seoul 135-710 (Korea, Republic of)

    2011-11-15

    Objective: The purposes of this study are to evaluate the ex vivo and in vivo efficacy of radiofrequency ablation (RFA) on different porcine tissues by the ablation of three different sites simultaneously. Materials and methods: A multichannel RFA system, enables three separate tumors to be ablated simultaneously, was used. RFA procedures were applied to normal porcine liver, kidney, and muscle together ex vivo (n = 12) and in vivo (n = 17). Pre-impedances, defined as baseline systemic impedances of tissues before beginning RFA, and the areas of ablation zones were measured and compared. Results: The areas of ablation zones among three organs had a significant difference in decreasing order as follows: liver, muscle, and kidney in the ex vivo study (p = 0.001); muscle, liver, and kidney in the in vivo study (p < 0.0001). The areas of ablation zones between ex vivo and in vivo had a significant difference in the liver and muscle (each p < 0.05). There was no significant correlation between the areas of ablation zones and pre-impedances in both studies. Conclusions: Renal RFA produced the smallest ablation zone in both in vivo and ex vivo studies. Muscular RFA demonstrated the largest ablation zone in the in vivo study, and hepatic RFA showed the largest ablation zone in the ex vivo study. This variability in the tissues should be considered for performing an optimized RFA for each organ site.

  3. Culture materials affect ex vivo expansion of hematopoietic progenitor cells.

    Science.gov (United States)

    LaIuppa, J A; McAdams, T A; Papoutsakis, E T; Miller, W M

    1997-09-01

    Ex vivo expansion of hematopoietic cells is important for applications such as cancer treatment, gene therapy, and transfusion medicine. While cell culture systems are widely used to evaluate the biocompatibility of materials for implantation, the ability of materials to support proliferation of primary human cells in cultures for reinfusion into patients has not been addressed. We screened a variety of commercially available polymer (15 types), metal (four types), and glass substrates for their ability to support expansion of hematopoietic cells when cultured under conditions that would be encountered in a clinical setting. Cultures of peripheral blood (PB) CD34+ cells and mononuclear cells (MNC) were evaluated for expansion of total cells and colony-forming unit-granulocyte monocyte (CFU-GM; progenitors committed to the granulocyte and/or monocyte lineage). Human hematopoietic cultures in serum-free medium were found to be extremely sensitive to the substrate material. The only materials tested that supported expansion at or near the levels of polystyrene were tissue culture polystyrene, Teflon perfluoroalkoxy, Teflon fluorinated ethylene propylene, cellulose acetate, titanium, new polycarbonate, and new polymethylpentene. MNC were less sensitive to the substrate materials than the primitive CD34+ progenitors, although similar trends were seen for expansion of the two cell populations on the substrates tested. CFU-GM expansion was more sensitive to substrate materials than was total cell expansion. The detrimental effects of a number of the materials on hematopoietic cultures appear to be caused by protein adsorption and/or leaching of toxins. Factors such as cleaning, sterilization, and reuse significantly affected the performance of some materials as culture substrates. We also used PB CD34+ cell cultures to examine the biocompatibility of gas-permeable cell culture and blood storage bags and several types of tubing commonly used with biomedical equipment

  4. Reduced ex Vivo Interleukin-6 Production by Dietary Fish Oil Is Not Modified by Linoleic Acid Intake in Healthy Men

    DEFF Research Database (Denmark)

    Damsgaard, C. T.; Lauritzen, L.; Calder, P. C.;

    2009-01-01

    production from cultures of whole blood, peripheral blood mononuclear cells (PBMC), and monocytes in healthy men. The study was a double-blinded, controlled, 2 X 2 factorial 8-wk intervention. Sixty-four healthy men were randomized to 5 mL/d FO or olive oil (00) provided in capsules and to spreads and oils......Fish oil (FO) is considered antiinflammatory, but evidence regarding its effect on human cytokine production is conflicting. High linoleic acid (LA) intake may impair any effects of FO. The aim of this study was to investigate how FO combined with high or low LA intake affected ex vivo cytokine...

  5. Two-photon excited spectroscopies of ex vivo human skin endogenous species irradiated by femtosecond laser pulses

    Institute of Scientific and Technical Information of China (English)

    Jianxin Chen; Shuangmu Zhuo; Tianshu Luo; Jingjun Zhao

    2006-01-01

    Two-photon excited spectroscopies from ex vivo human skin are investigated by using a femtosecond laser and a confocal microscope (Zeiss LSM 510 META). In the dermis, collagen is responsible for second harmonic generation (SHG); elastin, nicotinamide adenine dinucleotide (NADH), melanin and porphyrin are the primary endogenous sources of two-photon excited autofluorescence. In the epidermis, keratin,NADH, melanin and porphyrins contribute to autofluorescence signals. The results also show that the SHG spectra have the ability to shift with the excitation wavelength and the autofluorescence spectra display a red shift of the spectral peaks when increasing the excitation wavelength. These results may have practical implications for diagnosis of skin diseases.

  6. Two-photon excited spectroscopies of ex vivo human skin endogenous species irradiated by femtosecond laser pulses

    Science.gov (United States)

    Chen, Jianxin; Zhuo, Shuangmu; Luo, Tianshu; Zhao, Jingjun

    2006-10-01

    Two-photon excited spectroscopies from ex vivo human skin are investigated by using a femtosecond laser and a confocal microscope (Zeiss LSM 510 META). In the dermis, collagen is responsible for second harmonic generation (SHG); elastin, nicotinamide adenine dinucleotide (NADH), melanin and porphyrin are the primary endogenous sources of two-photon excited autofluorescence. In the epidermis, keratin, NADH, melanin and porphyrins contribute to autofluorescence signals. The results also show that the SHG spectra have the ability to shift with the excitation wavelength and the autofluorescence spectra display a red shift of the spectral peaks when increasing the excitation wavelength. These results may have practical implications for diagnosis of skin diseases.

  7. An optical biopsy system with miniaturized Raman and spectral imaging probes; in vivo animal and ex vivo clinical application studies

    Science.gov (United States)

    Sato, Hidetoshi; Suzuki, Toshiaki; Andriana, Bibin B.; Morita, Shin'ichi; Maruyama, Atsushi; Shinzawa, Hideyuki; Komachi, Yuichi; Kanai, Gen'ichi; Ura, Nobuo; Masutani, Koji; Matsuura, Yuji; Toi, Masakazu; Shimosegawa, Toru; Ozaki, Yukihiro

    2009-02-01

    An optical biopsy system which equips miniaturized Raman probes, a miniaturized endoscope and a fluorescent image probe has been developed for in vivo studies of live experimental animals. The present report describes basic optical properties of the system and its application studies for in vivo cancer model animals and ex vivo human cancer tissues. It was developed two types of miniaturized Raman probes, micro Raman probe (MRP) made of optical fibers and ball lens hollow optical fiber Raman probe (BHRP) made of single hollow optical fiber (HOF) with a ball lens. The former has rather large working distance (WD), up to one millimeter. The latter has small WD (~300μm) which depends on the focal length of the ball lens. Use of multiple probes with different WD allows one to obtain detailed information of subsurface tissues in the totally noninvasive manner. The probe is enough narrow to be inserted into a biopsy needle (~19G), for observations of the lesion at deeper inside bodies. The miniaturized endoscope has been applied to observe progression of a stomach cancer in the same rat lesion. It was succeeded to visualize structure of non-stained cancer tissue in live model animals by the fluorescent image technique. The system was also applied to ex vivo studies of human breast and stomach cancers.

  8. High-resolution ex vivo magnetic resonance angiography: a feasibility study on biological and medical tissues

    Directory of Open Access Journals (Sweden)

    Boel Lene WT

    2010-03-01

    Full Text Available Abstract Background In biomedical sciences, ex vivo angiography is a practical mean to elucidate vascular structures three-dimensionally with simultaneous estimation of intravascular volume. The objectives of this study were to develop a magnetic resonance (MR method for ex vivo angiography and to compare the findings with computed tomography (CT. To demonstrate the usefulness of this method, examples are provided from four different tissues and species: the human placenta, a rice field eel, a porcine heart and a turtle. Results The optimal solution for ex vivo MR angiography (MRA was a compound containing gelatine (0.05 g/mL, the CT contrast agent barium sulphate (0.43 mol/L and the MR contrast agent gadoteric acid (2.5 mmol/L. It was possible to perform angiography on all specimens. We found that ex vivo MRA could only be performed on fresh tissue because formalin fixation makes the blood vessels permeable to the MR contrast agent. Conclusions Ex vivo MRA provides high-resolution images of fresh tissue and delineates fine structures that we were unable to visualise by CT. We found that MRA provided detailed information similar to or better than conventional CTA in its ability to visualize vessel configuration while avoiding interfering signals from adjacent bones. Interestingly, we found that vascular tissue becomes leaky when formalin-fixed, leading to increased permeability and extravascular leakage of MR contrast agent.

  9. Expression patterns of intestinal calcium transport factors and ex-vivo absorption of calcium in horses

    Directory of Open Access Journals (Sweden)

    Sprekeler Nele

    2011-10-01

    Full Text Available Abstract Background In many species, the small intestine is the major site of calcium (Ca2+ absorption. The horse differs considerably from most other species with regard to the physiology of its Ca2+ metabolism and digestion. Thus, this study was performed to get more information about the transcellular Ca2+ absorption in the horse. Two mechanisms of intestinal Ca2+ absorption are described: the passive paracellular pathway and the active, vitamin D-dependent transcellular pathway. The latter involves the following elements: vitamin D receptors (VDR, transient receptor potential vanilloid channel members 5 and 6 (TRPV5/6, calbindin-D9k (CB, the Na/Ca exchanger (NCX1 and the plasma membrane Ca-ATPase (PMCA. The aim of the present study was to investigate the protein and mRNA expression patterns of VDR, CB and TRPV6 and the ex-vivo Ca2+ absorption in horses, assessed by qualitative and quantitative RT-PCR, western blot, immunohistochemistry and the Ussing chamber technique. Results Highest CB and TRPV6 mRNA levels were detected in the duodenum as compared to the middle parts of the jejunum and ileum and several sites of the large intestine. VDR mRNA levels did not change significantly throughout the intestine. TRPV5 mRNA was not detectable in the horse intestine. The highest VDR and CB protein levels were measured in the duodenum. Ussing chamber studies revealed ex-vivo Ca2+ absorption only in the duodenum, but not in cecum and specific sites of the colon. Conclusion The present findings suggest that TRPV6, CB and VDR may be involved in active intestinal Ca2+ absorption in horses, as described for other mammals. TRPV5 may not play a major role in this process. Furthermore, the expression patterns of these Ca2+ transport elements and the results of the Ussing chamber procedure indicate that a significant part of active intestinal Ca2+ absorption occurs in the duodenum in this species.

  10. Functional testing of topical skin formulations using an optimised ex vivo skin organ culture model.

    Science.gov (United States)

    Sidgwick, G P; McGeorge, D; Bayat, A

    2016-07-01

    A number of equivalent-skin models are available for investigation of the ex vivo effect of topical application of drugs and cosmaceuticals onto skin, however many have their drawbacks. With the March 2013 ban on animal models for cosmetic testing of products or ingredients for sale in the EU, their utility for testing toxicity and effect on skin becomes more relevant. The aim of this study was to demonstrate proof of principle that altered expression of key gene and protein markers could be quantified in an optimised whole tissue biopsy culture model. Topical formulations containing green tea catechins (GTC) were investigated in a skin biopsy culture model (n = 11). Punch biopsies were harvested at 3, 7 and 10 days, and analysed using qRT-PCR, histology and HPLC to determine gene and protein expression, and transdermal delivery of compounds of interest. Reduced gene expression of α-SMA, fibronectin, mast cell tryptase, mast cell chymase, TGF-β1, CTGF and PAI-1 was observed after 7 and 10 days compared with treated controls (p numbers in treated biopsies compared with untreated controls at day 7 and day 10 (p animal models in this context, prior to study in a clinical trial environment. PMID:27086034

  11. Effects of ex-vivo and in-vivo treatment with probiotics on the inflammasome in dogs with chronic enteropathy.

    Directory of Open Access Journals (Sweden)

    Silke Schmitz

    Full Text Available Inflammasomes coordinate the maturation of IL-1β and IL-18 in response to danger signals. They are vital for maintenance of intestinal homeostasis and have been linked to chronic intestinal inflammation in humans. Probiotics have been advocated as treatment in intestinal inflammation. So far, no study has investigated the role of the inflammasome in canine chronic enteropathy (CE. In this study the intestinal expression of inflammasome components was assessed in CE dogs compared to controls, when treated with probiotic Enterococcus faecium (EF ex-vivo and in-vivo. RNA extraction from endoscopic biopsies and reverse-transcriptase quantitative PCR was performed for NLRP3, casp-1, IL-1β and IL-18. Immunohistochemistry was performed to investigate protein expression in tissues. Gene expression of casp-1 and NLRP3 was lower in CE samples than controls. Ex-vivo treatment with EF reduced NLRP3 expression in control samples. Treatment of CE dogs with EF alongside dietary intervention had no effect on gene expression. In contrast, IL-1β protein expression in CE decreased with dietary treatment (but not with probiotics. The results of this study suggest that the inflammasome or its components may be partially involved in the inflammatory process seen in CE, but distinct from intestinal inflammation in humans.

  12. The role of Hibiscus sabdariffa L. (Roselle) in maintenance of ex vivo murine bone marrow-derived hematopoietic stem cells.

    Science.gov (United States)

    Abdul Hamid, Zariyantey; Lin Lin, Winnie Hii; Abdalla, Basma Jibril; Bee Yuen, Ong; Latif, Elda Surhaida; Mohamed, Jamaludin; Rajab, Nor Fadilah; Paik Wah, Chow; Wak Harto, Muhd Khairul Akmal; Budin, Siti Balkis

    2014-01-01

    Hematopoietic stem cells- (HSCs-) based therapy requires ex vivo expansion of HSCs prior to therapeutic use. However, ex vivo culture was reported to promote excessive production of reactive oxygen species (ROS), exposing HSCs to oxidative damage. Efforts to overcome this limitation include the use of antioxidants. In this study, the role of Hibiscus sabdariffa L. (Roselle) in maintenance of cultured murine bone marrow-derived HSCs was investigated. Aqueous extract of Roselle was added at varying concentrations (0-1000 ng/mL) for 24 hours to the freshly isolated murine bone marrow cells (BMCs) cultures. Effects of Roselle on cell viability, reactive oxygen species (ROS) production, glutathione (GSH) level, superoxide dismutase (SOD) activity, and DNA damage were investigated. Roselle enhanced the survival (P < 0.05) of BMCs at 500 and 1000 ng/mL, increased survival of Sca-1(+) cells (HSCs) at 500 ng/mL, and maintained HSCs phenotype as shown from nonremarkable changes of surface marker antigen (Sca-1) expression in all experimental groups. Roselle increased (P < 0.05) the GSH level and SOD activity but the level of reactive oxygen species (ROS) was unaffected. Moreover, Roselle showed significant cellular genoprotective potency against H2O2-induced DNA damage. Conclusively, Roselle shows novel property as potential supplement and genoprotectant against oxidative damage to cultured HSCs. PMID:25405216

  13. Ex Vivo Lung Perfusion and Transplant: State of the Art and View to the Future.

    Science.gov (United States)

    Mohamed, Mohamed S A

    2015-12-01

    After the first clinical application of ex vivo lung perfusion in 2001, the technique has been used in many lung transplant centers worldwide. In addition, many modifications have been tested, leading to the development of various ex vivo lung perfusion systems and application protocols. Currently, the Lund protocol, the Toronto protocol, and Organ Care System Lung protocol are the clinically applied ex vivo lung perfusion protocols, based on the favorable results of the safety studies. Accordingly, the comparison among these EVLP systems and protocols should be an important research target, in order to provide the evidence based medical data that would recommend one protocol over the others. In this manuscript, the current experience with EVLP is reviewed and some molecular and clinical targets, that could be used to compare the various protocols of the technique, are introduced.

  14. Dermal absorption and skin damage following hydrofluoric acid exposure in an ex vivo human skin model.

    Science.gov (United States)

    Dennerlein, Kathrin; Kiesewetter, Franklin; Kilo, Sonja; Jäger, Thomas; Göen, Thomas; Korinth, Gintautas; Drexler, Hans

    2016-04-25

    The wide industrial use of hydrofluoric acid (HF) poses a high risk for accidental dermal exposure. Despite local and systemic hazards associated with HF, information on percutaneous penetration and tissue damage is rare. In the present ex vivo study, the dermal absorption of HF (detected in terms of fluoride ions) was quantified and the skin damaging potential as a function of concentration and exposure duration was assessed. Percutaneous penetration of HF (c=5, 30, and 50%) at 3 exposure durations (3, 5, and 10 min) was investigated in a static diffusion cell model using freshly excised human skin. Alterations of skin were histologically evaluated. HF rapidly penetrated through skin under formation of a considerable intradermal reservoir (∼ 13-67% of total absorbed fluoride). Histologically, epidermal alterations were detected already after exposure to 5% HF for 3 min. The degree of skin damage increased with rising concentration and exposure duration leading to coagulation necrosis. For HF concentrations of ≥ 30%, skin damage progressed into deeper skin layers. Topically applied HF concentration was the principal parameter determining HF induced skin effects. The intradermal HF retention capacity associated with progression and prolongation of HF induced skin effects must be considered in the review of skin decontamination procedures.

  15. Ex vivo expansion and pluripotential differentiation of cryopreserved human bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    XIANG Ying; ZHENG Qiang; JIA Bing-bing; HUANG Guo-ping; Xu Yu-lin; WANG Jin-fu; PAN Zhi-jun

    2007-01-01

    This study is aimed at investigating the potentials of ex vivo expansion and pluri-differentiation of cryopreservation of adult human bone marrow mesenchymal stem cells (hMSCs) into chondrocytes, adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced into chondrocytes, adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and detected for expressions of type II collagen, triglyceride or neuron-specific enolase and nestin. The result showed that the resuscitated cells could differentiate into chondrocytes after exposure to transforming growth factor β1 (TGF-β1), insulin-like growth factor I (IGF-I) and vitamin C (Vc), and uniformly changed morphologically from a spindle-like fibroblastic appearance to a polygonal shape in three weeks. The induced cells were heterochromatic to safranin O and expressed cartilage matrix-procollagenal (II) mRNA. The resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin and IGF-I showed adipogenesis, and lipid vacuoles accumulation was detectable after 21 d. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron specific endolase (NSE) that were special surface markers associated with neural cells at different stage. This study suggested that the resuscitated hMSCs should be still a population of pluripotential cells and that it could be used for establishing an abundant hMSC reservoir for further experiment and treatment of various clinical diseases.

  16. Spectral domain optical coherence tomography for ex vivo brain tumor analysis

    Science.gov (United States)

    Lenz, Marcel; Krug, Robin; Jaedicke, Volker; Stroop, Ralf; Schmieder, Kirsten; Hofmann, Martin R.

    2015-07-01

    Non-contact imaging methods to distinguish between healthy tissue and brain tumor tissue during surgery would be highly desirable but are not yet available. Optical Coherence Tomography (OCT) is a non-invasive imaging technology with a resolution around 1-15 μm and a penetration depth of 1-2 mm that may satisfy the demands. To analyze its potential, we measured ex vivo human brain tumor tissue samples from 10 patients with a Spectral Domain OCT system (Thorlabs Callisto: center wavelength of 930 nm) and compared the results with standard histology. In detail, three different measurements were made for each sample. First the sample was measured directly after surgery. Then it was embedded in paraffin (also H and E staining) and examined for the second time. At last, the slices of each paraffin block cut by the pathology were measured. Each time a B-scan was created and for a better comparison with the histology a 3D image was generated, in order to get the corresponding en face images. In both, histopathological diagnosis and the analysis of the OCT images, different types of brain tumor showed difference in structure. This has been affirmed by two blinded investigators. Nevertheless the difference between two images of samples taken directly after surgery is less distinct. To enhance the contrast in the images further, we employ Spectroscopic OCT and pattern recognition algorithms and compare these results to the histopathological standard.

  17. Thermal analysis of laser interstitial thermotherapy in ex vivo fibro-fatty tissue using exponential functions

    Energy Technology Data Exchange (ETDEWEB)

    Salas, Nelson Jr. [Biomedical Optics and Laser Laboratory, Department of Biomedical Engineering, University of Miami College of Engineering, PO Box 248294, Coral Gables, FL 33124 (United States); Manns, Fabrice [Biomedical Optics and Laser Laboratory, Department of Biomedical Engineering, University of Miami College of Engineering, PO Box 248294, Coral Gables, FL 33124 (United States); Milne, Peter J [Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, University of Miami School of Medicine, 1638 NW 10th Ave, McKnight Bldg, Miami, FL 33136 (United States); Denham, David B [Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, University of Miami School of Medicine, 1638 NW 10th Ave, McKnight Bldg, Miami, FL 33136 (United States); Minhaj, Ahmed M [Biomedical Optics and Laser Laboratory, Department of Biomedical Engineering, University of Miami College of Engineering, PO Box 248294, Coral Gables, FL 33124 (United States); Parel, Jean-Marie [Biomedical Optics and Laser Laboratory, Department of Biomedical Engineering, University of Miami College of Engineering, PO Box 248294, Coral Gables, FL 33124 (United States); Robinson, David S [Center for Breast Care, St Luke' s Hospital of Kansas City, 4400 Broadway, Suite 509, Kansas City, MO 64111 (United States)

    2004-05-07

    A therapeutic procedure to treat small, surface breast tumours up to 10 mm in radius plus a 5 mm margin of healthy, surrounding tissue using laser interstitial thermotherapy (LITT) is currently being investigated. The purpose of this study is to analyse and model the thermal and coagulative response of ex vivo fibro-fatty tissue, a model for breast tissue, during experimental laser interstitial thermotherapy at 980 nm. Laser radiation at 980 nm was delivered interstitially through a diffusing tip optical fibre inserted into a fibro-fatty tissue model to produce controlled heating at powers ranging from 3.2 to 8.0 W. Tissue temperature was measured with thermocouples placed at 15 positions around the fibre. The induced coagulation zone was measured on gross anatomical sections. Thermal analysis indicates that a finite sum of exponential functions is an approximate solution to the heat conduction equation that more accurately predicts the time-temperature dependence in tissue prior to carbonization (T < 100 deg. C) during LITT than the traditional model using a single exponential function. Analysis of the ellipsoid coagulation volume induced in tissue indicates that the 980 nm wavelength does not penetrate deep enough in fibro-fatty tissue to produce a desired 30 mm diameter (14.1 x 10{sup 3} mm{sup 3}) coagulation volume without unwanted tissue liquefaction and carbonization.

  18. Thermal analysis of laser interstitial thermotherapy in ex vivo fibro-fatty tissue using exponential functions

    Science.gov (United States)

    Salas, Nelson, Jr.; Manns, Fabrice; Milne, Peter J.; Denham, David B.; Minhaj, Ahmed M.; Parel, Jean-Marie; Robinson, David S.

    2004-05-01

    A therapeutic procedure to treat small, surface breast tumours up to 10 mm in radius plus a 5 mm margin of healthy, surrounding tissue using laser interstitial thermotherapy (LITT) is currently being investigated. The purpose of this study is to analyse and model the thermal and coagulative response of ex vivo fibro-fatty tissue, a model for breast tissue, during experimental laser interstitial thermotherapy at 980 nm. Laser radiation at 980 nm was delivered interstitially through a diffusing tip optical fibre inserted into a fibro-fatty tissue model to produce controlled heating at powers ranging from 3.2 to 8.0 W. Tissue temperature was measured with thermocouples placed at 15 positions around the fibre. The induced coagulation zone was measured on gross anatomical sections. Thermal analysis indicates that a finite sum of exponential functions is an approximate solution to the heat conduction equation that more accurately predicts the time-temperature dependence in tissue prior to carbonization (T deep enough in fibro-fatty tissue to produce a desired 30 mm diameter (14.1 × 103 mm3) coagulation volume without unwanted tissue liquefaction and carbonization.

  19. Thiolated chitosan nanoparticles for enhancing oral absorption of docetaxel: preparation, in vitro and ex vivo evaluation

    Science.gov (United States)

    Saremi, Shahrooz; Atyabi, Fatemeh; Akhlaghi, Seyedeh Parinaz; Ostad, Seyed Nasser; Dinarvand, Rassoul

    2011-01-01

    The aim of this study was to prepare and evaluate mucoadhesive core-shell nanoparticles based on copolymerization of thiolated chitosan coated on poly methyl methacrylate cores as a carrier for oral delivery of docetaxel. Docetaxel-loaded nanoparticles with various concentrations were prepared via a radical emulsion polymerization method using cerium ammonium nitrate as an initiator. The physicochemical properties of the obtained nanoparticles were characterized by: dynamic light-scattering analysis for their mean size, size distribution, and zeta potential; scanning electron microscopy and transmission electron microscopy for surface morphology; and differential scanning calorimetry analysis for confirmation of molecular dispersity of docetaxel in the nanoparticles. Nanoparticles were spherical with mean diameter below 200 nm, polydispersity of below 0.15, and positive zeta potential values. The entrapment efficiency of the nanoparticles was approximately 90%. In vitro release studies showed a sustained release characteristic for 10 days after a burst release at the beginning. Ex vivo studies showed a significant increase in the transportation of docetaxel from intestinal membrane of rat when formulated as nanoparticles. Cellular uptake of nanoparticles was investigated using fluoresceinamine-loaded nanoparticles. Docetaxel nanoparticles showed a high cytotoxicity effect in the Caco-2 and MCF-7 cell lines after 72 hours. It can be concluded that by combining the advantages of both thiolated polymers and colloidal particles, these nanoparticles can be proposed as a drug carrier system for mucosal delivery of hydrophobic drugs. PMID:21289989

  20. ESTUDIO EX VIVO DE LA LIBERACIÓN TRANSDÉRMICA DE ENALAPRIL

    Directory of Open Access Journals (Sweden)

    Lucía Lhez

    2010-01-01

    Full Text Available En el presente trabajo se estudió el comportamiento ex vivo de una formulación de enalapril maleato para su administración por vía transdérmica, utilizando piel de oreja de cerdo. Los experimentos se realizaron en celdas de difusión vertical tipo Franz. El principio activo fue formulado en una dispersión de carbopol. A partir de las cantidades acumuladas de enalapril en el compartimento receptor, se evaluó el flujo y el coeficiente de permeabilidad. Con el fin de optimizar la penetración del principio activo, se investigó el efecto de L-mentol como potenciador de la permeabildiad. Se determinó que este compuesto contribuye favorablemente a la penetración de enalapril a través de la piel y que los parámetros de permeabilidad, flujo y coeficiente de permeabilidad, alcanzan sus máximos valores cuando L-mentol está presente en un 3,82% en la formulación enalapril/carbopol.

  1. Influence of massage and occlusion on the ex vivo skin penetration of rigid liposomes and invasomes.

    Science.gov (United States)

    Trauer, Sindy; Richter, Heike; Kuntsche, Judith; Büttemeyer, Rolf; Liebsch, Manfred; Linscheid, Michael; Fahr, Alfred; Schäfer-Korting, Monika; Lademann, Jürgen; Patzelt, Alexa

    2014-02-01

    Liposomes are frequently described as drug delivery systems for dermal and transdermal applications. Recently, it has been shown that particulate substances penetrate effectively into hair follicles and that the follicular penetration depth can be increased by massaging the skin, which simulates the in vivo movement of hairs in the hair follicles. In the present study, massage was applied to skin mounted to Franz diffusion cells. By means of confocal laser scanning microscopy, the influence of massage and occlusion on the follicular penetration depths of rigid and flexible liposomes loaded with a hydrophilic and lipophilic dye was investigated. The application of massage increased follicular penetration significantly. Occlusion resulted in an increased follicular penetration depth only for rigid liposomes, whereas invasomes did not penetrate more effectively if occlusion was applied. The results confirm that massage is an important tool for increasing follicular penetration in ex vivo studies using Franz diffusion cells. Occlusion may reduce the efficacy of follicular penetration depending on the specific liposomal preparation. Rigidity in particular appears to be a relevant parameter.

  2. Functional physico-chemical, ex vivo permeation and cell viability characterization of omeprazole loaded buccal films for paediatric drug delivery.

    Science.gov (United States)

    Khan, Sajjad; Trivedi, Vivek; Boateng, Joshua

    2016-03-16

    Buccal films were prepared from aqueous and ethanolic Metolose gels using the solvent casting approach (40°C). The hydration (PBS and simulated saliva), mucoadhesion, physical stability (20°C, 40°C), in vitro drug (omeprazole) dissolution (PBS and simulated saliva), ex vivo permeation (pig buccal mucosa) in the presence of simulated saliva, ex vivo bioadhesion and cell viability using MTT of films were investigated. Hydration and mucoadhesion results showed that swelling capacity and adhesion was higher in the presence of PBS than simulated saliva (SS) due to differences in ionic strength. Omeprazole was more stable at 20°C than 40°C whilst omeprazole release reached a plateau within 1h and faster in PBS than in SS. Fitting release data to kinetic models showed that Korsmeyer-Peppas equation best fit the dissolution data. Drug release in PBS was best described by zero order via non-Fickian diffusion but followed super case II transport in SS attributed to drug diffusion and polymer erosion. The amount of omeprazole permeating over 2h was 275 ug/cm(2) whilst the formulations and starting materials showed cell viability values greater than 95%, confirming their safety for potential use in paediatric buccal delivery. PMID:26802493

  3. Novel approaches to expanding the lung donor pool: donation after cardiac death and ex vivo conditioning.

    Science.gov (United States)

    Cypel, Marcelo; Yeung, Jonathan C; Keshavjee, Shaf

    2011-06-01

    Two novel approaches have been developed to potentially increase the availability of donor lungs for lung transplantation. In the first approach, lungs from donation after cardiac death (DCD) donors are used to increase the quantity of organ donors. In the second approach, a newly developed normothermic ex vivo lung perfusion (EVLP) technique is used as a means of reassessing the adequacy of lung function from DCD and from high-risk brain death donors prior to transplantation. This EVLP technique can also act as a platform for the delivery of novel therapies to repair injured organs ex vivo. PMID:21511086

  4. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies.

    OpenAIRE

    Witkowski, Benoit; Amaratunga, Chanaki; Khim, Nimol; Sreng, Sokunthea; Chim, Pheaktra; Kim, Saorin; Lim, Pharath; Mao, Sivanna; Sopha, Chantha; Sam, Baramey; Anderson, Jennifer M; Duong, Socheat; Chuor, Char Meng; Walter R J Taylor; Suon, Seila

    2013-01-01

    BACKGROUND: Artemisinin resistance in Plasmodium falciparum lengthens parasite clearance half-life during artemisinin monotherapy or artemisinin-based combination therapy. Absence of in-vitro and ex-vivo correlates of artemisinin resistance hinders study of this phenotype. We aimed to assess whether an in-vitro ring-stage survival assay (RSA) can identify culture-adapted P falciparum isolates from patients with slow-clearing or fast-clearing infections, to investigate the stage-dependent susc...

  5. Photoacoustic tomography of joints aided by an Etanercept-conjugated gold nanoparticle contrast agent—an ex vivo preliminary rat study

    Science.gov (United States)

    Chamberland, David L.; Agarwal, Ashish; Kotov, Nicholas; Fowlkes, J. Brian; Carson, Paul L.; Wang, Xueding

    2008-03-01

    Monitoring of anti-rheumatic drug delivery in experimental models and in human diseases would undoubtedly be very helpful for both basic research and clinical management of inflammatory diseases. In this study, we have investigated the potential of an emerging hybrid imaging technology—photoacoustic tomography—in noninvasive monitoring of anti-TNF drug delivery. After the contrast agent composed of gold nanorods conjugated with Etanercept molecules was produced, ELISA experiments were performed to prove the conjugation and to show that the conjugated anti-TNF-α drug was biologically active. PAT of ex vivo rat tail joints with the joint connective tissue enhanced by intra-articularly injected contrast agent was conducted to examine the performance of PAT in visualizing the distribution of the gold-nanorod-conjugated drug in articular tissues. By using the described system, gold nanorods with a concentration down to 1 pM in phantoms or 10 pM in biological tissues can be imaged with good signal-to-noise ratio and high spatial resolution. This study demonstrates the feasibility of conjugating TNF antagonist pharmaceutical preparations with gold nanorods, preservation of the mechanism of action of TNF antagonist along with preliminary evaluation of novel PAT technology in imaging optical contrast agents conjugated with anti-rheumatic drugs. Further in vivo studies on animals are warranted to test the specific binding between such conjugates and targeted antigen in joint tissues affected by inflammation.

  6. Rye bran bread intake elevates urinary excretion of ferulic acid in humans, but does not affect the susceptibility of LDL to oxidation ex vivo

    DEFF Research Database (Denmark)

    Harder, H.; Tetens, I.; Let, Mette Bruni;

    2004-01-01

    women after a dietary intake of rye bran or an inert wheat bran (control) in a crossover study (2 x 6 weeks with 4 weeks washout). The potential antioxidative effect of the rye bran intervention was investigated by measuring low-density lipoprotein (LDL) susceptibility to copper oxidation ex vivo...... had no influence on lag time or propagation rate of the LDL oxidation ex vivo. Conclusions The present study demonstrated that ferulic acid from rye bran is bioavailable and that the urinary concentration of ferulic acid reflects the dietary intake of this hydroxycinnamic acid. Within the period...... of intervention, the elevated ferulic acid did not produce a measurable antioxidative effect on the subjects' LDL. It is suggested that the determination of ferulic acid in urine is a useful biomarker to assess the intake of ferulic acid from a regular diet....

  7. [Lymph node preparation in colorectal cancer. Ex vivo methylene blue injection as a novel technique to improve lymph node visualization].

    Science.gov (United States)

    Märkl, B; Kerwel, T; Jähnig, H; Anthuber, M; Arnholdt, H

    2008-07-01

    The UICC requires investigation of a minimum of 12 lymph nodes for adequate lymph node staging in colorectal cancer. Despite that, many authors recommend investigation of a larger number, and different techniques, such as fat clearance, have therefore been developed. In this study we introduce a novel technique involving ex vivo lymph node staining with intraarterial methylene blue injection in colon cancer. We compared 14 cases in which methylene injection was used with 14 cases from our records in which conventional investigation techniques were applied. The lymph node harvest differed highly significantly (pmethylene blue group and the unstained group, respectively. The largest difference occurred in the size group 2-4 mm (191 vs 70 lymph nodes). In 6 cases in the unstained group additional embedding of fatty tissue was necessary to reach an adequate number of investigated lymph nodes. Methylene blue injection is a novel and highly effective method that will improve lymph node preparation in colorectal cancer.

  8. Ex vivo model for pre-clinical evaluation of dialyzers containing new membranes.

    Science.gov (United States)

    Mahiout, A; Meinhold, H; Jörres, A; Krieg, R; Kessel, M; Tretzel, J; Baurmeister, U

    1985-01-01

    The ex vivo model which reflects hemodialysis modulating factors during the first twenty minutes of blood membrane interaction, is applicable as a pre-clinical test for new membranes. The biocompatibility of a new cellulosic membrane (MC) proved to be superior to regenerated cellulose and comparable to synthetic membranes such as PAN regarding complement activation.

  9. Bacterial transmission from contact lenses to porcine corneas : An ex vivo study

    NARCIS (Netherlands)

    Vermeltfoort, PBJ; van Kooten, TG; Bruinsma, GM; Hooymans, AMM; van der Mei, HC; Busscher, HJ

    2005-01-01

    PURPOSE. To quantify the transmission to ex vivo porcine eyes of Staphylococcus aureus 835 and Pseudomonas aeruginosa 3 from three types of contact lenses - one daily wear and two extended wear - differing in hydrophobicity and roughness. METHODS. One daily wear lens (etafilcon) and two extended-wea

  10. Development of an Ex Vivo, Beating Heart Model for CT Myocardial Perfusion

    NARCIS (Netherlands)

    Pelgrim, Gert Jan; Das, Marco; Haberland, Ulrike; Slump, Cees; Handayani, Astri; van Tuijl, Sjoerd; Stijnen, Marco; Klotz, Ernst; Oudkerk, Matthijs; Wildberger, Joachim E.; Vliegenthart, Rozemarijn

    2015-01-01

    Objective. To test the feasibility of a CT-compatible, ex vivo, perfused porcine heart model for myocardial perfusion CT imaging. Methods. One porcine heart was perfused according to Langendorff. Dynamic perfusion scanning was performed with a second-generation dual source CT scanner. Circulatory pa

  11. Surgical sealant in the prevention of early vein graft injury in an ex vivo model

    NARCIS (Netherlands)

    Stooker, W; Niessen, HWM; Jansen, EK; Fritz, J; Wildevuur, WR; Van Hinsbergh, VWM; Wildevuur, CRH; Eijsman, L

    2003-01-01

    Background: The amelioration of the adaptation process (arterialisation) of the vein graft wall to the arterial circulation in coronary artery bypass surgery by using extravascular support is clearly established in animal models and in in vitro and ex vivo set-ups. This support consists of some form

  12. Characterization of a novel brain barrier ex vivo insect-based P-glycoprotein screening model

    DEFF Research Database (Denmark)

    Andersson, O.; Badisco, L.; Hansen, A. H.;

    2014-01-01

    locust brains demonstrated differences in permeation of high and low permeability compounds. The vertebrate Pgp inhibitor verapamil did not affect the uptake of passively diffusing compounds but significantly increased the brain uptake of Pgp substrates in the ex vivo model. In addition, studies at 2°C...

  13. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell...

  14. Gravitational physiology of human immune cells: a review of in vivo, ex vivo and in vitro studies

    Science.gov (United States)

    Cogoli, A.

    1996-01-01

    The study of the function of immune cells in microgravity has been studied for more than 20 years in several laboratories. It is clear today that the immune system is depressed in more than 50% of the astronauts during and after space flight and that the activation of T lymphocytes by mitogens in vitro changes dramatically. This article gives an overview of the gravitational studies conducted by our laboratory in Spacelab, in MIR station, in sounding rockets and on the ground in the clinostat and the centrifuge. Three experimental approaches are followed in our work: (i) Ex vivo studies are performed with blood samples drawn from astronauts; (ii) in vivo studies are based on the application of seven antigens to the skin of the astronauts; (iii) in vitro studies are carried out with immune cells purified from the blood of healthy donors (not astronauts). The data from our in vivo and ex vivo studies are in agreement with those of other laboratories and show that the immunological function is depressed in the majority of astronauts as a consequence of the stress of space flight rather than by a direct influence of gravity on the cell. Immune depression may become a critical hazard on long duration flights on space stations or to other planets. In vitro experiments show that cultures of free-floating lymphocytes and monocytes undergo a dramatic depression of activation by the mitogen concanavalin A, while activation is more than doubled when the cells are attached to microcarrier beads. Such effects may be attributed to both direct and indirect effects of gravitational unloading on basic biological mechanisms of the cell. While the in vitro data are very important to clarify certain aspects of the biological mechanism of T cells activation, they are not descriptive of the changes of the immunological function of the astronauts.

  15. The Response of RIF-1 Fibrosarcomas to the Vascular-Disrupting Agent ZD6126 Assessed by In Vivo and Ex Vivo1H Magnetic Resonance Spectroscopy

    Directory of Open Access Journals (Sweden)

    Basetti Madhu

    2006-07-01

    Full Text Available The response of radiation-induced fibrosarcoma1 (RIF-1 tumors treated with the vascular-disrupting agent (VDA ZD6126 was assessed by in vivo and ex vivo1H magnetic resonance spectroscopy (MRS methods. Tumors treated with 200 mg/kg ZD6126 showed a significant reduction in total choline (tCho in vivo 24 hours after treatment, whereas control tumors showed a significant increase in tCho. This response was investigated further within both ex vivo unprocessed tumor tissues and tumor tissue metabolite extracts. Ex vivo high-resolution magic angle spinning (HRMAS and 1H MRS of metabolite extracts revealed a significant reduction in phosphocholine and glycerophosphocholine in biopsies of ZD6126-treated tumors, confirming in vivo tCho response. ZD6126-induced reduction in choline compounds is consistent with a reduction in cell membrane turnover associated with necrosis and cell death following disruption of the tumor vasculature. In vivo tumor tissue water diffusion and lactate measurements showed no significant changes in response to ZD6126. Spin-spin relaxation times (T2 of water and metabolites also remained unchanged. Noninvasive 1H MRS measurement of tCho in vivo provides a potential biomarker of tumor response to VDAs in RIF-1 tumors.

  16. An ex vivo continuous passive motion model in a porcine knee for assessing primary stability of cell-free collagen gel plugs

    Directory of Open Access Journals (Sweden)

    El-Zayat Bilal

    2010-12-01

    Full Text Available Abstract Background Primary stability of cartilage repair constructs is of the utmost importance in the clinical setting but few continuous passive motion (CPM models are available. Our study aimed to establish a novel ex vivo CPM animal model and to evaluate the required motion cycles for testing the mechanical properties of a new cell-free collagen type I gel plug (CaReS®-1S. Methods A novel ex vivo CPM device was developed. Full-thickness cartilage defects (11 mm diameter by 6 mm deep were created on the medial femoral condyle of porcine knee specimens. CaReS®-1S was implanted in 16 animals and each knee underwent continuous passive motion. After 0, 2000, 4000, 6000, and 8000 motions, standardized digital pictures of the grafts were taken, focusing on the worn surfaces. The percentage of worn surface on the total CaReS®-1S surface was evaluated with image processing software. Results Significant differences in the worn surface were recorded between 0 and 2000 motion cycles (p ®-1S with an empty defect site was recorded. Conclusion The ex vivo CPM animal model is appropriate in investigating CaReS®-1S durability under continuous passive motion. 2000 motion cycles appear adequate to assess the primary stability of type I collagen gels used to repair focal chondral defects.

  17. Investigating Complexity Using Excel and Visual Basic.

    Science.gov (United States)

    Zetie, K. P.

    2001-01-01

    Shows how some of the simple ideas in complexity can be investigated using a spreadsheet and a macro written in Visual Basic. Shows how the sandpile model of Bak, Chao, and Wiesenfeld can be simulated and animated. The model produces results that cannot easily be predicted from its properties. (Author/MM)

  18. Effects of mycophenolate mofetil on key pattern of coronary restenosis: a cascade of in vitro and ex vivo models

    Directory of Open Access Journals (Sweden)

    Baur Regine

    2005-05-01

    Full Text Available Abstract Background Mycophenolate mofetil (MMF, the prodrug of mycophenolic acid (MPA, is a rationally designed immunosuppressive drug. The current study investigates the effect of MMF on key pattern of restenosis in a cascade of in vitro and ex vivo models. Methods Part I of the study investigated in northern blot and cytoflow studies the effect of MMF (50, 100, 150, 200, 250, and 300 μg/mL on TNF-α induced expression of intercellular adhesion molecule 1 (ICAM-1 in human coronary endothelial cells (HCAEC and human coronary medial smooth muscle cells (HCMSMC. Part II of the study applied a human coronary 3D model of leukocyte attack, the 3DLA-model. HCAEC and HCMSMC were cultured on both sides of a polycarbonate filters, mimicking the internal elastic membrane. Leukocyte attack (LA was carried out by adding human monocytes (MC on the endothelial side. The effect of MMF (50 μg/mL on adhesion and chemotaxis (0.5, 1, 2, 3, 4, 6, and 24 h after LA and the effect on proliferation of co-cultured HCMSMC (24 h after LA was studied. In part III of the study a porcine coronary organ culture model of restenosis (POC-model was used. After ex vivo ballooning MMF (50 μg/mL was added to the cultures for a period of 1, 2, 3, 4, 5, 6, and 7 days. The effect on reactive cell proliferation and neointimal thickening was studied at day 7 and day 28 after ballooning. Results Expression of ICAM-1 in northern blot and cytoflow studies was neither clearly inhibited nor stimulated after administration of MMF in the clinical relevant concentration of 50 μg/mL. In the 3DLA-model 50 μg/mL of MMF caused a significant antiproliferative effect (p Conclusion Thus, the data demonstrate a significant antiproliferative effect of clinical relevant levels of MMF (50 μg/mL in the 3DLA-model. The antiproliferative effect was a direct antiproliferative effect that was not triggered via reduced expression of ICAM-1 or via an inhibition of MC-adhesion and chemotaxis. Probably due

  19. Exploring Optical Contrast in Ex-Vivo Breast Tissue Using Diffuse Reflectance Spectroscopy and Tissue Morphology

    Science.gov (United States)

    Kennedy, Stephanie Ann

    In this research, ex-vivo breast tissue is evaluated to determine which sources of optical contrast have the potential to detect malignancy at the margins in women of differing breast composition. Then, H&E images of ex-vivo breast tissue sites are quantified to further deconstruct the relationship between optical scattering and the underlying tissue morphology. H&E images were taken of the malignant and benign sites and quantified to describe the % adipose, % collagen and % glands. Adipose sites, images at 10x, were predominantly fatty and quantified according to adipocyte morphology. H&E-stained adipose tissue sections were analyzed with an automated image processing algorithm to extract average cell area and cell density. Non-adipose sites were imaged with a 2.5x objective. Grids of 200µm boxes corresponding to the 3mm x 2mm area were overlaid on each non-adipose image. The non-adipose images were classified as the following: adipose and collagen (fibroadipose); collagen and glands (fibroglandular); adipose, collagen and glands (mixed); and malignant sites. Correlations between and % collagen in were determined in benign sites. Age, BMI, and MBD were then correlated to in the adipose and non-adipose sites. Variability in was determined to be related to collagen and not adipose content. In order to further investigate this relationship, the importance of age, BMI and MBD was analyzed after adjusting for the % collagen. Lastly, the relationship between % collagen and % glands was analyzed to determine the relative contributions of % collagen and % glands . Statistics were calculated using Wilcoxon rank-sum tests, Pearson correlation coefficients and linear fits in R. Further deconstructing the relationship between optical scattering and tissue morphology resulted in a positive relationship between and % collagen. Increased variability was observed in sites with a higher percentage of collagen. In adipose tissues MBD was negatively correlated with age, BMI and

  20. Removing biofilms from microstructured titanium ex vivo: a novel approach using atmospheric plasma technology.

    Science.gov (United States)

    Rupf, Stefan; Idlibi, Ahmad Nour; Marrawi, Fuad Al; Hannig, Matthias; Schubert, Andreas; von Mueller, Lutz; Spitzer, Wolfgang; Holtmann, Henrik; Lehmann, Antje; Rueppell, Andre; Schindler, Axel

    2011-01-01

    The removal of biofilms from microstructured titanium used for dental implants is a still unresolved challenge. This experimental study investigated disinfection and removal of in situ formed biofilms from microstructured titanium using cold atmospheric plasma in combination with air/water spray. Titanium discs (roughness (Ra): 1.96 µm) were exposed to human oral cavities for 24 and 72 hours (n = 149 each) to produce biofilms. Biofilm thickness was determined using confocal laser scanning microscopy (n = 5 each). Plasma treatment of biofilms was carried out ex vivo using a microwave-driven pulsed plasma source working at temperatures from 39 to 43°C. Following plasma treatment, one group was air/water spray treated before re-treatment by second plasma pulses. Vital microorganisms on the titanium surfaces were identified by contact culture (Rodac agar plates). Biofilm presence and bacterial viability were quantified by fluorescence microscopy. Morphology of titanium surfaces and attached biofilms was visualized by scanning electron microscopy (SEM). Total protein amounts of biofilms were colorimetrically quantified. Untreated and air/water treated biofilms served as controls. Cold plasma treatment of native biofilms with a mean thickness of 19 µm (24 h) to 91 µm (72 h) covering the microstructure of the titanium surface caused inactivation of biofilm bacteria and significant reduction of protein amounts. Total removal of biofilms, however, required additional application of air/water spray, and a second series of plasma treatment. Importantly, the microstructure of the titanium discs was not altered by plasma treatment. The combination of atmospheric plasma and non-abrasive air/water spray is applicable for complete elimination of oral biofilms from microstructured titanium used for dental implants and may enable new routes for the therapy of periimplant disease.

  1. Removing biofilms from microstructured titanium ex vivo: a novel approach using atmospheric plasma technology.

    Directory of Open Access Journals (Sweden)

    Stefan Rupf

    Full Text Available The removal of biofilms from microstructured titanium used for dental implants is a still unresolved challenge. This experimental study investigated disinfection and removal of in situ formed biofilms from microstructured titanium using cold atmospheric plasma in combination with air/water spray. Titanium discs (roughness (Ra: 1.96 µm were exposed to human oral cavities for 24 and 72 hours (n = 149 each to produce biofilms. Biofilm thickness was determined using confocal laser scanning microscopy (n = 5 each. Plasma treatment of biofilms was carried out ex vivo using a microwave-driven pulsed plasma source working at temperatures from 39 to 43°C. Following plasma treatment, one group was air/water spray treated before re-treatment by second plasma pulses. Vital microorganisms on the titanium surfaces were identified by contact culture (Rodac agar plates. Biofilm presence and bacterial viability were quantified by fluorescence microscopy. Morphology of titanium surfaces and attached biofilms was visualized by scanning electron microscopy (SEM. Total protein amounts of biofilms were colorimetrically quantified. Untreated and air/water treated biofilms served as controls. Cold plasma treatment of native biofilms with a mean thickness of 19 µm (24 h to 91 µm (72 h covering the microstructure of the titanium surface caused inactivation of biofilm bacteria and significant reduction of protein amounts. Total removal of biofilms, however, required additional application of air/water spray, and a second series of plasma treatment. Importantly, the microstructure of the titanium discs was not altered by plasma treatment. The combination of atmospheric plasma and non-abrasive air/water spray is applicable for complete elimination of oral biofilms from microstructured titanium used for dental implants and may enable new routes for the therapy of periimplant disease.

  2. Hydrogen sulfide activates the carotid body chemoreceptors in cat, rabbit and rat ex vivo preparations.

    Science.gov (United States)

    Jiao, Yingfu; Li, Qian; Sun, Biying; Zhang, Guohua; Rong, Weifang

    2015-03-01

    We and others previously reported experimental evidence suggesting an important role for hydrogen sulfide (H2S) in oxygen sensing in murine carotid body chemoreceptors. More recent data implicated abnormal H2S-mediated chemoreceptor signaling in pathological conditions such as chronic heart failure and hypertension. However, the idea of H2S as a mediator of oxygen-sensing in chemoreceptors has been challenged. In particular, it was shown that exogenous H2S inhibited the release of neurotransmitters (ACh and ATP) from the cat carotid body, raising the possibility that there exists significant species difference in H2S-mediated signaling in chemoreceptors. This study was designed specifically to determine the effect of H2S on chemoreceptors in different species. We conducted multiunit extracellular recordings of the sinus nerve in the ex vivo carotid body preparation taken from the rat, the cat and the rabbit. As observed in the mouse carotid body, H2S donors (NaHS or Na2S) evoked qualitatively similar excitatory responses of the afferent sinus nerves of the species studied here. The excitatory effects of the H2S donors were concentration-dependent and reversible. The sinus nerve responses to H2S donors were prevented by blockade of the transmission between type I cells and the afferent terminals, as was the response to hypoxia. These results demonstrate that exogenous H2S exerts qualitatively similar excitatory effects on chemoreceptor afferents of different species. The role of endogenous H2S-mediated signaling in carotid body function in different species awaits further investigation.

  3. Use of formalin-fixed tissues for ex-vivo imaging with optical coherence tomography (OCT)

    Science.gov (United States)

    Shortkroff, Sonya; Goodwin, Alicia; Giattina, Susanne; Liu, Bin; Brezinski, Mark E.

    2006-02-01

    Structural and compositional analysis of normal and pathological tissues by OCT often is performed ex vivo and subsequently compared to the histology. Many of the tissues of interest require immediate fixation to prevent degradation of the sample. Frequently, samples are obtained up to a week prior to procuring images by OCT. We investigated whether fixation affects OCT image analysis by acquiring images of freshly isolated bovine ligament samples and repeating OCT imaging of the same area after fixation at 24 hours and at one week. Samples were divided into two groups: group one was fixed in 10% neutral buffered formalin for 24 hours and placed in normal saline while group two remained in formalin for one week. Tissue samples were processed for paraffin embedment and stained with Masson's trichrome or with picrosirius red. The banding pattern contrast ratio of the OCT images before and after fixation for both groups was measured and compared for possible differences. Histology was evaluated for tissue integrity and compared to the OCT images. The mean contrast ratio at time 0 was 5.41 +/- 1.1 and 5.31 +/- 0.6 for groups 1 and 2, respectively. Results at 1 week were slightly lower with 5.11 +/- 0.3 and 5.20 +/- 0.7, respectively. Statistical analysis of the data by ANOVA showed no difference in the contrast ratios with time or with treatment. This data indicates that 24 hours in formalin is sufficient to fix these small ligament samples with little effect on imaging up to one week after fixation.

  4. Search for PET probes for imaging the globus pallidus studied with rat brain ex vivo autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Ishiwata, Kiichi; Ogi, Nobuo; Wang, Wei-Fang; Ishii, Kenji; Senda, Michio [Tokyo Metropolitan Inst. of Gerontology (Japan). Positron Medical Center; Shimada, Junichi; Tanaka, Akira; Suzuki, Fumio

    2000-12-01

    We have evaluated the feasibility of using four positron emission tomography (PET) tracers for imaging the globus pallidus by ex vivo autoradiography in rats. The tracers investigated were [{sup 11}C]KF18446, [{sup 11}C]SCH23390 and [{sup 11}C]raclopride for mapping adenosine A{sub 2A}, dopamine D{sub 1} and dopamine D{sub 2} receptors, respectively, and [{sup 18}F]FDG. The highest uptake by the globus pallidus was found for [{sup 11}C]SCH23390, followed by [{sup 18}F]FDG, [{sup 11}C]KF18446 and [{sup 11}C]raclopride. The receptor-specific uptake by the globus pallidus was observed in [{sup 11}C]KF18446 and [{sup 11}C]SCH23390, but not in [{sup 11}C]raclopride. Uptake ratios of globus pallidus to the striatum for [{sup 18}F]FDG and [{sup 11}C]KF18446 were approximately 0.6, which was twice as large as that for [{sup 11}C]SCH 23390. In a rat model of degeneration of striatopallidal {gamma}-aminobutyric acid-ergic-enkephalin neurons induced by intrastriatal injection of quinolinic acid, the uptake of [{sup 11}C]KF18446 by the striatum and globus pallidus was remarkably reduced. To prove the visualization of the globus pallidus by PET with [{sup 18}F]FDG and [{sup 11}C]KF18446, PET-MRI registration technique and advances in PET technologies providing high-resolution PET scanner will be required. The metabolic activity of the globus pallidus could then be measured by PET with [{sup 18}F]FDG, and [{sup 11}C]KF18446 may be a candidate tracer for imaging the pallidal terminals projecting from the striatum. (author)

  5. Impact of AQP3 inducer treatment on cultured human keratinocytes, ex vivo human skin and volunteers.

    Science.gov (United States)

    Garcia, N; Gondran, C; Menon, G; Mur, L; Oberto, G; Guerif, Y; Dal Farra, C; Domloge, N

    2011-10-01

    One of the main functions of the skin is to protect the organism against environmental threats, such as thermal stress. Aquaporin-3 (AQP3) facilitates water and glycerol transport across cell membranes and therefore regulates osmotic balance in different situations of stress. This mechanism seems to be particularly important for the resistance of different organisms to cold stress. Consequently, we were interested in investigating the effect of cold and osmotic stress on AQP3 expression in normal human keratinocytes. We developed a new active ingredient to stimulate aquaporins in skin and demonstrated the partial restoration of AQP3 expression in keratinocytes transfected with AQP3 siRNA. Moreover, we examined the effect of cold stress on cell morphology and the impact of a pre-treatment with the active ingredient. Our results indicated that induction of AQP3 helped maintain a correct organization of the actin cytoskeleton, preserving cell morphology and preventing cells from rounding. Immunofluorescent staining revealed cytoplasmic localization of AQP3 and its translocation to the cell membrane following osmotic stress. Histological ex vivo studies of skin under different conditions, such as cold environment and tape-stripping, indicated that increase in AQP3 expression appears to be involved in skin protection and showed that the pattern of AQP3 expression was more enhanced in the active ingredient-treated samples. In vivo confocal microscopy by Vivascope showed a generally healthier appearance of the skin in the treated areas. These results attest to the potential value of the active ingredient in optimizing environmental stress resistance and protecting the skin from stratum corneum damage.

  6. Laser-assisted cryosurgery in ex vivo mice hepatic tissue: viability assays using green fluorescent protein.

    Science.gov (United States)

    Martínez-Suástegui, L; Duperray, B; Godinez, F; Guillén, G; Slade, A; Aguilar, G

    2011-02-01

    An experimental investigation is carried out to develop a novel approach to cryosurgery, where laser heating counteracts tissue freezing to better confine damage to the targeted cancerous tissue within a lethal low-temperature isothermal boundary-an approach we refer to as laser-assisted cryosurgery (LAC). The advantage of this procedure relative to conventional cryosurgery assisted with urethral warmers or cryoheaters is that laser heating provides volumetric rather than superficial heating, which leads to deeper penetration, more homogeneous tissue protection and better demarcation of the destructive freezing effect to a well-defined targeted volume. Tissue viability assays are performed using green fluorescence protein (GFP) as a viability marker and correlated with temperature history after performing LAC procedures on ex vivo mice hepatic tissue. The limit for cell denaturation at the irradiated surface predicted by GFP analysis is further confirmed using reverse transcription polymerase chain reaction (RT-PCR). In addition, the correlation between GFP fluorescence and cell viability and loss of GFP fluorescence in non-viable cells has been tested and validated by histological analysis using a standard cell viability measuring method (hematoxylin and eosin staining). Analysis of our experimental measurements show that reproducible thermal gradients (of 236 °C/cm) and predictable tissue necrosis can be reliably produced by LAC without exceeding temperature thresholds for cell denaturation (of T (surf) ≈ 48 °C) beyond preset tissue boundaries (with resolution of 0.1 °C/mm). The results have shown the feasibility of controlling temperatures at specified tissue locations to prevent hyperthermal or freezing damage. PMID:20963494

  7. Impact of AQP3 inducer treatment on cultured human keratinocytes, ex vivo human skin and volunteers.

    Science.gov (United States)

    Garcia, N; Gondran, C; Menon, G; Mur, L; Oberto, G; Guerif, Y; Dal Farra, C; Domloge, N

    2011-10-01

    One of the main functions of the skin is to protect the organism against environmental threats, such as thermal stress. Aquaporin-3 (AQP3) facilitates water and glycerol transport across cell membranes and therefore regulates osmotic balance in different situations of stress. This mechanism seems to be particularly important for the resistance of different organisms to cold stress. Consequently, we were interested in investigating the effect of cold and osmotic stress on AQP3 expression in normal human keratinocytes. We developed a new active ingredient to stimulate aquaporins in skin and demonstrated the partial restoration of AQP3 expression in keratinocytes transfected with AQP3 siRNA. Moreover, we examined the effect of cold stress on cell morphology and the impact of a pre-treatment with the active ingredient. Our results indicated that induction of AQP3 helped maintain a correct organization of the actin cytoskeleton, preserving cell morphology and preventing cells from rounding. Immunofluorescent staining revealed cytoplasmic localization of AQP3 and its translocation to the cell membrane following osmotic stress. Histological ex vivo studies of skin under different conditions, such as cold environment and tape-stripping, indicated that increase in AQP3 expression appears to be involved in skin protection and showed that the pattern of AQP3 expression was more enhanced in the active ingredient-treated samples. In vivo confocal microscopy by Vivascope showed a generally healthier appearance of the skin in the treated areas. These results attest to the potential value of the active ingredient in optimizing environmental stress resistance and protecting the skin from stratum corneum damage. PMID:21401652

  8. Correlation of In Vivo and Ex Vivo ADC and T2 of In Situ and Invasive Murine Mammary Cancers.

    Directory of Open Access Journals (Sweden)

    Xiaobing Fan

    Full Text Available Ex vivo MRI may aid in the evaluation of surgical specimens, and provide valuable information regarding the micro-anatomy of mammary/breast cancer. The use of ex vivo MRI to study mouse mammary cancer would be enhanced if there is a strong correlation between parameters derived from in vivo and ex vivo scans. Here, we report the correlation between apparent diffusion coefficient (ADC and T2 values measured in vivo and ex vivo in mouse mammary glands with in situ cancers (mammary intraepithelial neoplasia (MIN and invasive cancers (those which spread outside the ducts into surrounding tissue. MRI experiments were performed on the Polyoma middle T oncoprotein breast cancer mouse model (n = 15 in a 9.4T scanner. For in vivo experiments, T2-weighted (T2W images were acquired to identify abnormal regions, then ADC and T2 values were measured for nine selected slices. For ex vivo experiments, a midline incision was made along the spine, and then skin, glands, and tumors were gently peeled from the body. Tissue was fixed in formalin, placed around a mouse-sized sponge, and sutured together mimicking the geometry of the gland when attached to the mouse. The same pulse sequences used for in vivo experiments were repeated for ex vivo scans at room temperature. Regions of interest were manually traced on T2W images defining features that could be identified on in vivo and ex vivo images. The results demonstrate a strong positive correlations between in vivo and ex vivo invasive cancers for ADC (r = 0.89, p <0.0001 and T2 (r = 0.89, p <0.0001 values; and weak to moderate positive correlations between in vivo and ex vivo in situ cancers for ADC (r = 0.61, p <0.0001 and T2 (r = 0.79, p <0.0001 values. The average ex vivo ADC value was about 54% of the in vivo value; and the average ex vivo T2 was similar to the in vivo value for cancers. Although motion, fixation, and temperature differences affect ADC and T2, these results show a reliable relationship

  9. Transfer studies of polystyrene nanoparticles in the ex vivo human placenta perfusion model: key sources of artifacts

    Science.gov (United States)

    Grafmueller, Stefanie; Manser, Pius; Diener, Liliane; Maurizi, Lionel; Diener, Pierre-André; Hofmann, Heinrich; Jochum, Wolfram; Krug, Harald F.; Buerki-Thurnherr, Tina; von Mandach, Ursula; Wick, Peter

    2015-08-01

    Nanotechnology is a rapidly expanding and highly promising new technology with many different fields of application. Consequently, the investigation of engineered nanoparticles in biological systems is steadily increasing. Questions about the safety of such engineered nanoparticles are very important and the most critical subject with regard to the penetration of biological barriers allowing particle distribution throughout the human body. Such translocation studies are technically challenging and many issues have to be considered to obtain meaningful and comparable results. Here we report on the transfer of polystyrene nanoparticles across the human placenta using an ex vivo human placenta perfusion model. We provide an overview of several challenges that can potentially occur in any translocation study in relation to particle size distribution, functionalization and stability of labels. In conclusion, a careful assessment of nanoparticle properties in a physiologically relevant milieu is as challenging and important as the actual study of nanoparticle-cell interactions itself.

  10. Formulation, and physical, in vitro and ex vivo evaluation of transdermal ibuprofen hydrogels containing turpentine oil as penetration enhancer.

    Science.gov (United States)

    Khan, N R; Khan, G M; Wahab, A; Khan, A R; Hussain, A; Nawaz, A; Akhlaq, M

    2011-11-01

    The aim of the present study was to investigate the transdermal permeation enhancing capability of turpentine oil for ibuprofen from hydrogels. Ibuprofen 1% w/v hydrogels were developed with carboxypolymethylene with and without turpentine oil. Turpentine oil was incorporated in increasing concentrations, i.e. 0.5, 1, 1.5, 2, 2.5 and 3% of the total gel formulation, and its permeation enhancing effect was examined. Gels were examined physically for pH, viscosity, spreadability, extrudability, smoothness and appearance. To study the in vitro and ex vivo permeation potential of formulated gels, permeation studies were performed with a Franz diffusion cell using cellulose membrane and excised rabbit abdominal skin. Ibuprofen hydrogel with 3% turpentine oil showed a maximum flux of 10.87 mg/cm2/h across artificial skin and 17.26 mg/cm2/h across rabbit abdominal skin.

  11. Ex vivo mucoadhesion of different zinc-pectinate hydrogel beads.

    Science.gov (United States)

    Hagesaether, Ellen; Bye, Ragnar; Sande, S Arne

    2008-01-22

    The objective of this study was to investigate the mucoadhesive properties of pre-swelled hydrogel beads made of six types of pectin from three manufacturers. The types of pectin differed mainly in the degree of methoxylation and degree of amidation. Zinc ions were used as cross-linking agent. The mucoadhesive properties were tested on an inverted fresh porcine small intestine attached to a rotating cylinder. Beads made of pectin with a high degree of methoxylation (70%) showed superior mucoadhesive results compared to the other formulations, which could be correlated to the lower amount of zinc in this formulation, subsequently leading to a lower amount of cross-linking and higher mobility of the polymer chains of these beads. This study therefore also indicated the importance of doing mucoadhesive measurements on relevant formulations, and not basing the understanding solely on investigating polymer solutions. Samples from different manufacturers produced the same results.

  12. Ex vivo brain tumor analysis using spectroscopic optical coherence tomography

    Science.gov (United States)

    Lenz, Marcel; Krug, Robin; Welp, Hubert; Schmieder, Kirsten; Hofmann, Martin R.

    2016-03-01

    A big challenge during neurosurgeries is to distinguish between healthy tissue and cancerous tissue, but currently a suitable non-invasive real time imaging modality is not available. Optical Coherence Tomography (OCT) is a potential technique for such a modality. OCT has a penetration depth of 1-2 mm and a resolution of 1-15 μm which is sufficient to illustrate structural differences between healthy tissue and brain tumor. Therefore, we investigated gray and white matter of healthy central nervous system and meningioma samples with a Spectral Domain OCT System (Thorlabs Callisto). Additional OCT images were generated after paraffin embedding and after the samples were cut into 10 μm thin slices for histological investigation with a bright field microscope. All samples were stained with Hematoxylin and Eosin. In all cases B-scans and 3D images were made. Furthermore, a camera image of the investigated area was made by the built-in video camera of our OCT system. For orientation, the backsides of all samples were marked with blue ink. The structural differences between healthy tissue and meningioma samples were most pronounced directly after removal. After paraffin embedding these differences diminished. A correlation between OCT en face images and microscopy images can be seen. In order to increase contrast, post processing algorithms were applied. Hence we employed Spectroscopic OCT, pattern recognition algorithms and machine learning algorithms such as k-means Clustering and Principal Component Analysis.

  13. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies

    Science.gov (United States)

    Witkowski, Benoit; Amaratunga, Chanaki; Khim, Nimol; Sreng, Sokunthea; Chim, Pheaktra; Kim, Saorin; Lim, Pharath; Mao, Sivanna; Sopha, Chantha; Sam, Baramey; Anderson, Jennifer M; Duong, Socheat; Chuor, Char Meng; Taylor, Walter R J; Suon, Seila; Mercereau-Puijalon, Odile; Fairhurst, Rick M; Menard, Didier

    2016-01-01

    Summary Background Artemisinin resistance in Plasmodium falciparum lengthens parasite clearance half-life during artemisinin monotherapy or artemisinin-based combination therapy. Absence of in-vitro and ex-vivo correlates of artemisinin resistance hinders study of this phenotype. We aimed to assess whether an in-vitro ring-stage survival assay (RSA) can identify culture-adapted P falciparum isolates from patients with slow-clearing or fast-clearing infections, to investigate the stage-dependent susceptibility of parasites to dihydroartemisinin in the in-vitro RSA, and to assess whether an ex-vivo RSA can identify artemisinin-resistant P falciparum infections. Methods We culture-adapted parasites from patients with long and short parasite clearance half-lives from a study done in Pursat, Cambodia, in 2010 (registered with ClinicalTrials.gov, number NCT00341003) and used novel in-vitro survival assays to explore the stage-dependent susceptibility of slow-clearing and fast-clearing parasites to dihydroartemisinin. In 2012, we implemented the RSA in prospective parasite clearance studies in Pursat, Preah Vihear, and Ratanakiri, Cambodia (NCT01736319), to measure the ex-vivo responses of parasites from patients with malaria. Continuous variables were compared with the Mann-Whitney U test. Correlations were analysed with the Spearman correlation test. Findings In-vitro survival rates of culture-adapted parasites from 13 slow-clearing and 13 fast-clearing infections differed significantly when assays were done on 0–3 h ring-stage parasites (10·88% vs 0·23%; p=0·007). Ex-vivo survival rates significantly correlated with in-vivo parasite clearance half-lives (n=30, r=0·74, 95% CI 0·50–0·87; p<0·0001). Interpretation The in-vitro RSA of 0–3 h ring-stage parasites provides a platform for the molecular characterisation of artemisinin resistance. The ex-vivo RSA can be easily implemented where surveillance for artemisinin resistance is needed. Funding Institut

  14. An ex vivo original test using radiotracers for evaluating haemocompatibility of tubular biomaterials

    Energy Technology Data Exchange (ETDEWEB)

    Janvier, G.; Caix, J.; Bordenave, L.; Revel, P.; Baquey, C.; Ducassou, D. (Institut National de la Sante et de la Recherche Medicale (INSERM), 33 - Bordeaux (France))

    1994-02-01

    The haemocompatibility of a vascular prosthesis can be estimated as the result of its interaction with blood components. The authors describe an ex vivo canine shunt for evaluating isotopic haemocompatibility in blood-wall interactions. Methods employing radioisotopic tracers can be used to dynamically monitor the adsorption of labelled blood cells and proteins on different biomaterial surfaces. This ex vivo test should enable materials to be assessed for quality according to two thrombogenic criteria: (i) number of adhered platelets mm[sup -2] s[sup -1], (ii) quantity of adsorbed fibrinogen expressed as [mu]g mm[sup -2] s[sup -1], which would provide the basis for a scale of haemocompatibility. (author).

  15. New technologies and possibilities of breast cancer staging by the criterion N ex vivo

    Directory of Open Access Journals (Sweden)

    Sh. Kh. Gantsev

    2010-01-01

    Full Text Available The study was undertaken to improve breast cancer (BC staging by the criterion N ex vivo, by applying the up-to-date ultrasound and microsurgical technologies. A LySonix 3000® with PulseSelectTM system was used to expose lymph nodes and vessels. Axillary adipose tissue classically exposed was examined in 70 patients with Stages II-III BC. Lymph vessels and nodes were separated by the sonolipodestruction technique. The findings permit sonolipodestruction to be used for ex vivo total lymph dissection for BC as a method that improves its N-staging. Sonolymphodissection opens up fresh opportunities for studying the anatomic structure of the lymphatic apparatus.

  16. Tumor associated antigen specific T-cell populations identified in ex vivo expanded TIL cultures

    DEFF Research Database (Denmark)

    Junker, Niels; Kvistborg, Pia; Køllgaard, Tania;

    2012-01-01

    Ex vivo expanded tumor infiltrating lymphocytes (TILs) from malignant melanoma (MM) and head & neck squamous cell carcinoma (HNSCC) share a similar oligoclonal composition of T effector memory cells, with HLA class I restricted lysis of tumor cell lines. In this study we show that ex vivo expanded...... TILs from MM and HNSCC demonstrate a heterogeneous composition in frequency and magnitude of tumor associated antigen specific populations by Elispot IFN¿ quantitation. TILs from MM and HNSCC shared reactivity towards NY ESO-1, cyclin B1 and Bcl-x derived peptides. Additionally we show that dominating...... the heterogeneous tumors upon adoptive transfer; increasing the probability of tumor control by minimizing immune evasion by tumor cell escape variants....

  17. Sustained ex vivo skin antiseptic activity of chlorhexidine in poly(epsilon-caprolactone) nanocapsule encapsulated form and as a digluconate.

    Science.gov (United States)

    Lboutounne, Hassan; Chaulet, Jean-François; Ploton, Christine; Falson, Françoise; Pirot, Fabrice

    2002-08-21

    In this work, the sustained bactericidal activity of chlorhexidine base loaded poly(epsilon-caprolactone), PCL, nanocapsules against Staphylococcus epidermidis inoculated onto porcine ear skin was investigated. Drug loaded nanocapsules were prepared by the interfacial polymer deposition following solvent displacement method, then characterized by photon correlation spectroscopy, electrophoretic measurements, transmission and scanning electron microscopy. Antimicrobial activity of these colloidal carriers was evaluated (i) in vitro against eight strains of bacteria, and (ii) ex vivo against Staphylococcus epidermidis inoculated for 12 h onto porcine ear skin surface treated for 3 min either with 0.6% chlorhexidine base loaded or unloaded nanocapsules suspended in hydrogel, or 1% chlorhexidine digluconate aqueous solution. Chlorhexidine absorption into the stratum corneum (SC) was evaluated by the tape-stripping method. The results showed that chlorhexidine nanocapsules in aqueous suspension having a 200-300 nm size and a positive charge exhibited similar minimum inhibitory concentrations against several bacteria with chlorhexidine digluconate aqueous solution. Ex vivo, there was a significant reduction in the number of colony forming units (CFUs) from 3-min treated skin with chlorhexidine nanocapsule suspension (5 to <1 log(10)) compared to chlorhexidine digluconate solution (5 to 2.02 log(10)) after a 8-h artificial contamination. After a 12-h artificial contamination, both formulations failed to achieve a 5 log(10) reduction. Furthermore, from a 3-min treatment with an identical applied dose and a subsequent 12-h artificial contamination, a residual chlorhexidine concentration in the SC was found to be three-fold higher with chlorhexidine nanocapsule suspension than with chlorhexidine digluconate solution. Interestingly, nanocapsules were shown in porcine skin follicles. Consequently, a topical application of chlorhexidine base-loaded positively charged

  18. Calcium Dynamics of Ex Vivo Long-Term Cultured CD8+ T Cells Are Regulated by Changes in Redox Metabolism

    Science.gov (United States)

    Gran, Margaret A.; Potnis, Anish; Hill, Abby; Lu, Hang

    2016-01-01

    T cells reach a state of replicative senescence characterized by a decreased ability to proliferate and respond to foreign antigens. Calcium release associated with TCR engagement is widely used as a surrogate measure of T cell response. Using an ex vivo culture model that partially replicates features of organismal aging, we observe that while the amplitude of Ca2+ signaling does not change with time in culture, older T cells exhibit faster Ca2+ rise and a faster decay. Gene expression analysis of Ca2+ channels and pumps expressed in T cells by RT-qPCR identified overexpression of the plasma membrane CRAC channel subunit ORAI1 and PMCA in older T cells. To test whether overexpression of the plasma membrane Ca2+ channel is sufficient to explain the kinetic information, we adapted a previously published computational model by Maurya and Subramaniam to include additional details on the store-operated calcium entry (SOCE) process to recapitulate Ca2+ dynamics after T cell receptor stimulation. Simulations demonstrated that upregulation of ORAI1 and PMCA channels is not sufficient to explain the observed alterations in Ca2+ signaling. Instead, modeling analysis identified kinetic parameters associated with the IP3R and STIM1 channels as potential causes for alterations in Ca2+ dynamics associated with the long term ex vivo culturing protocol. Due to these proteins having known cysteine residues susceptible to oxidation, we subsequently investigated and observed transcriptional remodeling of metabolic enzymes, a shift to more oxidized redox couples, and post-translational thiol oxidation of STIM1. The model-directed findings from this study highlight changes in the cellular redox environment that may ultimately lead to altered T cell calcium dynamics during immunosenescence or organismal aging. PMID:27526200

  19. MSP is a negative regulator of inflammation and lipogenesis in ex vivo models of non-alcoholic steatohepatitis.

    Science.gov (United States)

    Chanda, Dipanjan; Li, Jieyi; Oligschlaeger, Yvonne; Jeurissen, Mike L J; Houben, Tom; Walenbergh, Sofie M A; Shiri-Sverdlov, Ronit; Neumann, Dietbert

    2016-01-01

    Non-alcoholic steatohepatitis (NASH), a metabolic disorder consisting of steatosis and inflammation, is considered the hepatic equivalent of metabolic syndrome and can result in irreversible liver damage. Macrophage-stimulating protein (MSP) is a hepatokine that potentially has a beneficial role in hepatic lipid and glucose metabolism via the activation of AMP-activated protein kinase (AMPK). In the current study, we investigated the regulatory role of MSP in the development of inflammation and lipid metabolism in various NASH models, both in vitro and ex vivo. We observed that MSP treatment activated the AMPK signaling pathway and inhibited lipopolysaccharide (LPS)- and palmitic acid (PA)-induced gene expression of pro-inflammatory cytokines in primary mouse hepatocytes. In addition, MSP treatment resulted in a significant reduction in PA-induced lipid accumulation and inhibited the gene expression of key lipogenic enzymes in HepG2 cells. Upon short hairpin RNA-induced knockdown of RON (the membrane-bound receptor for MSP), the anti-inflammatory and anti-lipogenic effects of MSP were markedly ablated. Finally, to mimic NASH ex vivo, we challenged bone marrow-derived macrophages with oxidized low-density lipoprotein (oxLDL) in combination with LPS. OxLDL+LPS exposure led to a marked inhibition of AMPK activity and a robust increase in inflammation. MSP treatment significantly reversed these effects by restoring AMPK activity and by suppressing pro-inflammatory cytokine gene expression and secretion under this condition. Taken together, these data suggest that MSP is an effective inhibitor of inflammation and lipid accumulation in the stressed liver, thereby indicating that MSP has a key regulatory role in NASH. PMID:27609031

  20. Expression of Plasmodium vivax crt-o Is Related to Parasite Stage but Not Ex Vivo Chloroquine Susceptibility.

    Science.gov (United States)

    Pava, Zuleima; Handayuni, Irene; Wirjanata, Grennady; To, Sheren; Trianty, Leily; Noviyanti, Rintis; Poespoprodjo, Jeanne Rini; Auburn, Sarah; Price, Ric N; Marfurt, Jutta

    2016-01-01

    Chloroquine (CQ)-resistant Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivax crt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax β-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5; P importance of development-stage composition for measuring pvcrt-o expression and suggest that pvcrt-o transcription is not a primary determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance. PMID:26525783

  1. Utilization of the organ care system as ex-vivo lung perfusion after cold storage transportation.

    Science.gov (United States)

    Mohite, P N; Maunz, O; Popov, A-F; Zych, B; Patil, N P; Simon, A R

    2015-11-01

    The Organ Care System (OCS) allows perfusion and ventilation of the donor lungs under physiological conditions. Ongoing trials to compare preservation with OCS Lung with standard cold storage do not include donor lungs with suboptimal gas exchange and donor lungs treated with OCS following cold storage transportation. We present a case of a 48-yr-old man who received such lungs after cold storage transportation treated with ex-vivo lung perfusion utilizing OCS.

  2. The use of ex vivo human skin tissue for genotoxicity testing

    Energy Technology Data Exchange (ETDEWEB)

    Reus, Astrid A.; Usta, Mustafa [TNO Triskelion BV, Utrechtseweg 48, 3704 HE, Zeist (Netherlands); Krul, Cyrille A.M., E-mail: cyrille.krul@tno.nl [TNO, Utrechtseweg 48, 3704 HE Zeist (Netherlands)

    2012-06-01

    As a result of the chemical legislation concerning the registration, evaluation, authorization and restriction of chemicals (REACH), and the Seventh Amendment to the Cosmetics Directive, which prohibits animal testing in Europe for cosmetics, alternative methods for safety evaluation of chemicals are urgently needed. Current in vitro genotoxicity assays are not sufficiently predictive for the in vivo situation, resulting in an unacceptably high number of misleading positives. For many chemicals and ingredients of personal care products the skin is the first site of contact, but there are no in vitro genotoxicity assays available in the skin for additional evaluation of positive or equivocal responses observed in regulatory in vitro genotoxicity assays. In the present study ex vivo human skin tissue obtained from surgery was used for genotoxicity evaluation of chemicals by using the comet assay. Fresh ex vivo human skin tissue was cultured in an air–liquid interface and topically exposed to 20 chemicals, including true positive, misleading positive and true negative genotoxins. Based on the results obtained in the present study, the sensitivity, specificity and accuracy of the ex vivo skin comet assay to predict in vivo genotoxicity were 89%, 90% and 89%, respectively. Donor and experimental variability were mainly reflected in the magnitude of the response and not the difference between the presence and absence of a genotoxic response. The present study indicates that human skin obtained from surgery is a promising and robust model for safety evaluation of chemicals that are in direct contact with the skin. -- Highlights: ► We use human skin obtained from surgery for genotoxicity evaluation of chemicals. ► We use the comet assay as parameter for genotoxicity in ex vivo human skin. ► Sensitivity, specificity and accuracy to predict in vivo genotoxins are determined. ► Sensitivity, specificity and accuracy are 89%, 90% and 90%, respectively. ► The method

  3. IL-12 directs further maturation of ex vivo differentiated NK cells with improved therapeutic potential.

    Directory of Open Access Journals (Sweden)

    Dorit Lehmann

    Full Text Available The possibility to modulate ex vivo human NK cell differentiation towards specific phenotypes will contribute to a better understanding of NK cell differentiation and facilitate tailored production of NK cells for immunotherapy. In this study, we show that addition of a specific low dose of IL-12 to an ex vivo NK cell differentiation system from cord blood CD34(+ stem cells will result in significantly increased proportions of cells with expression of CD62L as well as KIRs and CD16 which are preferentially expressed on mature CD56(dim peripheral blood NK cells. In addition, the cells displayed decreased expression of receptors such as CCR6 and CXCR3, which are typically expressed to a lower extent by CD56(dim than CD56(bright peripheral blood NK cells. The increased number of CD62L and KIR positive cells prevailed in a population of CD33(+NKG2A(+ NK cells, supporting that maturation occurs via this subtype. Among a series of transcription factors tested we found Gata3 and TOX to be significantly downregulated, whereas ID3 was upregulated in the IL-12-modulated ex vivo NK cells, implicating these factors in the observed changes. Importantly, the cells differentiated in the presence of IL-12 showed enhanced cytokine production and cytolytic activity against MHC class I negative and positive targets. Moreover, in line with the enhanced CD16 expression, these cells exhibited improved antibody-dependent cellular cytotoxicity for B-cell leukemia target cells in the presence of the clinically applied antibody rituximab. Altogether, these data provide evidence that IL-12 directs human ex vivo NK cell differentiation towards more mature NK cells with improved properties for potential cancer therapies.

  4. The ex vivo neurotoxic, myotoxic and cardiotoxic activity of cucurbituril-based macrocyclic drug delivery vehicles

    OpenAIRE

    Oun, Rabbab; Floriano, Rafael S.; Isaacs, Lyle; Rowan, Edward G.; Wheate, Nial J.

    2014-01-01

    The cucurbituril family of drug delivery vehicles have been examined for their tissue specific toxicity using ex vivo models. Cucurbit[6]uril (CB[6]), cucurbit[7]uril (CB[7]) and the linear cucurbituril-derivative Motor2 were examined for their neuro-, myo- and cardiotoxic activity and compared with β-cyclodextrin. The protective effect of drug encapsulation by CB[7] was also examined on the platinum-based anticancer drug cisplatin. The results show that none of the cucurbiturils have statist...

  5. Animal models of ex vivo lung perfusion as a platform for transplantation research

    OpenAIRE

    Nelson, Kevin; Bobba, Christopher; Ghadiali, Samir; Jr, Don Hayes; Black, Sylvester M; Whitson, Bryan A

    2014-01-01

    Ex vivo lung perfusion (EVLP) is a powerful experimental model for isolated lung research. EVLP allows for the lungs to be manipulated and characterized in an external environment so that the effect of specific ventilation/perfusion variables can be studied independent of other confounding physiologic contributions. At the same time, EVLP allows for normal organ level function and real-time monitoring of pulmonary physiology and mechanics. As a result, this technique provides unique advantage...

  6. EFFECT OF PERMEATION ENHANCER ON EX-VIVO PERMEATION OF ONDANSETRON HCl BUCCAL TABLETS

    OpenAIRE

    M. Praveen Kumar et al.

    2011-01-01

    The objective of the paper is to study the effect of a permeation enhancer, sodium taurocholate on permeation of Ondansetron HCl from bioadhesive buccal tablet formulation by performing ex-vivo permeation experiments using porcine buccal mucosa. Optimized formulation has selected based on in-vitro drug release studies of bilayered bioadhesive buccal tablets. To the optimized formulation, 10mM sodium taurocholate was added to increase the permeation of poorly permeable ondansetron HCl. It is w...

  7. Assessment of proarrhythmic activity of chloroquine in in vivo and ex vivo rabbit models

    Directory of Open Access Journals (Sweden)

    Shailaja B Khobragade

    2013-01-01

    Full Text Available Objectives: To evaluate the prolongation of ventricular repolarization and proarrhythmic activity of antimalarial drug chloroquine in two rabbit proarrhythmia models viz., in vivo α1 adrenoceptor-stimulated anesthetized rabbit and ex vivo isolated Langendorff rabbit heart using clofilium as standard proarrhythmic agent. Materials and Methods: In the in vivo model, three groups of rabbits, anesthetized by pentobarbitone sodium and α-chloralose, sensitized with α1 agonist methoxamine followed by either continuous infusion of saline (control or clofilium (3 mg/kg or chloroquine (21 mg/kg for 30 min. In ex vivo model, rabbit hearts were perfused with clofilium (10 μM or chloroquine (300 μM continuously after priming along with methoxamine, acetylcholine chloride and propranolol hydrochloride. Results: In these models, prolongation of repolarization during α1 -adrenoceptor stimulation produced early after depolarization (EAD and Torsade de pointes (TdP. Saline infusion did not induce any abnormality in the animals. Clofilium caused expected changes in the electrocardiogram in both the models including TdP (50.0% in in vivo and 66.67% in ex vivo. Chloroquine caused decrease in heart rate and increase in the corrected QT (QTc interval in both the models. Further, apart from different stages of arrhythmia, TdP was evident in 33.33% in ex vivo model, whereas no TdP was observed in in vivo model. Conclusions: The results indicated that proarrhythmic potential of chloroquine and clofilium was well evaluated in both the models; moreover, both the models can be used to assess the proarrhythmic potential of the new drug candidates.

  8. Criteria for viability assessment of discarded human donor livers during ex vivo normothermic machine perfusion.

    Directory of Open Access Journals (Sweden)

    Michael E Sutton

    Full Text Available Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37 °C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1 steadily increasing bile production, resulting in a cumulative output of ≥ 30 g after 6 h (high bile output group, and (2 a cumulative bile production <20 g in 6 h (low bile output group. Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial.

  9. The use of ex vivo human skin tissue for genotoxicity testing

    International Nuclear Information System (INIS)

    As a result of the chemical legislation concerning the registration, evaluation, authorization and restriction of chemicals (REACH), and the Seventh Amendment to the Cosmetics Directive, which prohibits animal testing in Europe for cosmetics, alternative methods for safety evaluation of chemicals are urgently needed. Current in vitro genotoxicity assays are not sufficiently predictive for the in vivo situation, resulting in an unacceptably high number of misleading positives. For many chemicals and ingredients of personal care products the skin is the first site of contact, but there are no in vitro genotoxicity assays available in the skin for additional evaluation of positive or equivocal responses observed in regulatory in vitro genotoxicity assays. In the present study ex vivo human skin tissue obtained from surgery was used for genotoxicity evaluation of chemicals by using the comet assay. Fresh ex vivo human skin tissue was cultured in an air–liquid interface and topically exposed to 20 chemicals, including true positive, misleading positive and true negative genotoxins. Based on the results obtained in the present study, the sensitivity, specificity and accuracy of the ex vivo skin comet assay to predict in vivo genotoxicity were 89%, 90% and 89%, respectively. Donor and experimental variability were mainly reflected in the magnitude of the response and not the difference between the presence and absence of a genotoxic response. The present study indicates that human skin obtained from surgery is a promising and robust model for safety evaluation of chemicals that are in direct contact with the skin. -- Highlights: ► We use human skin obtained from surgery for genotoxicity evaluation of chemicals. ► We use the comet assay as parameter for genotoxicity in ex vivo human skin. ► Sensitivity, specificity and accuracy to predict in vivo genotoxins are determined. ► Sensitivity, specificity and accuracy are 89%, 90% and 90%, respectively. ► The method

  10. Training gastroenterology fellows to perform gastric polypectomy using a novel ex vivo model

    Institute of Scientific and Technical Information of China (English)

    Ming-Jen Chen; Ching-Chung Lin; Chia-Yuan Liu; Chih-Jen Chen; Chen-Wang Chang; Ching-Wei Chang; Chien-Wei Lee; Shou-Chuan Shih; Horng-Yuan Wang

    2011-01-01

    AIM: To evaluate the effect of hands-on training of gastroenterology fellows in gastric polypectomy using an ex vivo simulator.METHODS: Eight gastroenterology fellows at Mackay Memorial Hospital, Taipei were evaluated in gastric polypectomy techniques using a pig stomach with artificial polyps created by a rubber band ligation device. The performance of four second year (year-2) fellows who had undergone one year of clinical training was compared with that of four first year (year-1) fellows both before and after a 4-h workshop using the ex vivo simulator. The workshop allowed for hands-on training in the removal of multiple artificial polyps and the placement of hemoclips at the excision site. Evaluation included observation of technical skills, procedure time, and the fellows' confidence scale.RESULTS: One week after the workshop, the year-1 fellows were re-evaluated and had significantly improved mean performance scores (from 17.9 ± 1.8 to 22.5 ± 0.7), confidence scale (from 4.5 ± 1.0 to 7.8 ± 0.5) and procedure time (from 615.0 ± 57.4 s to 357.5 ± 85.0 s) compared with their baseline performance. After 4 h of training using the ex vivo simulator, the skills of the year-1 fellows were statistically similar to those of the year-2 fellows.CONCLUSION: Use of this ex vivo simulator significantly improved the endoscopic gastric polypectomy skills of gastroenterology fellows who had not had previous clinical training in gastric polypectomy.

  11. Mass transfer trends occurring in engineered ex vivo tissue scaffolds.

    Science.gov (United States)

    Moore, Marc; Sarntinoranont, Malisa; McFetridge, Peter

    2012-08-01

    In vivo the vasculature provides an effective delivery system for cellular nutrients; however, artificial scaffolds have no such mechanism, and the ensuing limitations in mass transfer result in limited regeneration. In these investigations, the regional mass transfer properties that occur through a model scaffold derived from the human umbilical vein (HUV) were assessed. Our aim was to define the heterogeneous behavior associated with these regional variations, and to establish if different decellularization technologies can modulate transport conditions to improve microenvironmental conditions that enhance cell integration. The effect of three decellularization methods [Triton X-100 (TX100), sodium dodecyl sulfate (SDS), and acetone/ethanol (ACE/EtOH)] on mass transfer, cellular migration, proliferation, and metabolic activity were assessed. Results show that regional variation in tissue structure and composition significantly affects both mass transfer and cell function. ACE/EtOH decellularization was shown to increase albumin mass flux through the intima and proximate-medial region (0-250 μm) when compared with sections decellularized with TX100 or SDS; although, mass flux remained constant over all regions of the full tissue thickness when using TX100. Scaffolds decellularized with TX100 were shown to promote cell migration up to 146% further relative to SDS decellularized samples. These results show that depending on scaffold derivation and expectations for cellular integration, specificities of the decellularization chemistry affect the scaffold molecular architecture resulting in variable effects on mass transfer and cellular response.

  12. Ex-vivo multi-modal microscopy of healthy skin

    Science.gov (United States)

    Guevara, Edgar; Gutiérrez-Hernández, José Manuel; Castonguay, Alexandre; Lesage, Frédéric; González, Francisco Javier

    2014-09-01

    The thorough characterization of skin samples is a critical step in investigating dermatological diseases. The combination of depth-sensitive anatomical imaging with molecular imaging has the potential to provide vast information about the skin. In this proof-of-concept work we present high-resolution mosaic images of skin biopsies using Optical Coherence Tomography (OCT) manually co-registered with standard microscopy, bi-dimensional Raman spectral mapping and fluorescence imaging. A human breast skin sample, embedded in paraffin, was imaged with a swept-source OCT system at 1310 nm. Individual OCT volumes were acquired in fully automated fashion in order to obtain a large field-of-view at high resolution (~10μm). Based on anatomical features, the other three modalities were manually co-registered to the projected OCT volume, using an affine transformation. A drawback is the manual co-registration, which may limit the utility of this method. However, the results indicate that multiple imaging modalities provide complementary information about the sample. This pilot study suggests that multi-modal microscopy may be a valuable tool in the characterization of skin biopsies.

  13. Digital Radiography for Determination of Primary Tooth Length: In Vivo and Ex Vivo Studies

    Directory of Open Access Journals (Sweden)

    Maria D. Basso

    2015-01-01

    Full Text Available Background. Methods for determining the root canal length of the primary tooth should yield accurate and reproducible results. In vitro studies show some limitations, which do not allow their findings to be directly transferred to a clinical situation. Aim. To compare the accuracy of radiographic tooth length obtained from in vivo digital radiograph with that obtained from ex vivo digital radiograph. Method. Direct digital radiographs of 20 upper primary incisors were performed in teeth (2/3 radicular resorption that were radiographed by an intraoral sensor, according to the long-cone technique. Teeth were extracted, measured, and mounted in a resin block, and then radiographic template was used to standardise the sensor-target distance (30 cm. The apparent tooth length (APTL was obtained from the computer screen by means of an electronic ruler accompanying the digital radiography software (CDR 2.0, whereas the actual tooth length (ACTL was obtained by means of a digital calliper following extraction. Data were compared to the ACTL by variance analysis and Pearson’s correlation test. Results. The values for APTL obtained from in vivo radiography were slightly underestimated, whereas those values obtained from ex vivo were slightly overestimated. No significance was observed (P≤0.48 between APTL and ACTL. Conclusion. The length of primary teeth estimated by in vivo and ex vivo comparisons using digital radiography was found to be similar to the actual tooth length.

  14. The use of ex vivo human skin tissue for genotoxicity testing.

    Science.gov (United States)

    Reus, Astrid A; Usta, Mustafa; Krul, Cyrille A M

    2012-06-01

    As a result of the chemical legislation concerning the registration, evaluation, authorization and restriction of chemicals (REACH), and the Seventh Amendment to the Cosmetics Directive, which prohibits animal testing in Europe for cosmetics, alternative methods for safety evaluation of chemicals are urgently needed. Current in vitro genotoxicity assays are not sufficiently predictive for the in vivo situation, resulting in an unacceptably high number of misleading positives. For many chemicals and ingredients of personal care products the skin is the first site of contact, but there are no in vitro genotoxicity assays available in the skin for additional evaluation of positive or equivocal responses observed in regulatory in vitro genotoxicity assays. In the present study ex vivo human skin tissue obtained from surgery was used for genotoxicity evaluation of chemicals by using the comet assay. Fresh ex vivo human skin tissue was cultured in an air-liquid interface and topically exposed to 20 chemicals, including true positive, misleading positive and true negative genotoxins. Based on the results obtained in the present study, the sensitivity, specificity and accuracy of the ex vivo skin comet assay to predict in vivo genotoxicity were 89%, 90% and 89%, respectively. Donor and experimental variability were mainly reflected in the magnitude of the response and not the difference between the presence and absence of a genotoxic response. The present study indicates that human skin obtained from surgery is a promising and robust model for safety evaluation of chemicals that are in direct contact with the skin. PMID:22507867

  15. Toxoplasma gondii inhibits R5 HIV-1 replication in human lymphoid tissues ex vivo

    Science.gov (United States)

    Sassi, Atfa; Brichacek, Beda; Hieny, Sara; Yarovinsky, Felix; Golding, Hana; Grivel, Jean-Charles; Sher, Alan; Margolis, Leonid

    2016-01-01

    Critical events of HIV-1 pathogenesis occur in lymphoid tissues where HIV-1 is typically accompanied by infections with other pathogens (HIV co-pathogens). Co-pathogens greatly affect the clinical course of the disease and the transmission of HIV. The apicomplexan parasite Toxoplasma gondii is a common HIV co-pathogen associated with AIDS development. Here, we examined the interaction of T. gondii and HIV in coinfected human lymphoid tissue ex vivo. Both pathogens readily replicate in ex vivo infected blocks of human tonsillar tissue. Surprisingly, we found that live T. gondii preferentially inhibits R5 HIV-1 replication in coinfected tissues. This effect is reproduced by treatment of the tissue blocks with recombinant C-18, a T. gondii -encoded cyclophilin that binds to CCR5. These ex vivo findings raise the possibility that, in addition to being a co-factor in HIV disease, T. gondii may influence the outcome of viral infection by preferentially suppressing R5 variants. PMID:19671446

  16. Apoptotic cell death, detected ex vivo in peripheral blood lymphocytes of HIV-1 infected persons

    Directory of Open Access Journals (Sweden)

    L. F. te Velde

    1996-01-01

    Full Text Available In HIV-1 infection the ongoing depletion of CD4+ T-lymphocytes is believed, to a large extent, to be due to apoptosis. Until now quantitative information about in vivo apoptosis of lymphocytes in HIV-patients is scarce because of the very nature of the apoptotic process. Successful detection of apoptosis ex vivo requires the recognition of the initial phase of this process, because at a later stage the cells may not remain any longer in the circulation. We measured quantitatively the amount of early apoptotic peripheral blood lymphocytes directly ex vivo in HIV-1 infected patients using a recently described flow cytometric assay. With this method we observed in an unselected heterogenous group of twelve HIV-infected individuals a median percentage of apoptotic lymphocytes to be significantly higher than in ten healthy controls. To the best of our knowledge this is the first report of ex vivo observed increased apoptosis of peripheral blood lymphocytes in HIV-infected persons.

  17. Ex vivo generation of a functional and regenerative wound epithelium from axolotl (Ambystoma mexicanum) skin.

    Science.gov (United States)

    Ferris, Donald R; Satoh, Akira; Mandefro, Berhan; Cummings, Gillian M; Gardiner, David M; Rugg, Elizabeth L

    2010-10-01

    Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs. Regeneration is a stepwise process that requires interactions between keratinocytes, nerves and fibroblasts. The formation of a wound epithelium covering the amputation site is an early and necessary event in the process but the molecular mechanisms that underlie the role of the wound epithelium in regeneration remain unclear. We have developed an ex vivo model that recapitulates many features of in vivo wound healing. The model comprises a circular explant of axolotl (Ambystoma mexicanum) limb skin with a central circular, full thickness wound. Re-epithelialization of the wound area is rapid (typically <11 h) and is dependent on metalloproteinase activity. The ex vivo wound epithelium is viable, responds to neuronal signals and is able to participate in ectopic blastema formation and limb regeneration. This ex vivo model provides a reproducible and tractable system in which to study the cellular and molecular events that underlie wound healing and regeneration.

  18. Synaptic signal streams generated by ex vivo neuronal networks contain non-random, complex patterns.

    Science.gov (United States)

    Lee, Sangmook; Zemianek, Jill M; Shultz, Abraham; Vo, Anh; Maron, Ben Y; Therrien, Mikaela; Courtright, Christina; Guaraldi, Mary; Yanco, Holly A; Shea, Thomas B

    2014-11-01

    Cultured embryonic neurons develop functional networks that transmit synaptic signals over multiple sequentially connected neurons as revealed by multi-electrode arrays (MEAs) embedded within the culture dish. Signal streams of ex vivo networks contain spikes and bursts of varying amplitude and duration. Despite the random interactions inherent in dissociated cultures, neurons are capable of establishing functional ex vivo networks that transmit signals among synaptically connected neurons, undergo developmental maturation, and respond to exogenous stimulation by alterations in signal patterns. These characteristics indicate that a considerable degree of organization is an inherent property of neurons. We demonstrate herein that (1) certain signal types occur more frequently than others, (2) the predominant signal types change during and following maturation, (3) signal predominance is dependent upon inhibitory activity, and (4) certain signals preferentially follow others in a non-reciprocal manner. These findings indicate that the elaboration of complex signal streams comprised of a non-random distribution of signal patterns is an emergent property of ex vivo neuronal networks.

  19. Ex vivo surface and mechanical properties of coated orthodontic archwires.

    Science.gov (United States)

    Elayyan, Firas; Silikas, Nick; Bearn, David

    2008-12-01

    This study examined the mechanical and physical properties of retrieved coated nickel-titanium (NiTi) archwires compared with unused samples. Ultraesthetic 0.016 inch coated archwires (G&H(R) Wire Company) were investigated. Ten as-received wires were subjected to a three-point bending test using conventional and self-ligating bracket systems. Surface roughness of the coating was measured with a contact stylus profilometer. Optical and scanning electron microscopes were used to assess surface topography. Ten archwires were used in vivo for a period of between 4 and 6 weeks. Retrieved archwires were subjected to the same tests. The percentage of the remaining coating was calculated using digital photography. Coated archwires were used in vivo for a mean period of 33 days. Differences between the mean values of the as-received and retrieved archwires were determined using t-tests. In the three-point bending test, with conventional elastomeric ligation, retrieved wires produced a lower unloading force (P self-ligating bracket system, retrieved and as-received coated archwires produced the same amount of force (P > 0.05). With surface profilometry, all measured roughness parameters (except R(sm)) had greater surface roughness for the retrieved coated archwires (P < 0.05). Under microscopy, retrieved coated archwires showed discolouration, ditching, and delamination. Only 75 per cent of the coating was present in retrieved coated archwires. Retrieved coated archwires produced lower unloading force values than as-received coated archwires with conventional ligation. Surface roughness of coated archwires increased after use. Coated archwires have a low aesthetic value, with 25 per cent of the coating lost within 33 days in vivo. PMID:19011166

  20. Recondicionamento pulmonar ex vivo: uma nova era para o transplante pulmonar Ex vivo lung reconditioning: a new era for lung transplantation

    Directory of Open Access Journals (Sweden)

    Alessandro Wasum Mariani

    2012-12-01

    Full Text Available O transplante pulmonar consolidou-se como a melhor opção terapêutica para diversas pneumopatias terminais. O baixo número de doadores viáveis ainda persiste como uma grande limitação ao aumento do número de transplantes de pulmão, causando alta mortalidade na lista de espera. Diferentemente do transplante de outros órgãos sólidos, a maior limitação do transplante pulmonar não é o número absoluto de doadores e sim a viabilidade desses órgãos, que é reduzida devido às agressões ao pulmão ocasionadas pela morte encefálica e aos cuidados na UTI. Diversas são as propostas para o aumento do número de doadores: intensificação das campanhas de doação, o uso de doadores com coração parado, transplante pulmonar lobar intervivos e maior flexibilidade dos critérios para aceitação de doadores de pulmão. Todavia, a proposta que atrai a atenção de diversos grupos de transplante pulmonar é a perfusão pulmonar ex vivo, principalmente pela perspectiva de recuperação de pulmões inicialmente descartados. Esse sistema consiste na reperfusão e ventilação do bloco pulmonar isolado em um circuito de circulação extracorpórea modificado. Devido aos bons resultados apresentados e à perspectiva de aumento no número de órgãos aptos a transplante, diversos grupos têm estudado a técnica. Pesquisadores na Suécia, Canadá, Áustria, Inglaterra, Espanha e Brasil já possuem experiência sólida com o método e introduziram algumas variações. O objetivo deste artigo foi revisar o desenvolvimento, o estado da arte e as perspectivas futuras do modelo ex vivo de perfusão e recondicionamento pulmonar.Lung transplantation has come to be viewed as the best treatment option for various end-stage lung diseases. The low number of viable donors continues to be a major obstacle to increasing the number of lung transplants, resulting in high mortality among patients on the waiting list. Unlike transplantation of other solid organs

  1. The Ex Vivo Human Placental Transfer of the Anti-HIV Nucleoside Inhibitor Abacavir and the Protease Inhibitor Amprenavir

    OpenAIRE

    Bawdon, R E

    1998-01-01

    Objective: The transfer of abacavir, a new nucleoside inhibitor, and amprenavir, a new protease inhibitor, used for the treatment of human immunodeficiency virus, has been studied in the ex vivo human placental model.Methods: The ex vivo human placental model used C14 antipyrine to determine the transport fraction and clearance index of these compounds at both the peak and trough serum concentrations. The clearance index accumulation and tissue concentrations were determined for each drug by ...

  2. The ex vivo human placental transfer of the anti-HIV nucleoside inhibitor abacavir and the protease inhibitor amprenavir.

    OpenAIRE

    Bawdon, R E

    1998-01-01

    OBJECTIVE: The transfer of abacavir, a new nucleoside inhibitor, and amprenavir, a new protease inhibitor, used for the treatment of human immunodeficiency virus, has been studied in the ex vivo human placental model. METHODS: The ex vivo human placental model used C14 antipyrine to determine the transport fraction and clearance index of these compounds at both the peak and trough serum concentrations. The clearance index accumulation and tissue concentrations were determined for each drug by...

  3. Detecting hepatic steatosis using ultrasound-induced thermal strain imaging: an ex vivo animal study

    International Nuclear Information System (INIS)

    ± 0.037%). Using histology as a gold standard to classify mouse livers, US-TSI had a sensitivity and specificity of 70% and 90%, respectively. The area under the receiver operating characteristic curve was 0.775. This ex vivo study demonstrates the feasibility of using US-TSI to detect fatty livers and warrants further investigation of US-TSI as a diagnostic tool for hepatic steatosis. (paper)

  4. Detecting hepatic steatosis using ultrasound-induced thermal strain imaging: an ex vivo animal study

    Science.gov (United States)

    Mahmoud, Ahmed M.; Ding, Xuan; Dutta, Debaditya; Singh, Vijay P.; Kim, Kang

    2014-02-01

    mouse livers, US-TSI had a sensitivity and specificity of 70% and 90%, respectively. The area under the receiver operating characteristic curve was 0.775. This ex vivo study demonstrates the feasibility of using US-TSI to detect fatty livers and warrants further investigation of US-TSI as a diagnostic tool for hepatic steatosis.

  5. Effects of TGF-beta signalling inhibition with galunisertib (LY2157299) in hepatocellular carcinoma models and in ex vivo whole tumor tissue samples from patients

    OpenAIRE

    Serova, Maria; Tijeras-Raballand, Annemilaï; Santos, Célia Dos; Albuquerque, Miguel; Paradis, Valerie; Neuzillet, Cindy; Benhadji, Karim A; Raymond, Eric; Faivre, Sandrine; De Gramont, Armand

    2015-01-01

    Galunisertib (LY2157299) is a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation currently under clinical investigation in hepatocellular carcinoma (HCC) patients. Our study explored the effects of galunisertib in vitro in HCC cell lines and ex vivo on patient samples. Galunisertib was evaluated in HepG2, Hep3B, Huh7, JHH6 and SK-HEP1 cells as well as in SK-HEP1-derived cells tolerant to sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines...

  6. Evaluation of the In Vivo and Ex Vivo Binding of Novel BC1 Cannabinoid Receptor Radiotracers

    Energy Technology Data Exchange (ETDEWEB)

    Miller, A.; Gatley, J.; Gifford, A.

    2002-01-01

    The primary active ingredient of marijuana, 9-tetrahydrocannabinol, exerts its psychoactive effects by binding to cannabinoid CB1 receptors. These receptors are found throughout the brain with high concentrations in the hippocampus and cerebellum. The current study was conducted to evaluate the binding of a newly developed putative cannabinoid antagonist, AM630, and a classical cannabinoid 8-tetrahydrocannabinol as potential PET and/or SPECT imaging agents for brain CB1 receptors. For both of these ligands in vivo and ex vivo studies in mice were conducted. AM630 showed good overall brain uptake (as measure by %IA/g) and a moderately rapid clearance from the brain with a half-clearance time of approximately 30 minutes. However, AM630 did not show selective binding to CB1 cannabinoid receptors. Ex vivo autoradiography supported the lack of selective binding seen in the in vivo study. Similar to AM630, 8-tetrahydrocanibol also failed to show selective binding to CB1 receptor rich brain areas. The 8-tetrahydrocanibol showed moderate overall brain uptake and relatively slow brain clearance as compared to AM630. Further studies were done with AM2233, a cannabinoid ligand with a similar structure as AM630. These studies were done to develop an ex vivo binding assay to quantify the displacement of [131I]AM2233 binding by other ligands in Swiss-Webster and CB1 receptor knockout mice. By developing this assay we hoped to determine the identity of an unknown binding site for AM2233 present in the hippocampus of CB1 knockout mice. Using an approach based on incubation of brain slices prepared from mice given intravenous [131I]AM2233 in either the presence or absence of AM2233 (unlabelled) it was possible to demonstrate a significant AM2233-displacable binding in the Swiss-Webster mice. Future studies will determine if this assay is appropriate for identifying the unknown binding site for AM2233 in the CB1 knockout mice.

  7. Using computed tomography scans to develop an ex-vivo gastric model

    Institute of Scientific and Technical Information of China (English)

    Jerome A Henry; Gerard O'Sullivan; Abhay S Pandit

    2007-01-01

    The objective of this research was to use abdominal computed tomography (CT) scans to non-invasively quantify anthropometrical data of the human stomach and to concomitantly create an anatomically correct and distensible ex-vivo gastric model. Thirty-three abdominal CT scans of human subjects were obtained and were imported into reconstruction software to generate 3D models of the stomachs. Anthropometrical data such as gastric wall thickness, gastric surface area and gastric volume were subsequently quantified. A representative 3D computer model was exported into a selective laser sintering (SLS) rapid prototyping machine to create an anatomically correct solid gastric model. Subsequently,a replica wax template of the SLS model was created. A negative mould was offset around the wax template such that the offset distance was equivalent to that of the gastric wall thickness. A silicone with similar mechanical properties to the human stomach was poured into the offset. The lost wax manufacturing technique was employed to create a hollow distensible stomach model.3D computer gastric models were generated from the CT scans. A hollow distensible silicone ex-vivo gastric model with similar compliance to that of the human stomach was created. The anthropometrical data indicated that there is no significant relationship between BMI and gastric surface area or gastric volume. There were interand intra-group differences between groups with respect to gastric wall thickness. This study demonstrates that abdominal CT scans can be used to both non-invasively determine gastric anthropometrical data as well as create realistic ex-vivo stomach models.

  8. Characterization of micro-invasive trabecular bypass stents by ex vivo perfusion and computational flow modeling

    Directory of Open Access Journals (Sweden)

    Hunter KS

    2014-03-01

    Full Text Available Kendall S Hunter,1 Todd Fjield,2 Hal Heitzmann,2 Robin Shandas,1 Malik Y Kahook3 1Department of Bioengineering, University of Colorado Denver, Aurora, CO, USA; 2Glaukos Corporation, Laguna Hills, CA, USA; 3University of Colorado Hospital Eye Center, Aurora, CO, USA Abstract: Micro-invasive glaucoma surgery with the Glaukos iStent® or iStent inject® (Glaukos Corporation, Laguna Hills, CA, USA is intended to create a bypass through the trabecular meshwork to Schlemm's canal to improve aqueous outflow through the natural physiologic pathway. While the iStent devices have been evaluated in ex vivo anterior segment models, they have not previously been evaluated in whole eye perfusion models nor characterized by computational fluid dynamics. Intraocular pressure (IOP reduction with the iStent was evaluated in an ex vivo whole human eye perfusion model. Numerical modeling, including computational fluid dynamics, was used to evaluate the flow through the stents over physiologically relevant boundary conditions. In the ex vivo model, a single iStent reduced IOP by 6.0 mmHg from baseline, and addition of a second iStent further lowered IOP by 2.9 mmHg, for a total IOP reduction of 8.9 mmHg. Computational modeling showed that simulated flow through the iStent or iStent inject is smooth and laminar at physiological flow rates. Each stent was computed to have a negligible flow resistance consistent with an expected significant decrease in IOP. The present perfusion results agree with prior clinical and laboratory studies to show that both iStent and iStent inject therapies are potentially titratable, providing clinicians with the opportunity to achieve lower target IOPs by implanting additional stents. Keywords: glaucoma, iStent, trabecular bypass, intraocular pressure, ab-interno, CFD

  9. Application of Gold Nanorods for Photothermal Therapy in Ex Vivo Human Oesophagogastric Adenocarcinoma.

    Science.gov (United States)

    Singh, Mohan; Harris-Birtill, David C C; Zhou, Yu; Gallina, Maria E; Cass, Anthony E G; Hanna, George B; Elson, Daniel S

    2016-03-01

    Gold nanoparticles are chemically fabricated and tuned to strongly absorb near infrared (NIR) light, enabling deep optical penetration and therapy within human tissues, where sufficient heating induces tumour necrosis. In our studies we aim to establish the optimal gold nanorod (GNR) concentration and laser power for inducing hyperthermic effects in tissues and test this photothermal effect on ex vivo human oesophagogastric adenocarcinoma. The ideal GNR concentration and NIR laser power that would elicit sufficient hyperthermia for tumour necrosis was pre-determined on porcine oesophageal tissues. Human ex vivo oesophageal and gastric adenocarcinoma tissues were incubated with GNR solutions and a GNR-free control solution with corresponding healthy tissues for comparison, then irradiated with NIR light for 10 minutes. Temperature rise was found to vary linearly with both the concentration of GNRs and the laser power. Human ex vivo oesophageal and gastric tissues consistently demonstrated a significant temperature rise when incubated in an optimally concentrated GNR solution (3 x 10(10) GNRs/ml) prior to NIR irradiation delivered at an optimal power (2 W/cm2). A mean temperature rise of 27 degrees C was observed in tissues incubated with GNRs, whereas only a modest 2 degrees C rise in tissues not exposed to any GNRs. This study evaluates the photothermal effects of GNRs on oesophagogastric tissue examines their application in the minimally invasive therapeutics of oesophageal and gastric adenocarcinomas. This could potentially be an effective method of clinically inducing irreversible oesophagogastric tumour photodestruction, with minimal collateral damage expected in (healthy) tissues free from GNRs.

  10. Plaque characterization in ex vivo MRI evaluated by dense 3D correspondence with histology

    DEFF Research Database (Denmark)

    Engelen, A. van; de Bruijne, Marleen; Klein, S.;

    2011-01-01

    registration of histology data with ex vivo MRI data, using non-rigid registration, both for training and evaluation. This is more objective than previously presented methods, as it eliminates selection bias that is introduced when 2D MRI slices are manually matched to histological slices before evaluation....... Histological slices of human atherosclerotic plaques were manually segmented into necrotic core, fibrous tissue and calcification. Classification of these three components was voxelwise evaluated. As features the intensity, gradient magnitude and Laplacian in four MRI sequences after different degrees...

  11. In vitro and ex vivo effects of cyclosporin A on phagocytic host defenses against Aspergillus fumigatus.

    OpenAIRE

    Roilides, E.; Robinson, T.; T Sein; Pizzo, P A; Walsh, T J

    1994-01-01

    Because cyclosporin A (CsA) is extensively used as an immunosuppressive agent, its effects on phagocytic defenses against Aspergillus fumigatus were studied in vitro and ex vivo. After incubation with 10 to 250 ng of CsA per ml at 37 degrees C for 60 min, polymorphonuclear leukocytes (PMNs) exhibited unaltered superoxide anion (O2-) production in response to phorbol myristate acetate and N-formylmethionyl leucyl phenylalanine, whereas > or = 500 ng/ml significantly suppressed it (P < 0.01). M...

  12. Effects of zinc ex vivo on taurine uptake in goldfish retinal cells

    OpenAIRE

    Nusetti, Sonia; Urbina, Mary; Lima, Lucimey

    2010-01-01

    Background Taurine and zinc exert neurotrophic effects in the central nervous system. Current studies demonstrate that Na+/Cl- dependent neurotransmitter transporters, similar to that of taurine, are modulated by micromolar concentrations of zinc. This study examined the effect of zinc sulfate ex vivo on [3H]taurine transport in goldfish retina. Methods Isolated cells were incubated in Ringer with zinc (0.1–100 µM). Taurine transport was done with 50 nM [3H]taurine or by isotopic dilution wit...

  13. Ex vivo protocol for testing virus survival on human skin: experiments with herpesvirus 2.

    OpenAIRE

    Graham, M. L.; Springthorpe, V S; Sattar, S A

    1996-01-01

    We report an ex vivo method, which uses pieces of human skin excised during routine plastic surgery, for testing survival of hazardous pathogens. Using this procedure, we compared the survival of human herpesvirus 2 on human skin and on metal disks. At the physiological skin temperature of 32 degrees C, the half-life of the virus on skin was 1.44 h while on metal disks it was 0.36 h. Even at ambient temperature (22 degrees C), the virus lost infectivity faster (half-life = 0.96 h) on metal di...

  14. Antibody Discovery Ex Vivo Accelerated by the LacO/LacI Regulatory Network

    OpenAIRE

    Munehisa Yabuki; W Jason Cummings; Leppard, John B.; Immormino, Robert M.; Christi L Wood; Daniel S Allison; Gray, Patrick W.; Tjoelker, Larry W; Nancy Maizels

    2012-01-01

    Monoclonal antibodies (mAbs) can be potent and highly specific therapeutics, diagnostics and research reagents. Nonetheless, mAb discovery using current in vivo or in vitro approaches can be costly and time-consuming, with no guarantee of success. We have established a platform for rapid discovery and optimization of mAbs ex vivo. This DTLacO platform derives from a chicken B cell line that has been engineered to enable rapid selection and seamless maturation of high affinity mAbs. We have va...

  15. Ex-vivo liver perfusion for organ preservation: Recent advances in the field.

    Science.gov (United States)

    Barbas, A S; Goldaracena, N; Dib, M J; Selzner, M

    2016-07-01

    Liver transplantation is the optimal treatment for end-stage liver disease but is limited by the severe shortage of donor organs. This shortage has prompted increased utilization of marginal grafts from DCD and extended criteria donors, which poorly tolerate cold storage in comparison to standard criteria grafts. Ex-vivo liver perfusion (EVLP) technology has emerged as a potential alternative to cold storage for organ preservation, but there is no consensus regarding the optimal temperature or conditions for EVLP. Herein, we review recent advances in both pre-clinical and clinical studies, organized by perfusion temperature (hypothermic, subnormothermic, normothermic). PMID:27158081

  16. Phosphodiesterase inhibition mediates matrix metalloproteinase activity and the level of collagen degradation fragments in a liver fibrosis ex vivo rat model

    Directory of Open Access Journals (Sweden)

    Veidal Sanne Skovgård

    2012-12-01

    Full Text Available Abstract Background Accumulation of extracellular matrix (ECM and increased matrix metalloproteinase (MMP activity are hallmarks of liver fibrosis. The aim of the present study was to develop a model of liver fibrosis combining ex vivo tissue culture of livers from CCl4 treated animals with an ELISA detecting a fragment of type III collagen generated in vitro by MMP-9 (C3M, known to be associated with liver fibrosis and to investigate cAMP modulation of MMP activity and liver tissue turnover in this model. Findings In vivo: Rats were treated for 8 weeks with CCl4/Intralipid. Liver slices were cultured for 48 hours. Levels of C3M were determined in the supernatants of slices cultured without treatment, treated with GM6001 (positive control or treated with IBMX (phosphodiesterase inhibitor. Enzymatic activity of MMP-2 and MMP-9 were studied by gelatin zymography. Ex vivo: The levels of serum C3M increased 77% in the CCl4-treated rats at week 8 (p 4-treated animals had highly increased MMP-9, but not MMP-2 activity, compared to slices derived from control animals. Conclusions We have combined an ex vivo model of liver fibrosis with measurement of a biochemical marker of collagen degradation in the condition medium. This technology may be used to evaluate the molecular process leading to structural fibrotic changes, as collagen species are the predominant structural part of fibrosis. These data suggest that modulation of cAMP may play a role in regulation of collagen degradation associated with liver fibrosis.

  17. Effect of maternal seaweed extract supplementation on suckling piglet growth, humoral immunity, selected microflora, and immune response after an ex vivo lipopolysaccharide challenge.

    Science.gov (United States)

    Leonard, S G; Sweeney, T; Bahar, B; O'Doherty, J V

    2012-02-01

    The present study was conducted to investigate the effect of maternal dietary supplementation (n = 10 sows/treatment) with seaweed extract (SWE: 0 vs. 10.0 g/d) from d 107 of gestation until weaning (d 26) on neonatal piglet growth, humoral immunity, intestinal morphology, selected intestinal microflora, and VFA concentrations. Furthermore, this study examined the effect of dietary treatment on the immune response after an ex vivo Escherichia coli lipopolysaccharide (LPS) tissue challenge at weaning in a 2 × 2 factorial arrangement. The main factors consisted of sow dietary treatment (SWE or control) and immunological challenge (yes or no). The SWE supplement (10.0 g/d) contained laminarin (1.0 g), fucoidan (0.8 g), and ash (8.2 g) and was extracted from a Laminaria spp. The SWE-supplemented sows had greater colostrum IgA (P Piglets suckling SWE-supplemented sows had greater serum IgG (P piglets suckling SWE-supplemented sows had a decreased colonic E. coli population at weaning (P Piglets suckling SWE-supplemented sows had greater TNF-α mRNA expression after ex vivo LPS challenge compared with non-SWE-supplemented sows (P Piglet BW at birth and weaning, and small intestinal morphology were unaffected by sow dietary treatment under current experimental conditions. In summary, these results demonstrate an important immunomodulatory role of SWE supplementation characterized by enhanced colostral IgA and IgG concentrations, greater piglet circulatory IgG concentrations on d 14 of lactation, and enhanced TNF-α mRNA expression in the ileum after an ex vivo LPS challenge. These results indicate that SWE supplementation enhanced piglet immune function and colonic microflora at weaning.

  18. A novel technique for impaction bone grafting in acetabular reconstruction of revision total hip arthroplasty using an ex vivo compaction device

    International Nuclear Information System (INIS)

    Impaction bone grafting allows restoration of the acetabular bone stock in revision hip arthroplasty. The success of this technique depends largely on achieving adequate initial stability of the component. To obtain well-compacted, well-graded allograft aggregates, we developed an ex vivo compaction device to apply it in revision total hip arthroplasty on the acetabular side, and characterized mechanical properties and putative osteoconductivity of allograft aggregates. Morselized allograft bone chips were compacted ex vivo using the creep technique and subsequent impaction technique to form the bone aggregates. Impaction allograft reconstruction of the acetabulum using an ex vivo compaction device was performed on eight hips. The mechanical properties and three-dimensional micro-CT-based structural characteristics of the bone aggregates were investigated. In clinical practice, this technique offered good reproducibility in reconstructing the cavity and the segmental defects of the acetabulum, with no migration and no loosening of the component. In vitro analysis showed that the aggregates generated from 25 g fresh-frozen bone chips gained compression stiffness of 13.5-15.4 MPa under uniaxial consolidation strain. The recoil of the aggregates after compaction was 2.6-3.9%. The compression stiffness and the recoil did not differ significantly from those measured using a variety of proportions of large- and small-sized bone chips. Micro-CT-based structural analysis revealed average pore sizes of 268-299 μm and average throat diameter of pores in the bone aggregates of more than 100 μm. These sizes are desirable for osteoconduction, although large interconnected pores of more than 500 μm were detectable in association with the proportion of large-sized bone chips. Cement penetration into the aggregates was related to the proportion of large-sized bone chips. This study introduces the value of an ex vivo compaction device in bone graft compaction in clinical

  19. Wound healing activity of the leaves of Artocarpus heterophyllus Lam. (Moraceae on ex-vivo porcine skin wound healing model

    Directory of Open Access Journals (Sweden)

    K Periyanayagam

    2013-05-01

    Full Text Available ABSTRACT Objective: To prescreen the ex- vivo wound healing activity of flavonoid rich fraction of ethyl acetate extract of the leaves of Artocarpus heterophyllus Lam. Family Moraceae using porcine skin wound healing model (PSWHM along with  phytochemical, XRF, HPTLC analysis. The aim of this present study is to provide pharmacological validation to the traditional claim for wound healing activity of Artocarpus heterophyllus leaves. Method: Total phenolic content by UV spectral methods and ursolic acid content by HPTLC, trace elements by X-ray fluorescence were determined.  The wound healing effect of the ethyl acetate extract of the leaves of A.heterophyllus (EAAH was evaluated using ex- vivo porcine skin wound healing model - a novel organ culture model system for evaluation of drugs in cell-cell junction in the wound healing process. Results: Total phenolic content by UV method, HPTLC determination of ursolic acid content of EAAH was found to be 376.5mg/g GAE, 134mg/g respectively. XRF study showed the presence of calcium (39.4%, potassium (29.6%, magnesium (2.06%, Iron (0.99%, sulphur (1.83%, zinc (0.083%, strontium (0.23%, manganese (0.13% and aluminium (0.005%.   Histopathological evaluation showed all treated wounds were sound with no signs of apoptosis, necrosis or bacterial contamination and no toxicity of the tested concentrations of EAAH of the leaves. Morphology of the wound margins, epidermis and dermis layer were found to be normal. Epidermal migration or keratinocyte migration distances from the edges of each wound were measured, normalized with the PBS control group and expressed as mean%. The result clearly showed EAAH (1.5% promoted statistically significant wound healing effect is comparable to the standard drug Mupirocin. Conclusion: This study indicates that the ethyl acetate extract of the leaves of A.heterophyllus possesses potential wound healing activity on ex-vivo porcine skin wound healing model. Wound healing

  20. Use of Ex Vivo Normothermic Perfusion for Quality Assessment of Discarded Human Donor Pancreases.

    Science.gov (United States)

    Barlow, A D; Hamed, M O; Mallon, D H; Brais, R J; Gribble, F M; Scott, M A; Howat, W J; Bradley, J A; Bolton, E M; Pettigrew, G J; Hosgood, S A; Nicholson, M L; Saeb-Parsy, K

    2015-09-01

    A significant number of pancreases procured for transplantation are deemed unsuitable due to concerns about graft quality and the associated risk of complications. However, this decision is subjective and some declined grafts may be suitable for transplantation. Ex vivo normothermic perfusion (EVNP) prior to transplantation may allow a more objective assessment of graft quality and reduce discard rates. We report ex vivo normothermic perfusion of human pancreases procured but declined for transplantation, with ABO-compatible warm oxygenated packed red blood cells for 1-2 h. Five declined human pancreases were assessed using this technique after a median cold ischemia time of 13 h 19 min. One pancreas, with cold ischemia over 30 h, did not appear viable and was excluded. In the remaining pancreases, blood flow and pH were maintained throughout perfusion. Insulin secretion was observed in all four pancreases, but was lowest in an older donation after cardiac death pancreas. Amylase levels were highest in a gland with significant fat infiltration. This is the first study to assess the perfusion, injury, as measured by amylase, and exocrine function of human pancreases using EVNP and demonstrates the feasibility of the approach, although further refinements are required.

  1. Preparation and evaluation of SEDDS of simvastatin by in vivo, in vitro and ex vivo technique.

    Science.gov (United States)

    Karim, Fahim Tamzeedul; Kalam, Azad; Anwar, Rafi; Miah, Muhammad Masum; Rahman, Md Shamim; Islam, S M Ashraful

    2015-01-01

    The objective of this work was to formulate a Self Emulsifying Drug Delivery System (SEDDS) of simvastatin, a poorly soluble drug and to evaluate by in vivo, in vitro and ex vivo techniques. Oils and surfactants were screened out depending upon their solubilizing capacity. Among all of the solvents, Capryol 90 showed good solubilizing capacity. It dissolved 105 mg/ml of simvastatin. Tween-80 also showed good solubilizing capacity which was 117 mg/ml. The two excipients were used to prepare simvastatin SEDDS. Formulations were initially checked for the color, clarity and sedimentation. The SEDDS formulations were transparent and clear. Formulation F2 containing 7:3 (m/m) mixture of Capryol 90/Tween-80 produced smallest micro-emulsion with particles size of 0.074 µm and drug release was higher than other formulation (102% within 20 min). Ex vivo study of the SEDDS formulation was evaluated using guinea pig intestinal sac. Drug diffused from F2 formulation was significantly higher than pure drug (p < 0.001). In vivo study of SEDDS was performed in albino mice using plasma cholesterol level as a pharmacodynamic marker parameter. The test formulation (F2) appeared remarkable reduction in plasma cholesterol level, after oral administration which showed that SEDDS may be an effective technique for the oral administration of simvastatin. PMID:25138349

  2. Esomeprazole immediate release tablets: Gastric mucosa ex vivo permeation, absorption and antisecretory activity in conscious rats.

    Science.gov (United States)

    Benetti, Camillo; Flammini, Lisa; Vivo, Valentina; Colombo, Paolo; Colombo, Gaia; Elviri, Lisa; Scarpignato, Carmelo; Buttini, Francesca; Bettini, Ruggero; Barocelli, Elisabetta; Rossi, Alessandra

    2016-10-10

    The aim of this work was to study the esomeprazole activity on the control of gastric secretion after administration of a novel immediate release tablet. The ex vivo permeation of esomeprazole across porcine gastric mucosa from immediate release tablets, containing sodium carbonate or magnesium oxide as alkalinizing agents, was firstly assessed. Pharmacokinetics and pharmacodynamics studies in conscious rats following the administration of immediate release tablets with sodium carbonate, in comparison with delayed-release tablets having the same formula, were also conducted. The results showed an important effect of sodium carbonate and magnesium oxide on the drug release, on the ex vivo trans-mucosal transport and the stability in acid environment. In particular, the presence of sodium carbonate in esomeprazole tablet formulation provided the maximum increase of the drug in vitro transport across the mucosa. Then, the absorption and the antisecretory activity of this proton pump inhibitor orally administered in rats as immediate release tablets containing Na2CO3, was superior but not significantly different compared to delayed-release tablets having the same formula. In the adopted animal model, an activity of esomeprazole from immediate release alkaline formulation was seen also in presence of partial gastric absorption allowing inhibition of proton pumps reached via systemic circulation. This esomeprazole immediate release formulation could be used for the on-demand treatment of acid-related disorders such as gastro-esophageal reflux disease.

  3. Ex vivo expansion of tumor-infiltrating lymphocytes from nasopharyngeal carcinoma patients for adoptive immunotherapy

    Institute of Scientific and Technical Information of China (English)

    Jia He; Xiao-Feng Tang; Qiu-Yan Chen; Hai-Qiang Mai; Zhou-Feng Huang; Jiang Li; Yi-Xin Zeng

    2012-01-01

    Establishing Epstein-Barr virus (EBV)-specific cytolytic T lymphocytes (EBV-CTLs) from peripheral blood mononuclear cells (PBMCs) for adoptive immunotherapy has been reported in EBV-associated malignancies including Hodgkin's lymphoma and nasopharyngeal carcinoma (NPC).In the current study,we performed ex vivo expansion of tumor-infiltrating lymphocytes (TILs) obtained from NPC biopsy specimens with a rapid expansion protocol using anti-CD3 monoclonal antibody (OKT3),recombinanthuman interleukin (IL)-2,and irradiated PBMCs from healthy donors to initiate the growth of TILs.Young TIL cultures comprised of more than 90% of CD3+ T cells,a variable percentage of CD3+CD8+ and CD3+CD4+ T cells,and less than 10% of CD3-CD16+ natural killer cells,a similar phenotype of EBV-CTL cultures from PBMCs.Interestingly,TIL cultures secreted high levels of the Th1 cytokines,interferon gamma (IFNγ) and tumor necrosis factor-alpha (TNF-α),and low levels of the Th2 cytokines,IL-4 and IL10.Moreover,young TILs could recognize autologous EBV-transformed B lymphoblast cell lines,but not autologous EBV-negative blast cells or allogeneic EBV-negative tumor cells.Taken together,these data suggest that ex vivo expansion of TILs from NPC biopsy tissue is an appealing alternative method to establish T cell-based immunotherapy for NPC.

  4. Normothermic ex vivo perfusion prevents lung injury compared to extended cold preservation for transplantation.

    Science.gov (United States)

    Cypel, M; Rubacha, M; Yeung, J; Hirayama, S; Torbicki, K; Madonik, M; Fischer, S; Hwang, D; Pierre, A; Waddell, T K; de Perrot, M; Liu, M; Keshavjee, S

    2009-10-01

    Treatment of injured donor lungs ex vivo to accelerate organ recovery and ameliorate reperfusion injury could have a major impact in lung transplantation. We have recently demonstrated a feasible technique for prolonged (12 h) normothermic ex vivo lung perfusion (EVLP). This study was performed to examine the impact of prolonged EVLP on ischemic injury. Pig donor lungs were cold preserved in Perfadex for 12 h and subsequently divided into two groups: cold static preservation (CSP) or EVLP at 37 degrees C with Steen solution for a further 12 h (total 24 h preservation). Lungs were then transplanted and reperfused for 4 h. EVLP preservation resulted in significantly better lung oxygenation (PaO(2) 531 +/- 43 vs. 244 +/- 49 mmHg, p < 0.01) and lower edema formation rates after transplantation. Alveolar epithelial cell tight junction integrity, evaluated by zona occludens-1 protein staining, was disrupted in the cell membranes after prolonged CSP but not after EVLP. The maintenance of integrity of barrier function during EVLP translates into significant attenuation of reperfusion injury and improved graft performance after transplantation. Integrity of functional metabolic pathways during normothermic perfusion was confirmed by effective gene transfer and GFP protein synthesis by lung alveolar cells. In conclusion, EVLP prevents ongoing injury associated with prolonged ischemia and accelerates lung recovery. PMID:19663886

  5. Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche.

    Directory of Open Access Journals (Sweden)

    Wenting Zhang

    Full Text Available We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC using an osteoblast (OSB-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1 optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2 replenishing OSB during culture as a practical means of prolonging MMC's viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses.

  6. Ex vivo complement protein adsorption on positively and negatively charged cellulose dialyser membranes.

    Science.gov (United States)

    Mahiout, A; Matata, B M; Vienken, J; Courtney, J M

    1997-05-01

    An ex vivo test system was used to measure complement protein C3 and factor B adsorption onto small dialyser modules made from regenerated and modified cellulosic hollow fibre membranes in which positive diethylaminoethyl (DEAE) or negative carboxymethyl (CM) groups were introduced into the cellulose matrix. The extracorporeal system, which included test-dialysers and the dialysis environment, allowed the use of labelled proteins without contaminating the blood donors which were connected in an open-loop fashion to the extracorporeal test system. The modules were removed at selected time points from the extracorporeal system for radioactivity counting. The results were used to evaluate the mechanisms involved in complement reactions to foreign surfaces. The system therefore allowed the analysis of complement protein adsorption occurring in the dialyser modules and its relationship to the complement generation rate in the extracorporeal system to be evaluated. It was possible to demonstrate that significant complement C3 and factor B adsorption occurred in the test modules made of cellulosic membranes. Complement adsorption as a function of the pH and the release reaction of the adsorbed C3 and factor B after membrane blood perfusion were therefore found to be variable according to the cellulosic membrane type and the presence of positive or negative charged groups within the cellulose matrix. The data obtained from the ex vivo model therefore provided additional evidence on the discussion of the mechanisms involved in the increased complement activation by regenerated cellulose and in its attenuation by DEAE- or CM-modified cellulose.

  7. In vivo and ex vivo evaluation of cosmetic properties of seedcakes.

    Science.gov (United States)

    Ratz-Łyko, Anna; Arct, Jacek; Pytkowska, Katarzyna; Majewski, Sławomir

    2015-04-01

    The seedcakes are a potential source of natural bioactive substances: antioxidants, protein, and carbohydrates. Thus, they may scavenge free radicals and have an effect on the stratum corneum hydration and epidermal barrier function. The aim of the study was to evaluate the in vivo and ex vivo properties of emulsions with the seedcake extracts using the pH meter, corneometer, tewameter, methyl nicotinate model of micro-inflammation in human skin, and tape stripping of the stratum corneum. The in vivo and ex vivo studies showed that the emulsions with Oenothera biennis, Borago officinalis, and Nigella sativa seedcake extracts have anti-inflammatory and antioxidant activity. The 6-week topical application of the emulsions with the B. officinalis and N. sativa seedcakes significantly reduced skin irritation and influenced the improvement of the skin hydration and epidermal barrier function compared with placebo. The seedcakes due to their antioxidant and anti-inflammatory activities have potential application in anti-aging, moisturizing, mitigating, and protective cosmetics. PMID:25415370

  8. Ex vivo expansion of tumor-infiltrating lymphocytes from nasopharyngeal carcinoma patients for adoptive immunotherapy

    Directory of Open Access Journals (Sweden)

    Jiang Li

    2012-06-01

    Full Text Available Establishing Epstein-Barr virus(EBV-specific cytolytic T lymphocytes(EBV-CTLs from peripheral blood mononuclear cells(PBMCs for adoptive immunotherapy has been reported in EBV-associated malignancies including Hodgkin's lymphoma and nasopharyngeal carcinoma(NPC. In the current study, we performed ex vivo expansion of tumor-infiltrating lymphocytes(TILs obtained from NPC biopsy specimens with a rapid expansion protocol using anti-CD3 monoclonal antibody(OKT3, recombinant human interleukin(IL-2, and irradiated PBMCs from healthy donors to initiate the growth of TILs. Young TIL cultures comprised of more than 90% of CD3+ T cells, a variable percentage of CD3+CD8+ and CD3+CD4+ T cells, and less than 10% of CD3-CD16+ natural killer cells, a similar phenotype of EBV-CTL cultures from PBMCs. Interestingly, TIL cultures secreted high levels of the Th1 cytokines, interferon gamma (IFNγ and tumor necrosis factor-alpha (TNF-α, and low levels of the Th2 cytokines, IL-4 and IL-10. Moreover, young TILs could recognize autologous EBV-transformed B lymphoblast cell lines, but not autologous EBV-negative blast cells or allogeneic EBV-negative tumor cells. Taken together, these data suggest that ex vivo expansion of TILs from NPC biopsy tissue is an appealing alternative method to establish T cell-based immunotherapy for NPC.

  9. In vivo prevention of transplant arteriosclerosis by ex vivo-expanded human regulatory T cells.

    Science.gov (United States)

    Nadig, Satish N; Wieckiewicz, Joanna; Wu, Douglas C; Warnecke, Gregor; Zhang, Wei; Luo, Shiqiao; Schiopu, Alexandru; Taggart, David P; Wood, Kathryn J

    2010-07-01

    Transplant arteriosclerosis is the hallmark of chronic allograft dysfunction (CAD) affecting transplanted organs in the long term. These fibroproliferative lesions lead to neointimal thickening of arteries in all transplanted allografts. Luminal narrowing then leads to graft ischemia and organ demise. To date, there are no known tolerance induction strategies that prevent transplant arteriosclerosis. Therefore, we designed this study to test the hypothesis that human regulatory T cells (T(reg) cells) expanded ex vivo can prevent transplant arteriosclerosis. Here we show the comparative capacity of T(reg) cells, sorted via two separate strategies, to prevent transplant arteriosclerosis in a clinically relevant chimeric humanized mouse system. We found that the in vivo development of transplant arteriosclerosis in human arteries was prevented by treatment of ex vivo-expanded human T(reg) cells. Additionally, we show that T(reg) cells sorted on the basis of low expression of CD127 provide a more potent therapy to conventional T(reg) cells. Our results demonstrate that human T(reg) cells can inhibit transplant arteriosclerosis by impairing effector function and graft infiltration. We anticipate our findings to serve as a foundation for the clinical development of therapeutics targeting transplant arteriosclerosis in both allograft transplantation and other immune-mediated causes of vasculopathy.

  10. The ex vivo purge of cancer cells using oncolytic viruses: recent advances and clinical implications

    Directory of Open Access Journals (Sweden)

    Tsang JJ

    2015-01-01

    Full Text Available Jovian J Tsang,1,2 Harold L Atkins2,3 1Department of Biochemistry, University of Ottawa, 2Cancer Therapeutics, Ottawa Hospital Research Institute, 3Blood and Marrow Transplant Program, The Ottawa Hospital, Ottawa, ON, Canada Abstract: Hematological malignancies are treated with intensive high-dose chemotherapy, with or without radiation. This is followed by hematopoietic stem cell (HSC transplantation (HSCT to rescue or reconstitute hematopoiesis damaged by the anticancer therapy. Autologous HSC grafts may contain cancer cells and purging could further improve treatment outcomes. Similarly, allogeneic HSCT may be improved by selectively purging alloreactive effector cells from the graft rather than wholesale immune cell depletion. Viral agents that selectively replicate in specific cell populations are being studied in experimental models of cancer and immunological diseases and have potential applications in the context of HSC graft engineering. This review describes preclinical studies involving oncolytic virus strains of adenovirus, herpes simplex virus type 1, myxoma virus, and reovirus as ex vivo purging agents for HSC grafts, as well as in vitro and in vivo experimental studies using oncolytic coxsackievirus, measles virus, parvovirus, vaccinia virus, and vesicular stomatitis virus to eradicate hematopoietic malignancies. Alternative ex vivo oncolytic virus strategies are also outlined that aim to reduce the risk of relapse following autologous HSCT and mitigate morbidity and mortality due to graft-versus-host disease in allogeneic HSCT. Keywords: hematopoietic stem cells, oncolytic virus, hematopoietic stem cell transplantation, stem cell graft purging, hematopoietic malignancy, graft vs host disease

  11. Proteomic Profiling of Ex Vivo Expanded CD34-Positive Haematopoetic Cells Derived from Umbilical Cord Blood

    Directory of Open Access Journals (Sweden)

    Heiner Falkenberg

    2013-01-01

    Full Text Available Ex vivo expansion of haematopoetic cells by application of specific cytokines is one approach to overcome boundaries in cord blood transplantation due to limited numbers of haematopoetic stem cells. While many protocols describe an effective increase of total cell numbers and the amount of CD34-positive cells, it still remains unclear if and how the procedure actually affects the cells’ properties. In the presented publications, CD34-positive cells were isolated from cord blood and expanded for up to 7 days in media supplemented with stem cell factor (SCF, thrombopoietin (THPO, interleukin 6 (IL-6, and fms-related tyrosine kinase 3 ligand (FLT3lg. At days 3 and 7, expanded cells were harvested and analyzed by flow cytometry and quantitative proteomics. 2970 proteins were identified, whereof proteomic analysis showed 440 proteins significantly changed in abundance during ex vivo expansion. Despite the fact that haematopoetic cells still expressed CD34 on the surface after 3 days, major changes in regard to the protein profile were observed, while further expansion showed less effect on the proteome level. Enrichment analysis of biological processes clearly showed a proteomic change toward a protein biosynthesis phenotype already within the first three days of expression.

  12. Effect of anticonvulsant drugs on (35S)t-butylbicyclophosphorothionate binding in vitro and ex vivo

    International Nuclear Information System (INIS)

    Using several concentrations of eight anticonvulsant drugs in clinical use (carbamazepine, clonazepam, phenytoin, phenobarbital, ethosuximide, primidone, sodium valproate, and D,L-γ-vinyl GABA), we studied their abilities in vitro to displace (35S)t-butylbicyclophosphorothionate (35S-TBPS) from its binding site in a homogenate of rat brain. Thereafter ethosuximide (150 mg/kg), phenobarbital (30 mg/kg), clonazepam (0.3 mg/kg), or phenytoin (100 mg/kg) was injected intraperitoneally into rats for 16-20 days; and the effect of drug administration on 35S-TBPS binding was studied in the cortex and hippocampus ex vivo. Phenobarbital (100 μM, P35S-TBPS binding in vitro by 10-16%. After drug administration of phenobarbital (concentration in plasma 168 μM), the number of binding sites decreased and the binding affinity (p35S-TBPS binding in vitro at the concentration analogous to therapeutic plasma levels or ex vivo at the dose used. These results suggest that the use of phenobarbital may modulate the TBPS binding site, but the role of the present findings in the anticonvulsant action of phenobarbital needs to be further studied. (author)

  13. White blood cell-based detection of asymptomatic scrapie infection by ex vivo assays.

    Directory of Open Access Journals (Sweden)

    Sophie Halliez

    Full Text Available Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods--in vitro, ex vivo and in vivo assays--to detect prion infectivity in cells prepared from blood samples obtained from scrapie infected sheep at different time points of the disease. Protein misfolding cyclic amplification (PMCA and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease (asymptomatic to terminally-ill stages. However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease.

  14. Clinical study of ex vivo photoacoustic imaging in endoscopic mucosal resection tissues

    Science.gov (United States)

    Lim, Liang; Streutker, Catherine J.; Marcon, Norman; Cirocco, Maria; Lakovlev, Vladimir V.; DaCosta, Ralph; Foster, F. S.; Wilson, Brian C.

    2015-03-01

    Accurate endoscopic detection and dysplasia in patients with Barrett's esophagus (BE) remains a major unmet clinical need. Current diagnosis use multiple biopsies under endoscopic image guidance, where up to 99% of the tissue remains unsampled, leading to significant risk of missing dysplasia. We conducted an ex vivo clinical trial using photoacoustic imaging (PAI) in patients undergoing endoscopic mucosal resection (EMR) with known high-grade dysplasia for the purpose of characterizing the esophageal microvascular pattern, with the long-term goal of performing in vivo endoscopic PAI for dysplasia detection and therapeutic guidance. EMR tissues were mounted immediately on an agar layer and covered with ultrasound gel. Digital photography guided the placement of the PAI transducer (40 MHz center frequency). The luminal side of the specimen was scanned over a field of view of 14 mm (width) by 15 mm (depth) at 680, 750, 824, 850 and 970 nm. Acoustic images were simultaneously acquired. Tissues were then sliced and fixed in formalin for histopathology with H and E staining. Analysis consisted of co-registration and correlation between the intrinsic PAI features and the histological images. The initial PAI + ultrasound images from 8 BE patients have demonstrated the technical feasibility of this approach and point to the potential of PAI to reveal the microvascular pattern within EMR specimens. There are several technical factors to be considered in rigorous interpretation of the PAI characteristics, including the loss of blood from the ex vivo specimens and the limited depth penetration of the photoacoustic signal.

  15. Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

    Energy Technology Data Exchange (ETDEWEB)

    Darvin, M E; Richter, H; Zhu, Y J; Meinke, M C; Knorr, F; Lademann, J [Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology, Venerology and Allergology, Charité - Universitätsmedizin Berlin (Germany); Gonchukov, S A [National Research Nuclear University ' ' MEPhI' ' (Russian Federation); Koenig, K [JenLab GmbH, Schillerstr. 1, 07745 Jena (Germany)

    2014-07-31

    Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted. (laser biophotonics)

  16. Phenolic compounds from red wine are protective against the DNA damaging effect of ionising radiation ex vivo

    International Nuclear Information System (INIS)

    Full text: Using the cytokinesis-block micronucleus assay we (a) investigated which compounds in red wine can prevent oxidative damage to DNA in vitro and (b) performed in vivo interventions with red wine (RW), de-alcoholised red wine (DEALC) and alcohol (ALC) to distinguish the effects of alcohol from the other fractions of RW in prevention of oxidative damage to DNA ex vivo. Cells were either challenged with ionising gamma radiation or hydrogen peroxide, two different forms of oxidative stress. The relative contribution of ethanol, glycerol, tartaric acid, catechin + caffeic acid, a mixture of all of these and phenolic stripped RW at in vivo relevant concentrations on spontaneous and oxidative stress induced DNA damage was evaluated in vitro. The results from these studies have shown that (a) only ethanol significantly increases spontaneous DNA damage, but this effect is eliminated when ethanol is included in a mixture of all the other wine components (P<0.05), (b) the strongest and only significant protective effect against hydrogen peroxide induced DNA damage was observed for the catechin + caffeic acid mixture (P<0.05) and (c) all compounds tested were significantly protective against ionising radiation-induced DNA damage in a dose dependent manner with the strongest protection being observed for the catechin + caffeic acid mixture and a mixture of all components (P < 0.0001). In the in vivo intervention studies with ex vivo challenge of whole blood showed that 1-2h after consumption of 300ml DEALC produced a significant protection against the DNA damaging effects of ionising radiation (P = 0.0002) but ALC significantly enhanced the damaging effects of ionising radiation (P 0.0002) while RW produced an intermediate effect. The data from these studies are particularly interesting because they clearly demonstrate the potential beneficial effects of wine phenolics in counteracting the harmful effects of ionising radiation. Furthermore they demonstrate that the

  17. Intra-Tissue Pressure Measurement in Ex Vivo Liver Undergoing Laser Ablation with Fiber-Optic Fabry-Perot Probe

    Directory of Open Access Journals (Sweden)

    Daniele Tosi

    2016-04-01

    Full Text Available We report the first-ever intra-tissue pressure measurement performed during 1064 nm laser ablation (LA of an ex vivo porcine liver. Pressure detection has been performed with a biocompatible, all-glass, temperature-insensitive Extrinsic Fabry-Perot Interferometry (EFPI miniature probe; the proposed methodology mimics in-vivo treatment. Four experiments have been performed, positioning the probe at different positions from the laser applicator tip (from 0.5 mm to 5 mm. Pressure levels increase during ablation time, and decrease with distance from applicator tip: the recorded peak parenchymal pressure levels range from 1.9 kPa to 71.6 kPa. Different pressure evolutions have been recorded, as pressure rises earlier in proximity of the tip. The present study is the first investigation of parenchymal pressure detection in liver undergoing LA: the successful detection of intra-tissue pressure may be a key asset for improving LA, as pressure levels have been correlated to scattered recurrences of tumors by different studies.

  18. The ex vivo and in vivo biological performances of graphene oxide and the impact of surfactant on graphene oxide's biocompatibility

    Institute of Scientific and Technical Information of China (English)

    Guangbo Qu; Xiaoyan Wang; Qian Liu; Rui Liu; Nuoya Yin; Juan Ma; Liqun Chen

    2013-01-01

    Graphene oxide (GO) displays promising properties for biomedical applications including drug delivery and cancer therapeutics.However,GO exposure also raises safety concerns such as potential side effects on health.Here,the biological effects of GO suspended in phosphate buffered saline (PBS) with or without 1% nonionic surfactant Tween 80 were investigated.Based on the ex vivo experiments,Tween 80 significantly affected the interaction between GO and peripheral blood from mice.GO suspension in PBS tended to provoke the aggregation of diluted blood cells,which could be prevented by the addition of Tween 80.After intravenous administration,GO suspension with or without 1% Tween 80 was quickly eliminated by the mononuclear phagocyte system.Nevertheless,GO suspension without Tween 80 showed greater accumulation in lungs than that containing 1% Tween 80.In contrast,less GO was found in livers for GO suspension compared to Tween 80 assisted GO suspension.Organs including hearts,livers,lungs,spleens,kidneys,brains,and testes did not reveal histological alterations.The indexes of peripheral blood showed no change upon GO exposure.Our results together demonstrated that Tween 80 could greatly alter GO's biological performance and determine the pattern of its biodistribution in mice.

  19. SERS-active Au/SiO2 clouds in powder for rapid ex vivo breast adenocarcinoma diagnosis.

    Science.gov (United States)

    Cepeda-Pérez, Elisa; López-Luke, Tzarara; Salas, Pedro; Plascencia-Villa, Germán; Ponce, Arturo; Vivero-Escoto, Juan; José-Yacamán, Miguel; de la Rosa, Elder

    2016-06-01

    In the present work, we report a dry-based application technique of Au/SiO2 clouds in powder for rapid ex vivo adenocarcinoma diagnosis through surface-enhanced Raman scattering (SERS); using low laser power and an integration time of one second. Several characteristic Raman peaks frequently used for the diagnosis of breast adenocarcinoma in the range of the amide III are successfully enhanced by breading the tissue with Au/SiO2 powder. The SERS activity of these Au/SiO2 powders is attributed to their rapid rehydration upon contact with the wet tissues, which promotes the formation of gold nanoparticle aggregates. The propensity of the Au/SiO2 cloud structures to adsorb biomolecules in the vicinity of the gold nanoparticle clusters promotes the necessary conditions for SERS detection. In addition, electron microscopy, together with elemental analysis, have been used to confirm the structure of the new Au/SiO2 cloud material and to investigate its distribution in breast tissues. PMID:27375955

  20. Intra-Tissue Pressure Measurement in Ex Vivo Liver Undergoing Laser Ablation with Fiber-Optic Fabry-Perot Probe.

    Science.gov (United States)

    Tosi, Daniele; Saccomandi, Paola; Schena, Emiliano; Duraibabu, Dinesh Babu; Poeggel, Sven; Leen, Gabriel; Lewis, Elfed

    2016-04-15

    We report the first-ever intra-tissue pressure measurement performed during 1064 nm laser ablation (LA) of an ex vivo porcine liver. Pressure detection has been performed with a biocompatible, all-glass, temperature-insensitive Extrinsic Fabry-Perot Interferometry (EFPI) miniature probe; the proposed methodology mimics in-vivo treatment. Four experiments have been performed, positioning the probe at different positions from the laser applicator tip (from 0.5 mm to 5 mm). Pressure levels increase during ablation time, and decrease with distance from applicator tip: the recorded peak parenchymal pressure levels range from 1.9 kPa to 71.6 kPa. Different pressure evolutions have been recorded, as pressure rises earlier in proximity of the tip. The present study is the first investigation of parenchymal pressure detection in liver undergoing LA: the successful detection of intra-tissue pressure may be a key asset for improving LA, as pressure levels have been correlated to scattered recurrences of tumors by different studies.

  1. Changes in metabolic proteins in ex vivo rat retina during glutamate-induced neural progenitor cell induction.

    Science.gov (United States)

    Tokuda, Kazuhiro; Kuramitsu, Yasuhiro; Baron, Byron; Kitagawa, Takao; Tokuda, Nobuko; Kobayashi, Masaaki; Kimura, Kazuhiro; Sonoda, Koh-Hei; Nakamura, Kazuyuki

    2016-08-01

    Understanding how energy metabolism and related proteins influence neural progenitor cells in adult tissues is critical for developing new strategies in clinical tissue regeneration therapy. We have recently reported that a subtoxic concentration of glutamate-induced neural progenitor cells in the mature ex vivo rat retina. We herein explore changes in the metabolic pathways during the process. We firstly observed an increase in lactate and lactate dehydrogenase concentration in the glutamate-treated retina. We then investigated the levels of glycolytic enzymes and confirmed significant upregulation of pyruvate kinase M type (PKM), especially PKM2, enolase, phosphoglycerate mutase 1 (PGAM1), and inosine-5'-monophosphate dehydrogenase (IMPDH1) in the glutamate-treated retina compared to the untreated retina. An analysis of the subcellular localization of PKM2 revealed nuclear translocation in the treated retina, which has been reported to regulate cell cycle proliferation and glycolytic enzymes. Our findings indicate that the mature rat retina undergoes an increase in aerobic glycolysis. PKM2, both in the cytoplasm and in the nucleus, may thus play an important role during neural progenitor cell induction, as it does in other proliferating cells. PMID:27421851

  2. Ex vivo multiscale quantitation of skin biomechanics in wild-type and genetically-modified mice using multiphoton microscopy

    Science.gov (United States)

    Bancelin, Stéphane; Lynch, Barbara; Bonod-Bidaud, Christelle; Ducourthial, Guillaume; Psilodimitrakopoulos, Sotiris; Dokládal, Petr; Allain, Jean-Marc; Schanne-Klein, Marie-Claire; Ruggiero, Florence

    2015-12-01

    Soft connective tissues such as skin, tendon or cornea are made of about 90% of extracellular matrix proteins, fibrillar collagens being the major components. Decreased or aberrant collagen synthesis generally results in defective tissue mechanical properties as the classic form of Elhers-Danlos syndrome (cEDS). This connective tissue disorder is caused by mutations in collagen V genes and is mainly characterized by skin hyperextensibility. To investigate the relationship between the microstructure of normal and diseased skins and their macroscopic mechanical properties, we imaged and quantified the microstructure of dermis of ex vivo murine skin biopsies during uniaxial mechanical assay using multiphoton microscopy. We used two genetically-modified mouse lines for collagen V: a mouse model for cEDS harboring a Col5a2 deletion (a.k.a. pN allele) and the transgenic K14-COL5A1 mice which overexpress the human COL5A1 gene in skin. We showed that in normal skin, the collagen fibers continuously align with stretch, generating the observed increase in mechanical stress. Moreover, dermis from both transgenic lines exhibited altered collagen reorganization upon traction, which could be linked to microstructural modifications. These findings show that our multiscale approach provides new crucial information on the biomechanics of dermis that can be extended to all collagen-rich soft tissues.

  3. Borrelia burgdorferi Induces TLR2-Mediated Migration of Activated Dendritic Cells in an Ex Vivo Human Skin Model

    Science.gov (United States)

    Wagemakers, Alex; van ‘t Veer, Cornelis; Oei, Anneke; van der Pot, Wouter J.; Ahmed, Kalam; van der Poll, Tom; Geijtenbeek, Teunis B. H.; Hovius, Joppe W. R.

    2016-01-01

    Borrelia burgdorferi is transmitted into the skin of the host where it encounters and interacts with two dendritic cell (DC) subsets; Langerhans cells (LCs) and dermal DCs (DDCs). These cells recognize pathogens via pattern recognition receptors, mature and migrate out of the skin into draining lymph nodes, where they orchestrate adaptive immune responses. In order to investigate the response of skin DCs during the early immunopathogenesis of Lyme borreliosis, we injected B. burgdorferi intradermally into full-thickness human skin and studied the migration of DCs out of the skin, the activation profile and phenotype of migrated cells. We found a significant increase in the migration of LCs and DDCs in response to B. burgdorferi. Notably, migration was prevented by blocking TLR2. DCs migrated from skin inoculated with higher numbers of spirochetes expressed significantly higher levels of CD83 and produced pro-inflammatory cytokines. No difference was observed in the expression of HLA-DR, CD86, CD38, or CCR7. To conclude, we have established an ex vivo human skin model to study DC-B. burgdorferi interactions. Using this model, we have demonstrated that B. burgdorferi-induced DC migration is mediated by TLR2. Our findings underscore the utility of this model as a valuable tool to study immunity to spirochetal infections. PMID:27695100

  4. Effects of TGF-beta signalling inhibition with galunisertib (LY2157299) in hepatocellular carcinoma models and in ex vivo whole tumor tissue samples from patients.

    Science.gov (United States)

    Serova, Maria; Tijeras-Raballand, Annemilaï; Dos Santos, Célia; Albuquerque, Miguel; Paradis, Valerie; Neuzillet, Cindy; Benhadji, Karim A; Raymond, Eric; Faivre, Sandrine; de Gramont, Armand

    2015-08-28

    Galunisertib (LY2157299) is a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation currently under clinical investigation in hepatocellular carcinoma (HCC) patients. Our study explored the effects of galunisertib in vitro in HCC cell lines and ex vivo on patient samples. Galunisertib was evaluated in HepG2, Hep3B, Huh7, JHH6 and SK-HEP1 cells as well as in SK-HEP1-derived cells tolerant to sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of p-Smad2 and p-Smad3 that was potently inhibited with galunisertib treatment at micromolar concentrations. Despite limited antiproliferative effects, galunisertib yielded potent anti-invasive properties. Tumor slices from 13 patients with HCC surgically resected were exposed ex vivo to 1 µM and 10 µM galunisertib, 5 µM sorafenib or a combination of both drugs for 48 hours. Galunisertib but not sorafenib decreased p-Smad2/3 downstream TGF-β signaling. Immunohistochemistry analysis of galunisertib and sorafenib-exposed samples showed a significant decrease of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3. In combination, galunisertib potentiated the effect of sorafenib efficiently by inhibiting proliferation and increasing apoptosis. Our data suggest that galunisertib may be active in patients with HCC and could potentiate the effects of sorafenib. PMID:26057634

  5. In vivo/ex vivo targeting of Langerhans cells after topical application of the immune response modifier TMX-202: confocal Raman microscopy and histology analysis

    Science.gov (United States)

    Darvin, Maxim E.; Thiede, Gisela; Ascencio, Saul Mujica; Schanzer, Sabine; Richter, Heike; Vinzón, Sabrina E.; Hasche, Daniel; Rösl, Frank; May, Roberto; Hazot, Yohan; Tamarkin, Dov; Lademann, Juergen

    2016-05-01

    The increased ability of TMX-202 (derivative of imiquimod) to penetrate the intact stratum corneum (SC) and the follicular orifices of porcine ear skin was shown ex vivo using confocal Raman microscopy and laser scanning microscopy. Moreover, to assess whether TMX-202 is able to reach the immune cells, Langerhans cells extracted from pretreated human skin were investigated ex vivo using confocal Raman microscopy combined with multivariate statistical methods. Tracking the Raman peak of dimethyl sulfoxide centered at 690 cm-1, the absorption of TMX-202 containing formulation by Langerhans cells was shown. To answer the question whether the TMX-202 active ingredient is able to reach Langerhans cells, the attraction of immune cells to TMX-202 containing formulation treated skin was measured in the in vivo rodent model Mastomys coucha. The results show that TMX-202 active ingredient is able to reach Langerhans cells after penetrating through the intact skin and subsequently attract immune cells. Both the intercellular/transcellular as well as the follicular pathways allow the penetration through the intact barrier of the SC.

  6. Nicotinamide enhances repair of arsenic and ultraviolet radiation-induced DNA damage in HaCaT keratinocytes and ex vivo human skin.

    Directory of Open Access Journals (Sweden)

    Benjamin C Thompson

    Full Text Available Arsenic-induced skin cancer is a significant global health burden. In areas with arsenic contamination of water sources, such as China, Pakistan, Myanmar, Cambodia and especially Bangladesh and West Bengal, large populations are at risk of arsenic-induced skin cancer. Arsenic acts as a co-carcinogen with ultraviolet (UV radiation and affects DNA damage and repair. Nicotinamide (vitamin B3 reduces premalignant keratoses in sun-damaged skin, likely by prevention of UV-induced cellular energy depletion and enhancement of DNA repair. We investigated whether nicotinamide modifies DNA repair following exposure to UV radiation and sodium arsenite. HaCaT keratinocytes and ex vivo human skin were exposed to 2μM sodium arsenite and low dose (2J/cm2 solar-simulated UV, with and without nicotinamide supplementation. DNA photolesions in the form of 8-oxo-7,8-dihydro-2'-deoxyguanosine and cyclobutane pyrimidine dimers were detected by immunofluorescence. Arsenic exposure significantly increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine in irradiated cells. Nicotinamide reduced both types of photolesions in HaCaT keratinocytes and in ex vivo human skin, likely by enhancing DNA repair. These results demonstrate a reduction of two different photolesions over time in two different models in UV and arsenic exposed cells. Nicotinamide is a nontoxic, inexpensive agent with potential for chemoprevention of arsenic induced skin cancer.

  7. Nicotinamide enhances repair of arsenic and ultraviolet radiation-induced DNA damage in HaCaT keratinocytes and ex vivo human skin.

    Science.gov (United States)

    Thompson, Benjamin C; Halliday, Gary M; Damian, Diona L

    2015-01-01

    Arsenic-induced skin cancer is a significant global health burden. In areas with arsenic contamination of water sources, such as China, Pakistan, Myanmar, Cambodia and especially Bangladesh and West Bengal, large populations are at risk of arsenic-induced skin cancer. Arsenic acts as a co-carcinogen with ultraviolet (UV) radiation and affects DNA damage and repair. Nicotinamide (vitamin B3) reduces premalignant keratoses in sun-damaged skin, likely by prevention of UV-induced cellular energy depletion and enhancement of DNA repair. We investigated whether nicotinamide modifies DNA repair following exposure to UV radiation and sodium arsenite. HaCaT keratinocytes and ex vivo human skin were exposed to 2μM sodium arsenite and low dose (2J/cm2) solar-simulated UV, with and without nicotinamide supplementation. DNA photolesions in the form of 8-oxo-7,8-dihydro-2'-deoxyguanosine and cyclobutane pyrimidine dimers were detected by immunofluorescence. Arsenic exposure significantly increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine in irradiated cells. Nicotinamide reduced both types of photolesions in HaCaT keratinocytes and in ex vivo human skin, likely by enhancing DNA repair. These results demonstrate a reduction of two different photolesions over time in two different models in UV and arsenic exposed cells. Nicotinamide is a nontoxic, inexpensive agent with potential for chemoprevention of arsenic induced skin cancer.

  8. Nicotinamide enhances repair of arsenic and ultraviolet radiation-induced DNA damage in HaCaT keratinocytes and ex vivo human skin.

    Science.gov (United States)

    Thompson, Benjamin C; Halliday, Gary M; Damian, Diona L

    2015-01-01

    Arsenic-induced skin cancer is a significant global health burden. In areas with arsenic contamination of water sources, such as China, Pakistan, Myanmar, Cambodia and especially Bangladesh and West Bengal, large populations are at risk of arsenic-induced skin cancer. Arsenic acts as a co-carcinogen with ultraviolet (UV) radiation and affects DNA damage and repair. Nicotinamide (vitamin B3) reduces premalignant keratoses in sun-damaged skin, likely by prevention of UV-induced cellular energy depletion and enhancement of DNA repair. We investigated whether nicotinamide modifies DNA repair following exposure to UV radiation and sodium arsenite. HaCaT keratinocytes and ex vivo human skin were exposed to 2μM sodium arsenite and low dose (2J/cm2) solar-simulated UV, with and without nicotinamide supplementation. DNA photolesions in the form of 8-oxo-7,8-dihydro-2'-deoxyguanosine and cyclobutane pyrimidine dimers were detected by immunofluorescence. Arsenic exposure significantly increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine in irradiated cells. Nicotinamide reduced both types of photolesions in HaCaT keratinocytes and in ex vivo human skin, likely by enhancing DNA repair. These results demonstrate a reduction of two different photolesions over time in two different models in UV and arsenic exposed cells. Nicotinamide is a nontoxic, inexpensive agent with potential for chemoprevention of arsenic induced skin cancer. PMID:25658450

  9. Immunization with Dendritic Cells Pulsed ex vivo with Recombinant Chlamydial Protease-Like Activity Factor Induces Protective Immunity Against Genital Chlamydia muridarum Challenge

    Directory of Open Access Journals (Sweden)

    Bernard eArulanandam

    2011-12-01

    Full Text Available We have shown that immunization with soluble recombinant (r chlamydial protease-like activity factor (rCPAF and a T helper (Th 1 type adjuvant can induce significantly enhanced bacterial clearance and protection against Chlamydia–induced pathological sequelae in the genital tract. In this study, we investigated the use of bone marrow derived dendritic cells (BMDCs pulsed ex vivo with rCPAF+CpG in an adoptive subcutaneous immunization for the ability to induce protective immunity against genital chlamydial infection. We found that BMDCs pulsed with rCPAF+CpG efficiently up-regulated the expression of activation markers CD86, CD80, CD40 and major histocompatibility complex class II (MHC II, and secreted interleukin-12, but not IL-10 and IL-4. Mice adoptively immunized with rCPAF+CpG-pulsed BMDCs or UV-EB+CpG-pulsed BMDCs produced elevated levels of antigen-specific IFN- and enhanced IgG1 and IgG2a antibodies. Moreover, mice immunized with rCPAF+CpG-pulsed BMDCs or UV-EB+CpG-pulsed BMDCs exhibited significantly reduced genital Chlamydia shedding, accelerated resolution of infection, and reduced oviduct pathology when compared to infected mock-immunized animals. These results suggest that adoptive subcutaneous immunization with ex vivo rCPAF-pulsed BMDCs is an effective approach, comparable to that induced by UV-EB-BMDCs, for inducing robust anti-Chlamydia immunity.

  10. Fasudil and SOD packaged in peptide-studded-liposomes: Properties, pharmacokinetics and ex-vivo targeting to isolated perfused rat lungs.

    Science.gov (United States)

    Gupta, Nilesh; Al-Saikhan, Fahad I; Patel, Brijeshkumar; Rashid, Jahidur; Ahsan, Fakhrul

    2015-07-01

    The present study investigated the feasibility of encapsulating two drugs, fasudil and superoxide dismutase (SOD), into liposomes for targeted and inhalational delivery to the pulmonary vasculature to treat pulmonary arterial hypertension (PAH). Nanosized liposomes were prepared by a thin-film formation and extrusion method, and the drugs were encapsulated by a modified freeze-thaw technique. The peptide CARSKNKDC (CAR), a pulmonary-specific targeting sequence, was conjugated on the surface of liposomes. Formulations were optimized for various physicochemical properties, tested for their ex-vivo and in-vivo drug absorption after intratracheal administration, and evaluated for short-term safety in healthy rats. The homogenous nanosized liposomes contained both SOD (~55% entrapment) and fasudil (~40% entrapment), and were stable at 4°C and after nebulization. Liposomes released the drugs in a controlled-release fashion. Compared with plain liposomes, CAR-liposomes increased the uptake by pulmonary endothelial and smooth muscle cells by ~2-fold. CAR-liposomes extended the biological half-lives of SOD and fasudil by ~3-fold. Ex-vivo studies demonstrated that CAR-liposomes were better retained in the lungs than plain liposomes. Bronchoalveolar lavage studies indicated the safety of peptide-equipped liposomes as pulmonary delivery carriers. Overall, this study demonstrates that CAR-liposomes may be used as inhalational carriers for SOD plus fasudil-based combination therapy for PAH. PMID:25888802

  11. Quantification of plaque lipids in the aortic root of ApoE-deficient mice by 3D DIXON magnetic resonance imaging in an ex vivo model

    Energy Technology Data Exchange (ETDEWEB)

    Dietel, Barbara; Kuehn, Constanze; Achenbach, Stephan [University Hospital of Erlangen-Nuremberg, Department of Cardiology and Angiology, Erlangen (Germany); Budinsky, Lubos [Campus Science Support Facilities (CSF), Vienna Biocenter (VBC), Vienna (Austria); Uder, Michael [University Hospital of Erlangen-Nuremberg, Department of Radiology, Erlangen (Germany); Hess, Andreas [University of Erlangen-Nuremberg, Department of Experimental and Clinical Pharmacology and Toxicology, Erlangen (Germany)

    2014-10-31

    To establish a dedicated protocol for the three-dimensional (3D) quantification of plaque lipids in apolipoprotein E-deficient (apoE{sup -/-}) mice using ex vivo MRI. ApoE{sup -/-} mice were fed a high-fat diet (n = 10) or normal food (n = 10) for 3 months. Subsequently, a 3D FLASH MRI sequence was used to view the anatomy of the aortic root in the isolated hearts, where a 3D double-echo two-excitation pulse sequence (DIXON sequence) was used to selectively image plaque lipids. The vessel wall, lumen and plaque lipid volumes were quantified by MRI and histology for correlation analysis. DIXON MRI allowed visualisation and accurate quantification of plaque lipids. When comparing the vessel wall, lumen and plaque lipid sizes in the aortic root, Bland-Altman and linear regression analysis revealed a close correlation between MRI results and the histological data both on a slice-by-slice basis and of the volumetric measurements (vessel wall: r{sup 2} = 0.775, p < 0.001; vessel lumen: r{sup 2} = 0.875; p = 0.002; plaque lipid: r{sup 2} = 0.819, p = 0.003). The combination of 3D FLASH and DIXON-sequence MRI permits an accurate ex vivo assessment of the investigated plaque parameters in the aortic root of mice, particularly the lipid content. (orig.)

  12. Safety and efficient ex vivo expansion of stem cells using platelet-rich plasma technology.

    Science.gov (United States)

    Anitua, Eduardo; Prado, Roberto; Orive, Gorka

    2013-09-01

    The goal of this Review is to provide an overview of the cell culture media supplements used in the ex vivo expansion of stem cells intended for cell therapy. Currently, the gold standard is the culture supplemented with fetal bovine serum, however, their use in cell therapy raises many concerns. The alternatives to its use are presented, ranging from the use of human serum to platelet-rich plasma (PRP), to serum-free media or extracellular matrix components. Finally, various growth factors present in PRP are described, which make it a safe and effective stem cell expansion supplement. These growth factors could be responsible for their efficiency, as they increase both stem cell proliferation and survival. The different PRP formulations are also discussed, as well as the need for protocol standardization.

  13. Ex vivo electrical impedance measurements on excised hepatic tissue from human patients with metastatic colorectal cancer

    International Nuclear Information System (INIS)

    Point-wise ex vivo electrical impedance spectroscopy measurements were conducted on excised hepatic tissue from human patients with metastatic colorectal cancer using a linear four-electrode impedance probe. This study of 132 measurements from 10 colorectal cancer patients, the largest to date, reports that the equivalent electrical conductivity for tumor tissue is significantly higher than normal tissue (p < 0.01), ranging from 2–5 times greater over the measured frequency range of 100 Hz–1 MHz. Difference in tissue electrical permittivity is also found to be statistically significant across most frequencies. Furthermore, the complex impedance is also reported for both normal and tumor tissue. Consistent with trends for tissue electrical conductivity, normal tissue has a significantly higher impedance than tumor tissue (p < 0.01), as well as a higher net capacitive phase shift (33° for normal liver tissue in contrast to 10° for tumor tissue). (paper)

  14. Ex vivo evaluation of the percutaneous penetration of proanthocyanidin extracts from Guazuma ulmifolia using photoacoustic spectroscopy.

    Science.gov (United States)

    Rocha, J C B; Pedrochi, F; Hernandes, L; de Mello, J C P; Baesso, M L

    2007-03-21

    In this work photoacoustic spectroscopy has been applied to determine ex vivo the percutaneous penetration of proanthocyanidins present in extracts obtained from Guazuma ulmifolia, in rats. Lotion formulations containing 0.0663 mg of procyanidin B2 day(-1)animal(-1) were topically applied during 7, 10 and 13 days in each group of the animals. After the end of treatment the animals were killed, the skin dissected to remove the basal content, and the measurements were carried out as a function of the period of time of treatment. The results showed that despite the very low concentration of the active principle (procyanidin B2) in the lotion, the photoacoustic method was able to show the presence of optical absorption bands from this substance in the dermis region, evidencing once again that this method may be useful for studies of topically applied formulations of interest in the pharmacokinetic area.

  15. An ex Vivo Model for Evaluating Blood-Brain Barrier Permeability, Efflux, and Drug Metabolism

    DEFF Research Database (Denmark)

    Hellman, Karin; Aadal Nielsen, Peter; Ek, Fredrik;

    2016-01-01

    The metabolism of drugs in the brain is difficult to study in most species because of enzymatic instability in vitro and interference from peripheral metabolism in vivo. A locust ex vivo model that combines brain barrier penetration, efflux, metabolism, and analysis of the unbound fraction...... in intact brains was evaluated using known drugs. Clozapine was analyzed, and its major metabolites, clozapine N-oxide (CNO) and N-desmethylclozapine (NDMC), were identified and quantified. The back-transformation of CNO into clozapine observed in humans was also observed in locusts. In addition......, risperidone, citalopram, fluoxetine, and haloperidol were studied, and one preselected metabolite for each drug was analyzed, identified, and quantified. Metabolite identification studies of clozapine and midazolam showed that the locust brain was highly metabolically active, and 18 and 14 metabolites...

  16. First Danish experience with ex vivo lung perfusion of donor lungs before transplantation

    DEFF Research Database (Denmark)

    Henriksen, Ian Sune Iversen; Møller-Sørensen, Hasse; Møller, Christian Holdfold;

    2014-01-01

    INTRODUCTION: The number of lung transplantations is limited by a general lack of donor organs. Ex vivo lung perfusion (EVLP) is a novel method to optimise and evaluate marginal donor lungs prior to transplantation. We describe our experiences with EVLP in Denmark during the first year after its...... introduction. MATERIAL AND METHODS: The study was conducted by prospective registration of donor offers and lung transplantations in Denmark from 1 May 2012 to 30 April 2013. Donor lungs without any contraindications were transplanted in the traditional manner. Taken for EVLP were donor lungs that were...... otherwise considered transplantable, but failed to meet the usual criteria due to possible contusions or because they were from donors with sepsis or unable to pass the oxygenation test. RESULTS: In the study period, seven of 33 Danish lung transplantations were made possible due to EVLP. One patient died...

  17. Ex vivo imaging of human thyroid pathology using integrated optical coherence tomography and optical coherence microscopy

    Science.gov (United States)

    Zhou, Chao; Wang, Yihong; Aguirre, Aaron D.; Tsai, Tsung-Han; Cohen, David W.; Connolly, James L.; Fujimoto, James G.

    2010-01-01

    We evaluate the feasibility of optical coherence tomography (OCT) and optical coherence microscopy (OCM) for imaging of benign and malignant thyroid lesions ex vivo using intrinsic optical contrast. 34 thyroid gland specimens are imaged from 17 patients, covering a spectrum of pathology ranging from normal thyroid to benign disease/neoplasms (multinodular colloid goiter, Hashimoto's thyroiditis, and follicular adenoma) and malignant thyroid tumors (papillary carcinoma and medullary carcinoma). Imaging is performed using an integrated OCT and OCM system, with papillary architecture, microfollicules, psammomatous calcifications, or replacement of normal follicular architecture with sheets/nests of tumor cells, can be identified from OCT and OCM images and are clearly differentiable from normal or benign thyroid tissues. With further development of needle-based imaging probes, OCT and OCM could be promising techniques to use for the screening of thyroid nodules and to improve the diagnostic specificity of fine needle aspiration evaluation.

  18. Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells.

    Science.gov (United States)

    Zhang, Cheng Cheng; Kaba, Megan; Ge, Guangtao; Xie, Kathleen; Tong, Wei; Hug, Christopher; Lodish, Harvey F

    2006-02-01

    Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3(+) cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.

  19. Ex vivo 3D diffusion tensor imaging and quantification of cardiac laminar structure.

    Science.gov (United States)

    Helm, Patrick A; Tseng, Hsiang-Jer; Younes, Laurent; McVeigh, Elliot R; Winslow, Raimond L

    2005-10-01

    A three-dimensional (3D) diffusion-weighted imaging (DWI) method for measuring cardiac fiber structure at high spatial resolution is presented. The method was applied to the ex vivo reconstruction of the fiber architecture of seven canine hearts. A novel hypothesis-testing method was developed and used to show that distinct populations of secondary and tertiary eigenvalues may be distinguished at reasonable confidence levels (P < or = 0.01) within the canine ventricle. Fiber inclination and sheet angles are reported as a function of transmural depth through the anterior, lateral, and posterior left ventricle (LV) free wall. Within anisotropic regions, two consistent and dominant orientations were identified, supporting published results from histological studies and providing strong evidence that the tertiary eigenvector of the diffusion tensor (DT) defines the sheet normal.

  20. Comparison of lung preservation solutions in human lungs using an ex vivo lung perfusion experimental model

    Directory of Open Access Journals (Sweden)

    Israel L. Medeiros

    2012-09-01

    Full Text Available OBJECTIVE: Experimental studies on lung preservation have always been performed using animal models. We present ex vivo lung perfusion as a new model for the study of lung preservation. Using human lungs instead of animal models may bring the results of experimental studies closer to what could be expected in clinical practice. METHOD: Brain-dead donors whose lungs had been declined by transplantation teams were used. The cases were randomized into two groups. In Group 1, Perfadex®was used for pulmonary preservation, and in Group 2, LPDnac, a solution manufactured in Brazil, was used. An ex vivo lung perfusion system was used, and the lungs were ventilated and perfused after 10 hours of cold ischemia. The extent of ischemic-reperfusion injury was measured using functional and histological parameters. RESULTS: After reperfusion, the mean oxygenation capacity was 405.3 mmHg in Group 1 and 406.0 mmHg in Group 2 (p = 0.98. The mean pulmonary vascular resistance values were 697.6 and 378.3 dyn·s·cm-5, respectively (p =0.035. The mean pulmonary compliance was 46.8 cm H20 in Group 1 and 49.3 ml/cm H20 in Group 2 (p =0.816. The mean wet/dry weight ratios were 2.06 and 2.02, respectively (p=0.87. The mean Lung Injury Scores for the biopsy performed after reperfusion were 4.37 and 4.37 in Groups 1 and 2, respectively (p = 1.0, and the apoptotic cell counts were 118.75/mm² and 137.50/mm², respectively (p=0.71. CONCLUSION: The locally produced preservation solution proved to be as good as Perfadex®. The clinical use of LPDnac may reduce costs in our centers. Therefore, it is important to develop new models to study lung preservation.

  1. Ex-vivo human lung tumor model. Use for temperature measurements during thermal ablation of NSCLC

    Energy Technology Data Exchange (ETDEWEB)

    Koch, Franziska; Vietze, A.; Hosten, N. [Ernst-Moritz-Arndt-Univ. Greifswald (Germany). Diagnostic Radiology and Neuroradiology; Laskowsi, U. [Maerkische Kliniken Luedenscheid (Germany). Thoracic Surgery; Ritter, C. [Ernst-Moritz-Arndt-Univ. Greifswald (Germany). Pharmacology; Linder, A. [Klinikum Bremen-Ost (Germany). Thoracic Surgery

    2011-03-15

    In the present study we used an ex-vivo human lung cancer model to compare temperature diffusion during thermal ablation using one laser fiber to that of a two-fiber approach. Furthermore, we examined whether there was a difference between temperature diffusion in normal lung tissue and tumor tissue during laser ablation. Materials and Methods: 48 resected lung specimens containing non-small cell lung cancer were connected to a perfusion/ventilation apparatus and treated with 1 (22 specimens, group 1) or, in a second experiment, with 1 (13 specimens, group 2) or 2 (13 specimens, group 3) laser fibers. During tumor ablation, temperatures were measured interstitially every 5 sec. Laser ablation was followed by the taking of samples of 13 specimens for histological examination. For comparison we performed laser ablation in 7 specimens with normal lung tissue. Results: Laser treatment and temperature control were technically feasible in all samples. Thirty min after starting laser ablation with 1 fiber, a temperature of 61{+-}17 C was achieved in group 1 at a distance of 10 mm from the laser fiber and a temperature of 74{+-}11 C was achieved in group 2 (p = 0.1). In the middle between two active laser fibers placed 20 mm apart, a temperature of 93{+-}7 C was achieved. The temperature reached in normal lung tissue after 20 min of laser ablation was 77{+-}15 C at a distance of 10 mm from the laser fiber. Conclusion: The ex-vivo model allowed performance of laser-induced thermal ablation in the perfused and ventilated lung. The use of two laser fibers increases the achieved temperatures significantly (p < 0.05). Temperatures reached in normal lung tissue were as high as in tumor tissue (p = 0.24). (orig.)

  2. Spatial distribution of niche and stem cells in ex vivo human limbal cultures.

    Science.gov (United States)

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

    2014-11-01

    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.

  3. Detection of thrombus size and protein content by ex vivo magnetization transfer and diffusion weighted MRI

    Directory of Open Access Journals (Sweden)

    Phinikaridou Alkystis

    2012-06-01

    Full Text Available Abstract Background To utilize a rabbit model of plaque disruption to assess the accuracy of different magnetic resonance sequences [T1-weighted (T1W, T2-weighted (T2W, magnetization transfer (MT and diffusion weighting (DW] at 11.7 T for the ex vivo detection of size and composition of thrombus associated with disrupted plaques. Methods Atherosclerosis was induced in the aorta of male New Zealand White rabbits (n = 17 by endothelial denudation and high-cholesterol diet. Subsequently, plaque disruption was induced by pharmacological triggering. Segments of infra-renal aorta were excised fixed in formalin and examined by ex vivo magnetic resonance imaging (MRI at 11.7 T and histology. Results MRI at 11.7 T showed that: (i magnetization transfer contrast (MTC and diffusion weighted images (DWI detected thrombus with higher sensitivity compared to T1W and T2W images [sensitivity: MTC = 88.2%, DWI = 76.5%, T1W = 66.6% and T2W = 43.7%, P P (ii MTC and DWI provided a more accurate detection of thrombus area with histology as the gold-standard [underestimation of 6% (MTC and 17.6% (DWI compared to an overestimation of thrombus area of 53.7% and 46.4% on T1W and T2W images, respectively]; (iii the percent magnetization transfer rate (MTR correlated with the fibrin (r = 0.73, P = 0.003 and collagen (r = 0.9, P = 0.004 content of the thrombus. Conclusions The conspicuity of the thrombus was increased on MTC and DW compared to T1W and T2W images. Changes in the %MTR and apparent diffusion coefficient can be used to identify the organization stage of the thrombus.

  4. Persistence of DNA studied in different ex vivo and in vivo rat models simulating the human gut situation

    DEFF Research Database (Denmark)

    Wilcks, Andrea; van Hoek, A.H.A.M.; Joosten, R.G.;

    2004-01-01

    This study aimed to evaluate the possibility of DNA sequences from genetically modified plants to persist in the gastrointestinal (GI) tract. PCR analysis and transformation assays were used to study DNA persistence and integrity in various ex vivo and in vivo systems using gnotobiotic rats. DNA...

  5. A new method for real-time quantification of irrigant extrusion during root canal irrigation ex vivo

    NARCIS (Netherlands)

    Psimma, Z.; Boutsioukis, C.; Vasiliadis, L.; Kastrinakis, E.

    2013-01-01

    Aim (i) To introduce a new method of quantifying extruded irrigant during root canal irrigation ex vivo. (ii) to evaluate the effect of periapical tissue simulation and pressure equalization and (iii) to determine the effect of needle type, apical preparation size and apical constriction diameter

  6. Photonic Characteristics and Ex Vivo Imaging of Escherichia coli-Xen14 Within the Bovine Reproductive Tract

    Science.gov (United States)

    The objectives of this study were to (1) characterize the photonic properties of Escherichia coli-Xen14 and (2) conduct photonic imaging of E. coli-Xen14 within bovine reproductive tract segments (RTS) ex vivo (Bos indicus). E. coli-Xen14 was grown for 24 h in Luria Bertani medium (LB), with or with...

  7. Guided access cavity preparation using cone-beam computed tomography and optical surface scans - an ex vivo study

    DEFF Research Database (Denmark)

    Buchgreitz, J; Buchgreitz, M; Mortensen, D;

    2016-01-01

    AIM: To evaluate ex vivo, the accuracy of a preparation procedure planned for teeth with pulp canal obliteration (PCO) using a guide rail concept based on a cone-beam computed tomography (CBCT) scan merged with an optical surface scan. METHODOLOGY: A total of 48 teeth were mounted in acrylic bloc...

  8. Ex vivo measures of muscle mitochondrial capacity reveal quantitative limits of oxygen delivery by the circulation during exercise

    DEFF Research Database (Denmark)

    Boushel, Robert; Saltin, Bengt

    2013-01-01

    Muscle mitochondrial respiratory capacity measured ex vivo provides a physiological reference to assess cellular oxidative capacity as a component in the oxygen cascade in vivo. In this article, the magnitude of muscle blood flow and oxygen uptake during exercise involving a small-to-large fracti...... capacity measured ex vivo underestimates the maximal in vivo oxygen uptake of muscle by up to ∼2-fold. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.......Muscle mitochondrial respiratory capacity measured ex vivo provides a physiological reference to assess cellular oxidative capacity as a component in the oxygen cascade in vivo. In this article, the magnitude of muscle blood flow and oxygen uptake during exercise involving a small-to-large fraction...... of the body mass will be discussed in relation to mitochondrial capacity measured ex vivo. These analyses reveal that as the mass of muscle engaged in exercise increases from one-leg knee extension, to 2-arm cranking, to 2-leg cycling and x-country skiing, the magnitude of blood flow and oxygen delivery...

  9. Adhesion of Plasmodium falciparum infected erythrocytes in ex vivo perfused placental tissue

    DEFF Research Database (Denmark)

    Pehrson, Caroline; Mathiesen, Line; Heno, Kristine K;

    2016-01-01

    expressing VAR2CSA accumulated in perfused placental tissue whereas the EPCR binding and the transgenic parasite did not. Soluble CSA and antibodies specific against VAR2CSA inhibited binding of infected erythrocytes. CONCLUSION: The ex vivo model provides a novel way of studying receptor-ligand interactions...

  10. The development and characterization of a competitive ELISA for measuring active ADAMTS-4 in a bovine cartilage ex vivo model

    DEFF Research Database (Denmark)

    He, Yi; Zheng, Qinlong; Simonsen, Ole;

    2013-01-01

    )) in a bovine cartilage ex vivo model. We found that after stimulation with catabolic factors, the cartilage initially released high levels of aggrecanase-derived aggrecan fragments into supernatant but subsequently decreased to background levels. The level of active ADAMTS-4 released into the supernatant...

  11. Ex vivo generated natural killer cells acquire typical natural killer receptors and display a cytotoxic gene expression profile similar to peripheral blood natural killer cells

    NARCIS (Netherlands)

    Lehmann, D.; Spanholtz, J.; Osl, M.; Tordoir, M.; Lipnik, K.; Bilban, M.; Schlechta, B.; Dolstra, H.; Hofer, E.

    2012-01-01

    Ex vivo differentiation systems of natural killer (NK) cells from CD34+ hematopoietic stem cells are of potential importance for adjuvant immunotherapy of cancer. Here, we analyzed ex vivo differentiation of NK cells from cord blood-derived CD34+ stem cells by gene expression profiling, real-time RT

  12. In Vivo Consumption of Cranberry Exerts ex Vivo Antiadhesive Activity against FimH-Dominated Uropathogenic Escherichia coli: A Combined in Vivo, ex Vivo, and in Vitro Study of an Extract from Vaccinium macrocarpon.

    Science.gov (United States)

    Rafsanjany, Nasli; Senker, Jandirk; Brandt, Simone; Dobrindt, Ulrich; Hensel, Andreas

    2015-10-14

    For investigation of the molecular interaction of cranberry extract with adhesins of uropathogenic Escherichia coli (UPEC), urine from four volunteers consuming standardized cranberry extract (proanthocyanidin content = 1.24%) was analyzed within ex vivo experiments, indicating time-dependent significant inhibition of 40-50% of bacterial adhesion of UPEC strain NU14 to human T24 bladder cells. Under in vitro conditions a dose-dependent increase in bacterial adhesion was observed with proanthocyanidin-enriched cranberry Vaccinium macrocarpon extract (proanthocyanidin content = 21%). Confocal laser scanning microscopy and scanning electron microscopy proved that V.m. extract led to the formation of bacterial clusters on the outer plasma membrane of the host cells without subsequent internalization. This agglomerating activity was not observed when a PAC-depleted extract (V.m. extract(≠PAC)) was used, which showed significant inhibition of bacterial adhesion in cases where type 1 fimbriae dominated and mannose-sensitive UPEC strain NU14 was used. V.m. extract(≠PAC) had no inhibitory activity against P- and F1C-fimbriae dominated strain 2980. Quantitative gene expression analysis indicated that PAC-containing as well as PAC-depleted cranberry extracts increased the fimH expression in NU14 as part of a feedback mechanism after blocking FimH. For strain 2980 the PAC-containing extract led to up-regulation of P- and F1C-fimbriae, whereas the PAC-depleted extract had no influence on gene expression. V.m. and V.m. extract(≠PAC) did not influence biofilm and curli formation in UPEC strains NU14 and 2980. These data lead to the conclusion that also proanthocyanidin-free cranberry extracts exert antiadhesive activity by interaction with mannose-sensitive type 1 fimbriae of UPEC.

  13. Development and validation of an ex vivo electron paramagnetic resonance fingernail bio-dosimetric method

    International Nuclear Information System (INIS)

    There is an imperative need to develop methods that can rapidly and accurately determine individual exposure to radiation for screening (triage) populations and guiding medical treatment in an emergency response to a large-scale radiological/nuclear event. To this end, a number of methods that rely on dose-dependent chemical and/or physical alterations in biomaterials or biological responses are in various stages of development. One such method, ex vivo electron paramagnetic resonance (EPR) nail dosimetry using human nail clippings, is a physical bio-dosimetry technique that takes advantage of a stable radiation-induced signal (RIS) in the keratin matrix of fingernails and toenails. This dosimetry method has the advantages of ubiquitous availability of the dosimetric material, easy and non-invasive sampling, and the potential for immediate and rapid dose assessment. The major challenge for ex vivo EPR nail dosimetry is the overlap of mechanically induced signals and the RIS. The difficulties of analysing the mixed EPR spectra of a clipped irradiated nail were addressed in the work described here. The following key factors lead to successful spectral analysis and dose assessment in ex vivo EPR nail dosimetry: (1) obtaining a thorough understanding of the chemical nature, the decay behaviour, and the microwave power dependence of the EPR signals, as well as the influence of variation in temperature, humidity, water content, and O2 level; (2) control of the variability among individual samples to achieve consistent shape and kinetics of the EPR spectra; (3) use of correlations between the multiple spectral components; and (4) use of optimised modelling and fitting of the EPR spectra to improve the accuracy and precision of the dose estimates derived from the nail spectra. In the work described here, two large clipped nail datasets were used to test the procedures and the spectral fitting model of the results obtained with it. A 15-donor nail set with 90 nail samples

  14. Zinc enhances bone metabolism in ovariectomized rats and exerts anabolic osteoblastic/adipocytic marrow effects ex vivo.

    Science.gov (United States)

    Li, Binbin; Liu, Hao; Jia, Shengnan

    2015-02-01

    Investigations of bone mass and marrow adiposity are critical for defining the role of zinc (Zn) in bone metabolism. Rats used for study were grouped as follows: control (sham), ovariectomy (OVX), ovariectomy + estradiol (OVX-E), ovariectomy + Zn treatment (OVX-Zn). Bone mineral density (BMD) was quantified (microCT); serum osteocalcin, adiponectin, RANKL, and TRAP levels were assayed (ELISA); and biochemical determinations of serum alkaline phosphatase (ALP), calcium (Ca), and phosphorus (P) were done. Cells derived from bone mesenchymal stem cell (BMSC) isolates of respective test groups were compared, identifying primary osteoblasts by MTT assay and adipocytes by Oil Red O stain. Osteocalcin and adiponectin levels in culture supernatants were determined by ELISA. Zn supplementation resulted in a modest increase in BMD, but serum osteocalcin and ALP activity increased significantly (P < 0.01, both). Serum levels of RANKL and TRAP were lower in OVX-Zn (vs OVX) rats (P < 0.01), whereas serum concentrations of adiponectin, Ca, and P did not differ by group. Osteocalcin level was significantly upregulated ex vivo (P < 0.01) in the supernatant of cultured OVX-Zn (vs OVX) cells, accompanied by a slight upturn in osteoblastic differentiation. However, Oil Red O uptake and adiponectin level in supernatant were sharply diminished in cultured OVX-Zn (vs OVX) cells (P < 0.01). Overall, we concluded that Zn contributes to bone mass by marginally stimulating differentiation and proliferation of osteoblasts and by effectively inhibiting osteoclastic and adipocytic differentiation of BMSCs.

  15. Nose to brain microemulsion-based drug delivery system of rivastigmine: formulation and ex-vivo characterization.

    Science.gov (United States)

    Shah, Brijesh M; Misra, Manju; Shishoo, Chamanlal J; Padh, Harish

    2015-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to irreversible loss of neurons, cognition and formation of abnormal protein aggregates. Rivastigmine, a reversible cholinesterase inhibitor used for the treatment of AD, undergoes extensive first-pass metabolism, thus limiting its absolute bioavailability to only 36% after 3-mg dose. Due to extreme aqueous solubility, rivastigmine shows poor penetration and lesser concentration in the brain thus requiring frequent oral dosing. This investigation was aimed to formulate microemulsion (ME) and mucoadhesive microemulsions (MMEs) of rivastigmine for nose to brain delivery and to compare percentage drug diffused for both systems using in-vitro and ex-vivo study. Rivastigmine-loaded ME and MMEs were prepared by titration method and characterized for drug content, globule size distribution, zeta potential, pH, viscosity and nasal ciliotoxicity study. Rivastigmine-loaded ME system containing 8% w/w Capmul MCM EP, 44% w/w Labrasol:Transcutol-P (1:1) and 48% w/w distilled water was formulated, whereas 0.3% w/w chitosan (CH) and cetyl trimethyl ammonium bromide (as mucoadhesive agents) were used to formulate MMEs, respectively. ME and MMEs formulations were transparent with drug content, globule size and zeta potential in the range of 98.59% to 99.43%, 53.8 nm to 55.4 nm and -2.73 mV to 6.52 mV, respectively. MME containing 0.3% w/w CH followed Higuchi model (r(2) = 0.9773) and showed highest diffusion coefficient. It was free from nasal ciliotoxicity and stable for three months. However, the potential of developed CH-based MME for nose to brain delivery of rivastigmine can only be established after in-vivo and biodistribution study.

  16. Measurement of ventilation- and perfusion-mediated cooling during laser ablation in ex vivo human lung tumors

    International Nuclear Information System (INIS)

    Purpose: Perfusion-mediated tissue cooling has often been described in the literature for thermal ablation therapies of liver tumors. The objective of this study was to investigate the cooling effects of both perfusion and ventilation during laser ablation of lung malignancies. Materials and methods: An ex vivo lung model was used to maintain near physiological conditions for the specimens. Fourteen human lung lobes containing only primary lung tumors (non-small cell lung cancer) were used. Laser ablation was carried out using a Nd:YAG laser with a wavelength of 1064 nm and laser fibers with 30 mm diffusing tips. Continuous invasive temperature measurement in 10 mm distance from the laser fiber was performed. Laser power was increased at 2 W increments starting at 10 W up to a maximum power of 12-20 W until a temperature plateau around 60 deg. C was reached at one sensor. Ventilation and perfusion were discontinued for 6 min each to assess their effects on temperature development. Results: The experiments lead to 25 usable temperature profiles. A significant temperature increase was observed for both discontinued ventilation and perfusion. In 6 min without perfusion, the temperature rose about 5.5 deg. C (mean value, P < 0.05); without ventilation it increased about 7.0 deg. C (mean value, P < 0.05). Conclusion: Ventilation- and perfusion-mediated tissue cooling are significant influencing factors on temperature development during thermal ablation. They should be taken into account during the planning and preparation of minimally invasive lung tumor treatment in order to achieve complete ablation.

  17. Measurement of ventilation- and perfusion-mediated cooling during laser ablation in ex vivo human lung tumors

    Energy Technology Data Exchange (ETDEWEB)

    Vietze, Andrea, E-mail: anvie@gmx.de [Department of Diagnostic Radiology and Neuroradiology, Ernst-Moritz-Arndt-Universitaet Greifswald, Sauerbruchstrasse, 17487 Greifswald (Germany); Koch, Franziska, E-mail: franzi_koch@hotmail.com [Department of Diagnostic Radiology and Neuroradiology, Ernst-Moritz-Arndt-Universitaet Greifswald, Sauerbruchstrasse, 17487 Greifswald (Germany); Laskowski, Ulrich, E-mail: ulrich.laskowski@klinikum-luedenscheid.de [Department of Vascular and Thoracic Surgery, Klinikum Luedenscheid, Paulmannshoeher Strasse 14, 58515 Luedenscheid (Germany); Linder, Albert, E-mail: albert.linder@klinikum-bremen-ost.de [Department of Thoracic Surgery, Klinikum Bremen-Ost, Zuericher Strasse 40, 28325 Bremen (Germany); Hosten, Norbert, E-mail: hosten@uni-greifswald.de [Department of Diagnostic Radiology and Neuroradiology, Ernst-Moritz-Arndt-Universitaet Greifswald, Sauerbruchstrasse, 17487 Greifswald (Germany)

    2011-11-15

    Purpose: Perfusion-mediated tissue cooling has often been described in the literature for thermal ablation therapies of liver tumors. The objective of this study was to investigate the cooling effects of both perfusion and ventilation during laser ablation of lung malignancies. Materials and methods: An ex vivo lung model was used to maintain near physiological conditions for the specimens. Fourteen human lung lobes containing only primary lung tumors (non-small cell lung cancer) were used. Laser ablation was carried out using a Nd:YAG laser with a wavelength of 1064 nm and laser fibers with 30 mm diffusing tips. Continuous invasive temperature measurement in 10 mm distance from the laser fiber was performed. Laser power was increased at 2 W increments starting at 10 W up to a maximum power of 12-20 W until a temperature plateau around 60 deg. C was reached at one sensor. Ventilation and perfusion were discontinued for 6 min each to assess their effects on temperature development. Results: The experiments lead to 25 usable temperature profiles. A significant temperature increase was observed for both discontinued ventilation and perfusion. In 6 min without perfusion, the temperature rose about 5.5 deg. C (mean value, P < 0.05); without ventilation it increased about 7.0 deg. C (mean value, P < 0.05). Conclusion: Ventilation- and perfusion-mediated tissue cooling are significant influencing factors on temperature development during thermal ablation. They should be taken into account during the planning and preparation of minimally invasive lung tumor treatment in order to achieve complete ablation.

  18. CO2 laser and/or fluoride enamel treatment against in situ/ex vivo erosive challenge

    Science.gov (United States)

    JORDÃO, Maísa Camillo; FORTI, Gustavo Manzano; NAVARRO, Ricardo Scarparo; FREITAS, Patrícia Moreira; HONÓRIO, Heitor Marques; RIOS, Daniela

    2016-01-01

    ABSTRACT Objective This in situ/ex vivo study investigated the effect of CO2 laser irradiation and acidulated phosphate fluoride gel (APF) application, separately and in combination, on enamel resistance to erosion. Material and Methods During 2 experimental 5-day crossover phases, 8 volunteers wore intraoral appliances containing bovine enamel blocks which were submitted to four groups: 1st phase - control, untreated and CO2 laser irradiation, 2nd phase - fluoride application and fluoride application before CO2 laser irradiation. Laser irradiation was performed at 10.6 µm wavelength, 5 µs pulse duration and 50 Hz frequency, with average power input and output of 2.3 W and 2.0 W, respectively (28.6 J/cm2). APF gel (1.23%F, pH 3.5) was applied on enamel surface with a microbrush and left on for 4 minutes. Then, the enamel blocks were fixed at the intraoral appliance level. The erosion was performed extraorally 4 times daily for 5 min in 150 mL of cola drink. Enamel loss was measured profilometrically after treatment and after the in situ phase. The data were tested using one-way Repeated Measures Anova and Tukey's test (pFluoride treated enamel, associated (1.50±0.30 µm) or not (1.47±0.63 µm) with laser irradiation, significantly differed from the control. Conclusion The APF application decreased enamel wear; however, CO2 laser irradiation did not enhance fluoride ability to reduce enamel wear. PMID:27383703

  19. CO2 laser and/or fluoride enamel treatment against in situ/ex vivo erosive challenge

    Science.gov (United States)

    JORDÃO, Maísa Camillo; FORTI, Gustavo Manzano; NAVARRO, Ricardo Scarparo; FREITAS, Patrícia Moreira; HONÓRIO, Heitor Marques; RIOS, Daniela

    2016-01-01

    ABSTRACT Objective This in situ/ex vivo study investigated the effect of CO2 laser irradiation and acidulated phosphate fluoride gel (APF) application, separately and in combination, on enamel resistance to erosion. Material and Methods During 2 experimental 5-day crossover phases, 8 volunteers wore intraoral appliances containing bovine enamel blocks which were submitted to four groups: 1st phase - control, untreated and CO2 laser irradiation, 2nd phase - fluoride application and fluoride application before CO2 laser irradiation. Laser irradiation was performed at 10.6 µm wavelength, 5 µs pulse duration and 50 Hz frequency, with average power input and output of 2.3 W and 2.0 W, respectively (28.6 J/cm2). APF gel (1.23%F, pH 3.5) was applied on enamel surface with a microbrush and left on for 4 minutes. Then, the enamel blocks were fixed at the intraoral appliance level. The erosion was performed extraorally 4 times daily for 5 min in 150 mL of cola drink. Enamel loss was measured profilometrically after treatment and after the in situ phase. The data were tested using one-way Repeated Measures Anova and Tukey's test (p<0.05). Results CO2 laser alone (2.00±0.39 µm) did not show any significantly preventive effect against enamel erosion when compared with the control group (2.41±1.20 µm). Fluoride treated enamel, associated (1.50±0.30 µm) or not (1.47±0.63 µm) with laser irradiation, significantly differed from the control. Conclusion The APF application decreased enamel wear; however, CO2 laser irradiation did not enhance fluoride ability to reduce enamel wear. PMID:27383703

  20. Infrared fluorescent imaging as a potent tool for in vitro, ex vivo and in vivo models of visceral leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Estefanía Calvo-Álvarez

    2015-03-01

    Full Text Available Visceral leishmaniasis (VL is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS appear to be suitable to achieve this goal in the coming years.We generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II. The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR, was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks.The near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.

  1. Ex vivo and in vivo neuroprotection induced by argon when given after an excitotoxic or ischemic insult.

    Directory of Open Access Journals (Sweden)

    Hélène N David

    Full Text Available In vitro studies have well established the neuroprotective action of the noble gas argon. However, only limited data from in vivo models are available, and particularly whether postexcitotoxic or postischemic argon can provide neuroprotection in vivo still remains to be demonstrated. Here, we investigated the possible neuroprotective effect of postexcitotoxic-postischemic argon both ex vivo in acute brain slices subjected to ischemia in the form of oxygen and glucose deprivation (OGD, and in vivo in rats subjected to an intrastriatal injection of N-methyl-D-aspartate (NMDA or to the occlusion of middle-cerebral artery (MCAO. We show that postexcitotoxic-postischemic argon reduces OGD-induced cell injury in brain slices, and further reduces NMDA-induced brain damage and MCAO-induced cortical brain damage in rats. Contrasting with its beneficial effect at the cortical level, we show that postischemic argon increases MCAO-induced subcortical brain damage and provides no improvement of neurologic outcome as compared to control animals. These results extend previous data on the neuroprotective action of argon. Particularly, taken together with previous in vivo data that have shown that intraischemic argon has neuroprotective action at both the cortical and subcortical level, our findings on postischemic argon suggest that this noble gas could be administered during but not after ischemia, i.e. before but not after reperfusion has occurred, in order to provide cortical neuroprotection and to avoid increasing subcortical brain damage. Also, the effects of argon are discussed as regards to the oxygen-like chemical, pharmacological, and physical properties of argon.

  2. Hexachlorobenzene and its metabolites pentachlorophenol and tetrachlorohydroquinone: interaction with thyroxine binding sites of rat thyroid hormone carriers ex vivo and in vitro.

    Science.gov (United States)

    van Raaij, J A; van den Berg, K J; Notten, W R

    1991-12-01

    Previous results have indicated that hexachlorobenzene (HCB)-induced hypothyroidism may be caused by its main metabolite pentachlorophenol (PCP), and by tetrachlorohydroquinone (TCHQ), rather than by the parent compound. In the present experiments it was investigated whether hormone displacement from serum carriers could be a factor in the development of this hypothyroidism. In an in vitro competition assay PCP was an effective competitor for the thyroxine (T4)-binding sites of serum carriers, whereas HCB was ineffective. Ex vivo experimental results demonstrated occupation of T4-binding sites in sera from PCP-exposed animals but not in sera from HCB- or TCHQ-treated animals. Competing ability for T4-binding sites was still present in sera of PCP-exposed animals but was absent in HCB- or TCHQ-exposed animals. The results suggest that thyroid hormone displacement by the major metabolite PCP may play a role in HCB-induced hypothyroidism. PMID:1755017

  3. [Lymph node staging in gastrointestinal cancer. Combination of methylene blue-assisted lymph node dissection and ex vivo sentinel lymph node mapping].

    Science.gov (United States)

    Märkl, B; Arnholdt, H

    2012-11-01

    The histopathological lymph node staging is of crucial importance for the prognosis estimation and therapy stratification in gastrointestinal cancer. However, the recommended numbers of lymph nodes that should be evaluated are often not reached in routine practice. Methylene blue assisted lymph node dissection was introduced as a new, simple and efficient technique to improve lymph node harvest in gastrointestinal cancer. This method is inexpensive, causes no delay and needs no toxic substances. All studies performed revealed a highly significantly improved lymph node harvest in comparison to the conventional technique. Moreover, this technique can be combined with a new ex vivo sentinel lymph node mapping that for the first time is based on histological sentinel lymph node detection. The success rate of this method is similar to conventional techniques and it enables an efficient application of extended investigation methods, such as immunohistochemistry or the polymerase chain reaction.

  4. A method to measure the hyperelastic parameters of ex vivo breast tissue samples

    Science.gov (United States)

    Samani, Abbas; Plewes, Donald

    2004-09-01

    Over the past decade, there has been increasing interest in modelling soft tissue deformation. This topic has several biomedical applications ranging from medical imaging to robotic assisted telesurgery. In these applications, tissue deformation can be very large due to low tissue stiffness and lack of physical constraints. As a result, deformation modelling of such organs often requires a treatment, which reflects nonlinear behaviour. While computational techniques such as nonlinear finite element methods are well developed, the required intrinsic nonlinear mechanical parameters of soft tissues that are critical to develop reliable tissue deformation models are not well known. To address this issue, we developed a system to measure the hyperelastic parameters of small ex vivo tissue samples. This measurement technique consists of indenting an unconfined small block of tissue using a computer controlled loading system while measuring the resulting indentation force. The nonlinear tissue force-displacement response is used to calculate the hyperelastic parameters via an appropriate inversion technique. This technique is based on a nonlinear least squares formulation that uses a nonlinear finite element model as the direct problem solver. The features of the system are demonstrated with two samples of breast tissue and typical hyperelastic results are presented.

  5. Inhibition of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase (Ex Vivo by Morus indica (Mulberry

    Directory of Open Access Journals (Sweden)

    Vanitha Reddy Palvai

    2014-01-01

    Full Text Available Phytochemicals are the bioactive components that contribute to the prevention of cardiovascular and other degenerative diseases. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA reductase would be an effective means of lowering plasma cholesterol in humans. The present study explores the HMG CoA reductase inhibitory effect of extracts from leaves of Morus indica varieties, M5, V1, and S36, compared with the statin, using an ex vivo method. The assay is based on the stoichiometric formation of coenzyme A during the reduction of microsomal HMG CoA to mevalonate. Dechlorophyllised extract of three varieties was studied at 300 µg. The coenzyme A released at the end of assay in control (100.31 nmoles and statins (94.46 nm was higher than the dechlorphyllised extracts of the samples. The coenzyme A released during the reduction of HMG CoA to mevalonate in dechlorophyllised extracts of the samples was as follows: S36 < M5 < V1. The results indicated that the samples were highly effective in inhibiting the enzyme compared to statins (standard drug. The results indicate the role of Morus varieties extracts in modulating the cholesterol metabolism by inhibiting the activity of HMG CoA reductase. These results provide scope for designing in vivo animal studies to confirm their effect.

  6. Ex Vivo and In Vivo Lentivirus-Mediated Transduction of Airway Epithelial Progenitor Cells.

    Science.gov (United States)

    Leoni, Giulia; Wasowicz, Marguerite Y; Chan, Mario; Meng, Cuixiang; Farley, Raymond; Brody, Steven L; Inoue, Makoto; Hasegawa, Mamoru; Alton, Eric W F W; Griesenbach, Uta

    2015-01-01

    A key challenge in pulmonary gene therapy for cystic fibrosis is to provide long-term correction of the genetic defect. This may be achievable by targeting airway epithelial stem/progenitor cells with an integrating vector. Here, we evaluated the ability of a lentiviral vector, derived from the simian immunodeficiency virus and pseudotyped with F and HN envelope proteins from Sendai virus, to transduce progenitor basal cells of the mouse nasal airways. We first transduced basal cell-enriched cultures ex vivo and confirmed efficient transduction of cytokeratin-5 positive cells. We next asked whether progenitor cells could be transduced in vivo. We evaluated the transduction efficiency in mice pretreated by intranasal administration of polidocanol to expose the progenitor cell layer. Compared to control mice, polidocanol treated mice demonstrated a significant increase in the number of transduced basal cells at 3 and 14 days post vector administration. At 14 days, the epithelium of treated mice contained clusters (4 to 8 adjacent cells) of well differentiated ciliated, as well as basal cells suggesting a clonal expansion. These results indicate that our lentiviral vector can transduce progenitor basal cells in vivo, although transduction required denudation of the surface epithelium prior to vector administration. PMID:26471068

  7. Metabolomics reveals the heterogeneous secretome of two entomopathogenic fungi to ex vivo cultured insect tissues.

    Directory of Open Access Journals (Sweden)

    Charissa de Bekker

    Full Text Available Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with ex vivo insect tissue culturing shows that two generalist isolates of the genus Metarhizium and Beauveria, commonly used as biological pesticides, employ significantly different arrays of secondary metabolites during infectious and saprophytic growth. It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied.

  8. First Ex-Vivo Validation of a Radioguided Surgery Technique with beta- Radiation

    CERN Document Server

    Camillocci, E Solfaroli; Bocci, V; Carollo, A; Chiodi, G; Colandrea, M; Collamati, F; Cremonesi, M; Donnarumma, R; Ferrari, M E; Ferroli, P; Ghielmetti, F; Grana, C M; Marafini, M; Morganti, S; Terracciano, C Mancini; Patanè, M; Pedroli, G; Pollo, B; Recchia, L; Russomando, A; Toppi, M; Traini, G; Faccini, R

    2016-01-01

    Purpose: A radio-guided surgery technique with beta- -emitting radio-tracers was suggested to overcome the effect of the large penetration of gamma radiation. The feasibility studies in the case of brain tumors and abdominal neuro-endocrine tumors were based on simulations starting from PET images with several underlying assumptions. This paper reports, as proof-of-principle of this technique, an ex-vivo test on a meningioma patient. This test allowed to validate the whole chain, from the evaluation of the SUV of the tumor, to the assumptions on the bio-distribution and the signal detection. Methods: A patient affected by meningioma was administered 300 MBq of 90Y-DOTATOC. Several samples extracted from the meningioma and the nearby Dura Mater were analyzed with a beta- probe designed specifically for this radio-guided surgery technique. The observed signals were compared both with the evaluation from the histology and with the Monte Carlo simulation. Results: we obtained a large signal on the bulk tumor (105...

  9. Successful emergent lung transplantation after remote ex vivo perfusion optimization and transportation of donor lungs.

    Science.gov (United States)

    Wigfield, C H; Cypel, M; Yeung, J; Waddell, T; Alex, C; Johnson, C; Keshavjee, S; Love, R B

    2012-10-01

    A recent clinical trial provided evidence that ex vivo lung perfusion (EVLP) results in optimized human donor lungs for transplantation. Excellent recipient outcomes were documented after 4 h of normothermic perfusion. We report a clinical case utilizing remote EVLP to assess and improve function of initially otherwise unacceptable injured donor lungs followed by transportation and subsequent bilateral lung transplantation in a patient with virally induced refractory respiratory failure supported with extracorporeal membrane oxygenation. This is the first lung transplantation with the application of remote EVLP, wherein the donor lungs were transported from the donor hospital to a center for EVLP and then transported to another hospital for transplantation. It is also the first case of lung transplantation in the United States utilizing EVLP for functional optimization leading to successful transplantation. Organ procurement data, EVLP assessment, and the pre- and postoperative course of the recipient are presented. The available evidence supporting EVLP, the humanitarian and cooperative utilization of lungs otherwise discarded, are discussed. PMID:23009140

  10. Ex Vivo Assessment of a Parabolic-Tip Inflow Cannula for Pediatric Continuous-Flow VADs.

    Science.gov (United States)

    Griffin, Michael T; Grzywinski, Matthew F; Voorhees, Hannah J; Kameneva, Marina V; Olia, Salim E

    2016-01-01

    To address the challenge of unloading the left ventricle during pediatric mechanical circulatory support using next-generation rotary blood pumps, a novel inflow cannula was developed. This unique inflow cannula for pediatric, continuous-flow, left ventricular assist devices (VADs) with a parabolic-shaped inlet entrance was evaluated alongside a bevel-tip and fenestrated-tip cannula via an ex vivo, isolated-heart experimental setup. Performance was characterized using two clinical scenarios of over-pumping and hypovolemia, created by varying pump speed and filling preload pressure, respectively, at ideal and off-axis cannula placement to assess ventricular unloading and positional sensitivity. Quantitative and qualitative assessments were performed on the resultant hemodynamics and intra-ventricular boroscopic images to classify conditions of nonsuction, partial, gradual or severe entrainment, and ventricular collapse. The parabolic-tip cannula was found to be significantly less sensitive to placement position (p pumping and hypovolemic studies, respectively. We conclude that future pediatric VAD designs may benefit from incorporating the parabolic-tip inflow cannula design to maximize unloading of the left ventricle in ideal and nonoptimal conditions. PMID:27442862

  11. The influence of small intestinal mucus structure on particle transport ex vivo.

    Science.gov (United States)

    Bajka, Balázs H; Rigby, Neil M; Cross, Kathryn L; Macierzanka, Adam; Mackie, Alan R

    2015-11-01

    Mucus provides a barrier to bacteria and toxins while allowing nutrient absorption and waste transport. Unlike colonic mucus, small intestinal mucus structure is poorly understood. This study aimed to provide evidence for a continuous, structured mucus layer and assess the diffusion of different sized particles through it. Mucus structure was assessed by histology and immunohistochemistry. Ultra-structure was assessed by scanning electron microscopy. Tracking of 100 nm and 500 nm latex beads was conducted using ex vivo porcine mucus. The porcine jejunum and ileum were filled with mucus. Layered MUC2 staining was visible throughout the small intestine, covering villus tips. Scanning electron microscopy showed net-like mucin sheets covering villi (211 ± 7 nm pore diameter). Particle tracking of 100 nm latex beads, showed no inhibition of diffusion through mucus while 500 nm beads displayed limited diffusion. These results suggest a continuous mucus layer exists throughout the small intestine, which is highly stratified adjacent to the epithelium. The network observed is consistent with previous observations and correlates with stratified MUC2 staining. Mucin pore size is consistent with free diffusion of 100 nm and limited diffusion of 500 nm particles. Small Intestinal mucus structure has important implications for drug delivery systems and prevention and treatment of conditions like mucositis and inflammatory bowel disease.

  12. Nonlinear acoustic properties of ex vivo bovine liver and the effects of temperature and denaturation

    Science.gov (United States)

    Jackson, E. J.; Coussios, C.-C.; Cleveland, R. O.

    2014-06-01

    Thermal ablation by high intensity focused ultrasound (HIFU) has a great potential for the non-invasive treatment of solid tumours. Due to the high pressure amplitudes involved, nonlinear acoustic effects must be understood and the relevant medium property is the parameter of nonlinearity B/A. Here, B/A was measured in ex vivo bovine liver, over a heating/cooling cycle replicating temperatures reached during HIFU ablation, adapting a finite amplitude insertion technique, which also allowed for measurement of sound-speed and attenuation. The method measures the nonlinear progression of a plane wave through liver and B/A was chosen so that numerical simulations matched the measured waveforms. To create plane-wave conditions, sinusoidal bursts were transmitted by a 100 mm diameter 1.125 MHz unfocused transducer and measured using a 15 mm diameter 2.25 MHz broadband transducer in the near field. Attenuation and sound-speed were calculated using a reflected pulse from the smaller transducer using the larger transducer as the reflecting interface. Results showed that attenuation initially decreased with heating then increased after denaturation, the sound-speed initially increased with temperature and then decreased, and B/A showed an increase with temperature but no significant post-heating change. The B/A data disagree with other reports that show a significant change and we suggest that any nonlinear enhancement in the received ultrasound signal post-treatment is likely due to acoustic cavitation rather than changes in tissue nonlinearity.

  13. In vivo, Ex Vivo, and In Vitro Approaches to Study Intermediate Filaments in the Eye Lens.

    Science.gov (United States)

    Jarrin, Miguel; Young, Laura; Wu, Weiju; Girkin, John M; Quinlan, Roy A

    2016-01-01

    The role of the eye lens is to focus light into the retina. To perform this unique function, the ocular lens must be transparent. Previous studies have demonstrated the expression of vimentin, BFSP1, and BFSP2 in the eye lens. These intermediate filament (IF) proteins are essential to the optical properties of the lens. They are also important to its biomechanical properties, to the shape of the lens fiber cells, and to the organization and function of the plasma membrane. The eye lens is an iconic model in developmental studies, as a result different vertebrate models, including zebrafish, have been developed to study lens formation. In the present chapter, we have summarized the new approaches and the more breakthrough models (e.g., iPSc) that can be used to study the function of IFs in the ocular lens. We have presented three different groups of models. The first group includes in vitro models, where IFs can be studied and manipulated in lens cell cultures. The second includes ex vivo models. These replicate better the complex lens cell differentiation processes and the role(s) played by IFs. The third class is the in vivo models, and here, we have focused on Zebrafish and new imaging approaches using selective plane illumination microscopy. Finally, we present protocols on how to use these lens models to study IFs.

  14. Repopulating Decellularized Kidney Scaffolds: An Avenue for Ex Vivo Organ Generation

    Directory of Open Access Journals (Sweden)

    Robert A. McKee

    2016-03-01

    Full Text Available Recent research has shown that fully developed organs can be decellularized, resulting in a complex scaffold and extracellular matrix (ECM network capable of being populated with other cells. This work has resulted in a growing field in bioengineering focused on the isolation, characterization, and modification of organ derived acellular scaffolds and their potential to sustain and interact with new cell populations, a process termed reseeding. In this review, we cover contemporary advancements in the bioengineering of kidney scaffolds including novel work showing that reseeded donor scaffolds can be transplanted and can function in recipients using animal models. Several major areas of the field are taken into consideration, including the decellularization process, characterization of acellular and reseeded scaffolds, culture conditions, and cell sources. Finally, we discuss future avenues based on the advent of 3D bioprinting and recent developments in kidney organoid cultures as well as animal models of renal genesis. The ongoing mergers and collaborations between these fields hold the potential to produce functional kidneys that can be generated ex vivo and utilized for kidney transplantations in patients suffering with renal disease.

  15. Ex vivo imaging of human thyroid pathology using integrated optical coherence tomography and optical coherence microscopy

    Science.gov (United States)

    Zhou, Chao; Wang, Yihong; Aguirre, Aaron D.; Tsai, Tsung-Han; Cohen, David W.; Connolly, James L.; Fujimoto, James G.

    2010-01-01

    We evaluate the feasibility of optical coherence tomography (OCT) and optical coherence microscopy (OCM) for imaging of benign and malignant thyroid lesions ex vivo using intrinsic optical contrast. 34 thyroid gland specimens are imaged from 17 patients, covering a spectrum of pathology ranging from normal thyroid to benign disease/neoplasms (multinodular colloid goiter, Hashimoto's thyroiditis, and follicular adenoma) and malignant thyroid tumors (papillary carcinoma and medullary carcinoma). Imaging is performed using an integrated OCT and OCM system, with <4 μm axial resolution (OCT and OCM), and 14 μm (OCT) and <2 μm (OCM) transverse resolution. The system allows seamless switching between low and high magnifications in a way similar to traditional microscopy. Good correspondence is observed between optical images and histological sections. Characteristic features that suggest malignant lesions, such as complex papillary architecture, microfollicules, psammomatous calcifications, or replacement of normal follicular architecture with sheets/nests of tumor cells, can be identified from OCT and OCM images and are clearly differentiable from normal or benign thyroid tissues. With further development of needle-based imaging probes, OCT and OCM could be promising techniques to use for the screening of thyroid nodules and to improve the diagnostic specificity of fine needle aspiration evaluation.

  16. Development of an Ex Vivo, Beating Heart Model for CT Myocardial Perfusion

    Directory of Open Access Journals (Sweden)

    Gert Jan Pelgrim

    2015-01-01

    Full Text Available Objective. To test the feasibility of a CT-compatible, ex vivo, perfused porcine heart model for myocardial perfusion CT imaging. Methods. One porcine heart was perfused according to Langendorff. Dynamic perfusion scanning was performed with a second-generation dual source CT scanner. Circulatory parameters like blood flow, aortic pressure, and heart rate were monitored throughout the experiment. Stenosis was induced in the circumflex artery, controlled by a fractional flow reserve (FFR pressure wire. CT-derived myocardial perfusion parameters were analysed at FFR of 1 to 0.10/0.0. Results. CT images did not show major artefacts due to interference of the model setup. The pacemaker-induced heart rhythm was generally stable at 70 beats per minute. During most of the experiment, blood flow was 0.9–1.0 L/min, and arterial pressure varied between 80 and 95 mm/Hg. Blood flow decreased and arterial pressure increased by approximately 10% after inducing a stenosis with FFR ≤ 0.50. Dynamic perfusion scanning was possible across the range of stenosis grades. Perfusion parameters of circumflex-perfused myocardial segments were affected at increasing stenosis grades. Conclusion. An adapted Langendorff porcine heart model is feasible in a CT environment. This model provides control over physiological parameters and may allow in-depth validation of quantitative CT perfusion techniques.

  17. Engineering a human bone marrow model: a case study on ex vivo erythropoiesis.

    Science.gov (United States)

    Mantalaris, A; Keng, P; Bourne, P; Chang, A Y; Wu, J H

    1998-01-01

    Bone marrow, with its intricate, three-dimensional tissue structure facilitating cell-cell interactions, provides a microenvironment supporting the production of hundreds of billions of multilineal blood cells everyday. We have developed a three-dimensional bone marrow culture system in which marrow cells are cultured in a reactor packed with porous microspheres. The culture supports a three-dimensional growth configuration and multilineal hemopoiesis mimicking the bone marrow in vivo. We studied ex vivo human erythropoiesis using the three-dimensional culture system. The system sustained extensive erythropoiesis at low erythropoietin concentrations (0.2 U/mL), plus stem cell factor, interleukin-3, granulocyte-macrophage colony-stimulating factor, and insulin-like growth factor-I. Erythroid cell production lasted for more than 5 weeks, and the percentage of erythroid cells in the nonadherent cell population was approximately 60%. Flow cytometric analysis using cell surface markers specific for erythroid cells (CD71 and glycophorin-A) indicated that the culture produced early, intermediate, and late erythroid cells. As the culture progressed, the erythroid cell population shifted gradually toward mature cell types. When compared to the three-dimensional culture, the traditional flask cultures failed to support extensive erythropoiesis under the same conditions. This indicates that the three-dimensional bone marrow culture system provides a microenvironment conducive to erythropoiesis under more physiological conditions and is a better bone marrow model.

  18. Ex vivo absorption of promestriene from oil-in-water emulsion into infant foreskin.

    Science.gov (United States)

    Salmon, D; Kassai, B; Roussel, L; Mouriquand, P; Gérard, C; Gorduza, D B; Serre, C; Falson, F; Pivot, C; Pirot, F

    2013-11-01

    Hypospadias is a birth defect in which the urinary tract opening is not at the tip of the penis. Hypospadias surgery is frequently complicated by healing deficiencies. Topical treatments with oestrogens were reported to improve healing. In the present study, ex vivo percutaneous absorption of promestriene, a synthetic oestrogen resulting of the double esterification of estradiol was conducted as a pre-requisite for further clinical trial in infants. Penetration of promestriene into infant foreskin treated with commercial oil in water emulsion (10 μg mg(-1)) for 24 h was characterized showing controlled release properties enabling epidermal concentration more than six times higher than dermal concentration (4.13±2.46 mg g(-1) versus 0.62±0.84 mg g(-1), respectively). Furthermore, apparent promestriene fluxes into and through the skin (i.e., 1.5 μg cm(-2) h(-1) and<0.89 μg cm(-2) h(-1), respectively) were calculated from (i) drug amount retained into epidermis and dermis, or (ii) the limit of detection into the receptor fluid. In conclusion, less than 2% of initial dose were absorbed within 24h which compared well with others steroids applied topically in colloidal systems. PMID:23968783

  19. Cholesterol-lowering properties of Ganoderma lucidum in vitro, ex vivo, and in hamsters and minipigs

    Directory of Open Access Journals (Sweden)

    Hajjaj H

    2004-02-01

    Full Text Available Abstract Introduction There has been renewed interest in mushroom medicinal properties. We studied cholesterol lowering properties of Ganoderma lucidum (Gl, a renowned medicinal species. Results Organic fractions containing oxygenated lanosterol derivatives inhibited cholesterol synthesis in T9A4 hepatocytes. In hamsters, 5% Gl did not effect LDL; but decreased total cholesterol (TC 9.8%, and HDL 11.2%. Gl (2.5 and 5% had effects on several fecal neutral sterols and bile acids. Both Gl doses reduced hepatic microsomal ex-vivo HMG-CoA reductase activity. In minipigs, 2.5 Gl decreased TC, LDL- and HDL cholesterol 20, 27, and 18%, respectively (P Conclusions Overall, Gl has potential to reduce LDL cholesterol in vivo through various mechanisms. Next steps are to: fully characterize bioactive components in lipid soluble/insoluble fractions; evaluate bioactivity of isolated fractions; and examine human cholesterol lowering properties. Innovative new cholesterol-lowering foods and medicines containing Gl are envisioned.

  20. Ex vivo microbial leakage after using different final irrigation regimens with chlorhexidine

    Directory of Open Access Journals (Sweden)

    Esther NAVARRO-ESCOBAR

    2013-01-01

    Full Text Available Objective To assess the influence of final irrigation protocols with chlorhexidine in the coronal leakage of Enterococcus faecalis in filled root canals. Material and Methods Seventy single-root canals from extracted teeth were prepared using ProTaper instruments. The irrigation protocol accomplished an alternating irrigation with 5 mL of 2.5% sodium hypochlorite (NaOCI and 17% EDTA between each file. The teeth were randomly divided into four experimental groups (n=15 according to the final irrigation regimen: group 1, without final irrigation; group 2, irrigation with 10 mL 2.0% chlorhexidine (CHX; group 3, with a final application of EC40™; and group 4, irrigation with the combination (1:1 of 0.2% CHX + 0.1% cetrimide (CTR. All the teeth were mounted in a two-chamber apparatus and the coronal access was exposed to E. faecalis. The presence of turbidity in the BHI broth over a period of 180 days was observed. The Friedman test was used for statistical analysis. Results EC40™ varnish showed the least leakage at 180 days, and was statistically similar to 2% CHX. No significant differences were observed between the group without final irrigation and the 2% CHX group or 0.2% CHX + 0.1% CTR. Conclusions In this ex vivo study, EC40™ showed the longest delayed coronal leakage of E. faecalis, although without significant differences from 2% CHX.

  1. EFFECT OF PERMEATION ENHANCER ON EX-VIVO PERMEATION OF ONDANSETRON HCl BUCCAL TABLETS

    Directory of Open Access Journals (Sweden)

    M. Praveen Kumar et al.

    2011-11-01

    Full Text Available The objective of the paper is to study the effect of a permeation enhancer, sodium taurocholate on permeation of Ondansetron HCl from bioadhesive buccal tablet formulation by performing ex-vivo permeation experiments using porcine buccal mucosa. Optimized formulation has selected based on in-vitro drug release studies of bilayered bioadhesive buccal tablets. To the optimized formulation, 10mM sodium taurocholate was added to increase the permeation of poorly permeable ondansetron HCl. It is well known that natural surfactants like bile salts increase the permeability of drugs by perturbation of intercellular lipids. The results indicated that, from pure drug solution (5 mg/mL about 88.63% cumulative percentage of drug permeated across porcine buccal mucosa with flux of 0.0235 mg.h-1cm-2. However, the optimized formulation with sodium taurocholate increased flux (0.0523 mg.h-1cm-2 and cumulative amount of drug permeated (65.44% in comparison to formulation without permeation enhancer (38.45%, 0.0186 mg.h-1cm-2 with enhancement ratio of 2.81.

  2. Ex Vivo Assessment of a Parabolic-Tip Inflow Cannula for Pediatric Continuous-Flow VADs.

    Science.gov (United States)

    Griffin, Michael T; Grzywinski, Matthew F; Voorhees, Hannah J; Kameneva, Marina V; Olia, Salim E

    2016-01-01

    To address the challenge of unloading the left ventricle during pediatric mechanical circulatory support using next-generation rotary blood pumps, a novel inflow cannula was developed. This unique inflow cannula for pediatric, continuous-flow, left ventricular assist devices (VADs) with a parabolic-shaped inlet entrance was evaluated alongside a bevel-tip and fenestrated-tip cannula via an ex vivo, isolated-heart experimental setup. Performance was characterized using two clinical scenarios of over-pumping and hypovolemia, created by varying pump speed and filling preload pressure, respectively, at ideal and off-axis cannula placement to assess ventricular unloading and positional sensitivity. Quantitative and qualitative assessments were performed on the resultant hemodynamics and intra-ventricular boroscopic images to classify conditions of nonsuction, partial, gradual or severe entrainment, and ventricular collapse. The parabolic-tip cannula was found to be significantly less sensitive to placement position (p parabolic-tip cannula showed complete resistance to entrainment, whereas the fenestrated-tip had partial entrainment in 90% and 87% of the over-pumping and hypovolemic studies, respectively. We conclude that future pediatric VAD designs may benefit from incorporating the parabolic-tip inflow cannula design to maximize unloading of the left ventricle in ideal and nonoptimal conditions.

  3. Bipolar Radiofrequency Ablation Using Dual Internally Cooled Wet Electrodes: Experimental Study in Ex Vivo Bovine Liver

    International Nuclear Information System (INIS)

    To determine the optimized protocol for bipolar radiofrequency ablation (RFA), using dual internally cooled wet (ICW) electrodes in the ex vivo bovine liver. RFA was applied to the explanted bovine liver, using two 3 cm active tip electrodes with 3.5 cm spacing. A total of 25 ablation zones were created by five groups; group A: 70 W-20 minute (min), group B: 70 W-25 min, group C: 90 W-15 min, group D: 90 W-20 min, and group E: 90 W-25 min. We measured the total energy and size of ablation zones with a color of grey or pink. Statistical analysis was done using Kruskal Wallis test and Mann Whitney U-test. The mean energy, mean volume of ablation zone with grey and pink color of groups A to E were 16.7, 23.9, 16.7, 21.8, 29.2 kcal, 25.7, 34.3, 29.5, 36.2, 45.2 cm3, and 60.0, 88.0, 71.5, 87.4, 104.5 cm3, respectively. Those were significantly different (p < 0.05). The volume of ablation zone of group E with grey color was larger than groups A, B and C (p < 0.05). Bipolar RFA, using dual ICW electrodes, can produce a large ablation zone with the protocol of 90 W-25 min.

  4. Interphase chromosome positioning in in vitro porcine cells and ex vivo porcine tissues

    Directory of Open Access Journals (Sweden)

    Foster Helen A

    2012-11-01

    Full Text Available Abstract Background In interphase nuclei of a wide range of species chromosomes are organised into their own specific locations termed territories. These chromosome territories are non-randomly positioned in nuclei which is believed to be related to a spatial aspect of regulatory control over gene expression. In this study we have adopted the pig as a model in which to study interphase chromosome positioning and follows on from other studies from our group of using pig cells and tissues to study interphase genome re-positioning during differentiation. The pig is an important model organism both economically and as a closely related species to study human disease models. This is why great efforts have been made to accomplish the full genome sequence in the last decade. Results This study has positioned most of the porcine chromosomes in in vitro cultured adult and embryonic fibroblasts, early passage stromal derived mesenchymal stem cells and lymphocytes. The study is further expanded to position four chromosomes in ex vivo tissue derived from pig kidney, lung and brain. Conclusions It was concluded that porcine chromosomes are also non-randomly positioned within interphase nuclei with few major differences in chromosome position in interphase nuclei between different cell and tissue types. There were also no differences between preferred nuclear location of chromosomes in in vitro cultured cells as compared to cells in tissue sections. Using a number of analyses to ascertain by what criteria porcine chromosomes were positioned in interphase nuclei; we found a correlation with DNA content.

  5. An ex Vivo Model for Evaluating Blood-Brain Barrier Permeability, Efflux, and Drug Metabolism.

    Science.gov (United States)

    Hellman, Karin; Aadal Nielsen, Peter; Ek, Fredrik; Olsson, Roger

    2016-05-18

    The metabolism of drugs in the brain is difficult to study in most species because of enzymatic instability in vitro and interference from peripheral metabolism in vivo. A locust ex vivo model that combines brain barrier penetration, efflux, metabolism, and analysis of the unbound fraction in intact brains was evaluated using known drugs. Clozapine was analyzed, and its major metabolites, clozapine N-oxide (CNO) and N-desmethylclozapine (NDMC), were identified and quantified. The back-transformation of CNO into clozapine observed in humans was also observed in locusts. In addition, risperidone, citalopram, fluoxetine, and haloperidol were studied, and one preselected metabolite for each drug was analyzed, identified, and quantified. Metabolite identification studies of clozapine and midazolam showed that the locust brain was highly metabolically active, and 18 and 14 metabolites, respectively, were identified. The unbound drug fraction of clozapine, NDMC, carbamazepine, and risperidone was analyzed. In addition, coadministration of drugs with verapamil or fluvoxamine was performed to evaluate drug-drug interactions in all setups. All findings correlated well with the data in the literature for mammals except for the stated fact that CNO is a highly blood-brain barrier permeant compound. Overall, the experiments indicated that invertebrates might be useful for screening of blood-brain barrier permeation, efflux, metabolism, and analysis of the unbound fraction of drugs in the brain in early drug discovery. PMID:26930271

  6. Ex-vivo evaluation of gene therapy vectors in human pancreatic (cancer) tissue slices

    Institute of Scientific and Technical Information of China (English)

    Michael A van Geer; Koert FD Kuhlmann; Conny T Bakker; Fibo JW ten Kate; Ronald PJ Oude Elferink; Piter J Bosma

    2009-01-01

    AIM: To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions.METHODS: Human pancreatic tissue samples (malignant and normal) were obtained from surgical specimens and processed immediately to tissue slices.Tissue slices were cultured ex vivo for 1-6 d in an incubator using 95% O2. Slices were subsequently analyzed for viability and morphology. In addition the slices were incubated with different viral vectors expressing the repor ter genes GFP or DsRed.Expression of these reporter genes was measured at 72 h after infection.RESULTS: With the Krumdieck tissue slicer, uniform slices could be generated from pancreatic tissue but only upon embedding the tissue in 3% low melting agarose. Immunohistological examination showed the presence of all pancreatic cell types. Pancreatic normal and cancer tissue slices could be cultured for up to 6 d, while retaining viability and a moderate to good morphology. Reporter gene expression indicated that the slices could be infected and transduced efficiently by adenoviral vectors and by adeno associated viral vectors, whereas transduction with lentiviral vectors was limited. For the adenoviral vector, the transduction seemed limited to the peripheral layers of the explants.CONCLUSION: The presented sys tem al lows reproducible processing of minimal amounts of pancreatic tissue into slices uniform in size, suitable for pre-clinical evaluation of gene therapy vectors.

  7. Resident Education in Principles and Technique of Bowel Surgery Using an Ex-Vivo Porcine Model

    Directory of Open Access Journals (Sweden)

    M. Bijoy Thomas

    2010-01-01

    Full Text Available Objective. improve competency of residents with lysis of adhesion (LOA and bowel surgery using a porcine model. Study Design. Pig bowel was removed at time of an anatomy laboratory, cleansed, and used to demonstrate surgical techniques and principles of LOA, repair of enterotomy, bowel resection, and anastomosis. Participants were surveyed pre- and posttraining session using 10 point Likert scale. Results. Thirty one residents at varying levels of training participated. After the training session, there was a significant improvement noted in mean scores for comfort level with LOA (6.3 versus 7.7, =.007, comfort level with enterotomy repair (2.8 versus 6.4, <.0001, understanding principles of LOA (5.0 versus 7.7, <.0001, understanding principles of enterotomy repair (3.5 versus 7.0, <.0001, and familiarity with instruments used (5.8 versus 7.3, =.01. Conclusion. Training sessions using ex-vivo porcine model improve resident perception of knowledge and comfort with LOA and enterotomy repair.

  8. Virulence diversity among bacteremic Aeromonas isolates: ex vivo, animal, and clinical evidences.

    Directory of Open Access Journals (Sweden)

    Po-Lin Chen

    Full Text Available BACKGROUND: The objective of this study was to compare virulence among different Aeromonas species causing bloodstream infections. METHODOLOGY/PRINCIPAL FINDINGS: Nine of four species of Aeromonas blood isolates, including A. dhakensis, A. hydrophila, A. veronii and A. caviae were randomly selected for analysis. The species was identified by the DNA sequence matching of rpoD. Clinically, the patients with A. dhakensis bacteremia had a higher sepsis-related mortality rate than those with other species (37.5% vs. 0%, P = 0.028. Virulence of different Aeromonas species were tested in C. elegans, mouse fibroblast C2C12 cell line and BALB/c mice models. C. elegans fed with A. dhakensis and A. caviae had the lowest and highest survival rates compared with other species, respectively (all P values <0.0001. A. dhakensis isolates also exhibited more cytotoxicity in C2C12 cell line (all P values <0.0001. Fourteen-day survival rate of mice intramuscularly inoculated with A. dhakensis was lower than that of other species (all P values <0.0001. Hemolytic activity and several virulence factor genes were rarely detected in the A. caviae isolates. CONCLUSIONS/SIGNIFICANCE: Clinical data, ex vivo experiments, and animal studies suggest there is virulence variation among clinically important Aeromonas species.

  9. Laparoscopic Nephrectomy, Ex Vivo Partial Nephrectomy, and Autotransplantation for the Treatment of Complex Renal Masses

    Directory of Open Access Journals (Sweden)

    Jasmir Gopal Nayak

    2014-01-01

    Full Text Available In the contemporary era of minimally invasive surgery, very few T1/T2 renal lesions are not amenable to nephron-sparing surgery. However, centrally located lesions continue to pose a clinical dilemma. We sought to describe our local experience with three cases of laparoscopic nephrectomy, ex vivo partial nephrectomy, and autotransplantation. Laparoscopic donor nephrectomy was performed followed by immediate renal cooling and perfusion with isotonic solution. Back-table partial nephrectomy, renorrhaphy, and autotransplantation were then performed. Mean warm ischemia (WIT and cold ischemic times (CIT were 2 and 39 minutes, respectively. Average blood loss was 267 mL. All patients preserved their renal function postoperatively. Final pathology confirmed pT1, clear cell renal cell carcinoma with negative margins in all. All are disease free at up to 39 months follow-up with stable renal function. In conclusion, the described approach remains a viable option for the treatment of complex renal masses preserving oncological control and renal function.

  10. Computed Tomographic Tenography of Normal Equine Digital Flexor Tendon Sheath: An Ex Vivo Study

    Directory of Open Access Journals (Sweden)

    Luca Lacitignola

    2015-01-01

    Full Text Available Aim of this study was to document the normal computed tomographic tenography findings of digital flexor tendon sheath. Six ex vivo normal equine forelimbs were used. An axial approach was used to inject 185 mg/mL of iopamidol in a total volume of 60 mL into the digital flexor tendon sheaths. Single-slice helical scans, with 5 mm thickness, spaced every 3 mm, for a pitch of 0.6, and with bone algorithm reconstruction, were performed before and after injections of contrast medium. To obtain better image quality for multiplanar reconstruction and 3D reformatting, postprocessing retroreconstruction was performed to reduce the images to submillimetre thickness. Computed tomographic tenography of digital flexor tendon sheaths could visualize the following main tendon structures for every forelimb in contrast-enhanced images as low densities surrounded by high densities: superficial digital flexor tendon, deep digital flexor tendon, manica flexoria, mesotendons, and synovial recess. Results of this study suggest that computed tomographic tenography can be used with accuracy and sensitivity to evaluate the common disorders of the equine digital flexor tendon sheath and the intrathecal structures.

  11. Ex vivo hepatic venography for hepatocellular carcinoma in livers explanted for liver transplantation

    Directory of Open Access Journals (Sweden)

    Takatsuki Mitsuhisa

    2011-09-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC is supposed to have a venous drainage system to a portal vein, which makes intrahepatic metastasis possible. However, the mechanism of extrahepatic recurrence, including the possibility of a direct route to the systemic circulation from the HCC nodules, remains unclear. Therefore, we performed retrograde hepatic venography for HCC in livers that had been explanted for liver transplantation in order to explore the possible direct connection between the hepatic vein and HCC nodules. Methods Of 105 living-donor liver transplantations (LDLT performed up to July, 2009 at the Department of Surgery, Nagasaki University Hospital, dynamic hepatic venography was performed with contrast media under fluoroscopy for the most recent 13 cases with HCC. The presence of a tumor stain for each HCC case was evaluated and compared with the histological findings of HCC. Results Hepatic venography revealed a tumor stain in 2 of 13 cases (15%. Neither showed any microscopic tumor invasion of HCC into the hepatic vein. In the other 11 cases, there were 4 microscopic portal venous invasions and 2 microscopic hepatic venous invasions. No patients have shown HCC recurrence in follow-up (median period, 13 months. Conclusion Using ex vivo hepatic venography, a direct connection to the hepatic vein from HCC in whole liver was revealed in 2 cases without demonstrated histopathological invasion to hepatic vein for the first time in the literature. The finding suggests that there is direct spillage of HCC cells into the systemic circulation via hepatic vein.

  12. The ex vivo neurotoxic, myotoxic and cardiotoxic activity of cucurbituril-based macrocyclic drug delivery vehicles.

    Science.gov (United States)

    Oun, Rabbab; Floriano, Rafael S; Isaacs, Lyle; Rowan, Edward G; Wheate, Nial J

    2014-11-01

    The cucurbituril family of drug delivery vehicles have been examined for their tissue specific toxicity using ex vivo models. Cucurbit[6]uril (CB[6]), cucurbit[7]uril (CB[7]) and the linear cucurbituril-derivative Motor2 were examined for their neuro-, myo- and cardiotoxic activity and compared with β-cyclodextrin. The protective effect of drug encapsulation by CB[7] was also examined on the platinum-based anticancer drug cisplatin. The results show that none of the cucurbiturils have statistically measurable neurotoxicity as measured using mouse sciatic nerve compound action potential. Cucurbituril myotoxicity was measured by nerve-muscle force of contraction through chemical and electrical stimulation. Motor2 was found to display no myotoxicity, whereas both CB[6] and CB[7] showed myotoxic activity via a presynaptic effect. Finally, cardiotoxicity, which was measured by changes in the rate and force of right and left atria contraction, was observed for all three cucurbiturils. Free cisplatin displays neuro-, myo- and cardiotoxic activity, consistent with the side-effects seen in the clinic. Whilst CB[7] had no effect on the level of cisplatin's neurotoxic activity, drug encapsulation within the macrocycle had a marked reduction in both the drug's myo- and cardiotoxic activity. Overall the results are consistent with the relative lack of toxicity displayed by these macrocycles in whole animal acute systemic toxicity studies and indicate continued potential of cucurbiturils as drug delivery vehicles for the reduction of the side effects associated with platinum-based chemotherapy.

  13. Ex vivo adenoviral vector gene delivery results in decreased vector-associated inflammation pre- and post-lung transplantation in the pig.

    Science.gov (United States)

    Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Rubacha, Matthew; Koike, Terumoto; Chun, Yi-Min; Hu, Jim; Waddell, Thomas K; Hwang, David M; Liu, Mingyao; Keshavjee, Shaf

    2012-06-01

    Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function. PMID:22453765

  14. Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

    Science.gov (United States)

    Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Rubacha, Matthew; Koike, Terumoto; Chun, Yi-Min; Hu, Jim; Waddell, Thomas K; Hwang, David M; Liu, Mingyao; Keshavjee, Shaf

    2012-01-01

    Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function. PMID:22453765

  15. Terbinafine hydrochloride nanovesicular gel: In vitro characterization, ex vivo permeation and clinical investigation.

    Science.gov (United States)

    AbdelSamie, Sara M; Kamel, Amany O; Sammour, Omaima A; Ibrahim, Shady M

    2016-06-10

    In this work, nanovesicular chitosan gels were prepared for dermal delivery of terbinafine hydrochloride (TBN HCl). Ethosomes and vesicles containing different types of penetration enhancers (PEs) viz. Terpenes (cineole and limonene), labrasol and transcutol were developed. The prepared vesicles were evaluated for physical characteristics as well as skin interaction. The selected vesicles were incorporated into chitosan gel. An in vivo animal study was done on rat induced superficial Candida infection model. Moreover, randomized double blind clinical study was done on patients to compare the effect of the selected nanovesicular gel against the market product. Results showed the formation of nearly spherical, mostly deformable vesicular systems with size range of 95.5-530nm, zeta potential range of -0.1 to 15mV and entrapment efficiency range of 20-96.7%. Penetration enhancer vesicles (PEVs) prepared with 4% limonene (ELI4) showed the highest percent of drug deposition in the skin (53%) and the highest local accumulation efficiency value (35.3). In vivo animal study showed that the lowest fungal burden produced with ELI4 chitosan gel. Clinical studies showed cure rate of 86% within 7days treatment in case of limonene nanovesicular gel compared to 20% for market product (Lamisil® cream). PMID:27072432

  16. An Ex Vivo Imaging Pipeline for Producing High- Quality and High-Resolution Diffusion-Weighted Imaging Datasets

    DEFF Research Database (Denmark)

    Dyrby, Tim Bjørn; Baaré, William F.C.; Alexander, Daniel C.;

    2011-01-01

    for consistent reconstruction of fiber directions; (iii) diffusivity measures in the postmortem brain tissue were stable over a 3‐year period. On the basis of these results, we established an optimized ex vivo pipeline for high‐quality and high‐resolution DWI. The pipeline produces DWI data sets with a high......Diffusion tensor (DT) imaging and related multifiber reconstruction algorithms allow the study of in vivo microstructure and, by means of tractography, structural connectivity. Although reconstruction algorithms are promising imaging tools, high‐quality diffusion‐weighted imaging (DWI) datasets...... complexity, to establish an ex vivo imaging pipeline for generating high‐quality DWI datasets. Perfusion fixation ensured that tissue characteristics were comparable to in vivo conditions. There were three main results: (i) heat conduction and unstable tissue mechanics accounted for time‐varying artefacts...

  17. Accurate in vivo dielectric properties of liver from 500 MHz to 40 GHz and their correlation to ex vivo measurements.

    Science.gov (United States)

    Farrugia, L; Wismayer, P Schembri; Mangion, L Zammit; Sammut, C V

    2016-01-01

    In this article, we report on the characterization of the dielectric properties of in vivo rat liver at 36.4°C from 500 MHz up to 40 GHz with less than 5% uncertainty. The measured data were fitted to a Cole-Cole model and dielectric parameters are presented together with their respective 95% confidence interval. The root mean square error is 0.42. Moreover, ex vivo measurements were conducted in situ at 1, 2, 4 and 6 min after animal death and are compared to in vivo measurements. The results show that immediate changes in [Formula: see text]and [Formula: see text] are within experimental uncertainty, and therefore changes between in vivo and published ex vivo dielectric properties can be attributed to tissue hydration.

  18. Visualization of luminal thrombosis and mural Iron accumulation in giant aneurysms with Ex vivo 4.7T magnetic resonance imaging

    Directory of Open Access Journals (Sweden)

    Petri Honkanen

    2014-01-01

    Full Text Available Background: Better diagnostic tools to identify rupture-prone saccular intracranial aneurysms (sIA are needed. Inflammation and luminal thrombus associate with degeneration and rupture of the sIA wall. Iron-uptake has been detected in the inflammatory cells of the sIA wall and thrombus is the likely source of this iron. We investigated ex vivo the use of magnetic resonance imaging (MRI to detect iron accumulation and luminal thrombus in giant sIAs. Methods: Giant sIAs (n = 3 were acquired from microsurgical operations, fixed with formalin, embedded in agar and imaged at 4.7T. Samples were sectioned maintaining the orientation of the axial plane of MRI scans, and stained (hematoxylin-eosin and Prussian blue. Results: All three giant sIAs showed a degenerated hypocellular wall with both mural and adventitial iron accumulation and displayed different degrees of luminal thrombus formation and thrombus organization. Signal intensity varied within the same sIA wall and associated with iron accumulation in all tested sequences. Wall areas with iron accumulation had significantly lower signal to noise ratio (SNR compared with areas without iron accumulation (P = 0.002. Fresh and organizing thrombus differed in their MRI presentation and differed in signal intensity of the aneurysm wall (P = 0.027. Conclusion: MRI can detect ex vivo the accumulation of iron in giant sIA wall, as well as fresh and organizing luminal thrombus. These features have been previously associated with fragile, rupture-prone aneurysm wall. Further studies of iron accumulation as a marker of rupture-prone aneurysm wall are needed.

  19. Heating drug delivery to vascular wall with Rhodamine B and fluorescence labeled Paclitaxel ranging 50 to 70°C: ex vivo study

    Science.gov (United States)

    Homma, R.; Shinozuka, M.; Shimazaki, N.; Arai, T.

    2016-03-01

    We studied heating drug delivery to vascular wall with Rhodamine B ranging 50 to 70°C ex vivo study. Porcine carotid artery was dipped in the heated Rhodamine B solution in 15 s and then cooled by 37°C saline. Rhodamine B concentration distribution in the vascular wall cross-section was measured by a fluorescence microscope using 550 nm for excitation and 620 nm emission for fluorescence detection. The total amount of measured fluorescence in the vascular wall was calculated as a indication of delivered Rhodamine B quantity. The delivered Rhodamine B quantity was increased with increasing heating temperature with 50 to 70°C. In the cases of 60 to 70°C heating, the delivered Rhodamine B quantity was 3.1 to 23.3 fold by that of 37°C. Defined penetration depth of the delivered Rhodamine B in the vascular wall was also significantly increased with 65°C and 70°C heating. We also studied heating drug delivery to the vascular wall with fluorescence labeled Paclitaxel with 70°C in 15 s and 60 s heating ex vivo. In both contact duration, the delivered Paclitaxel quantity was increased. To understand these drug delivery enhancement effects, we investigated the vascular cross-sectional structure change by the heating. Some holes over 50 nm in diameter appeared on the internal elastic lamina with 70°C heating. We prospected that vascular surface structure change by the heating might enhance drug delivery to the vascular wall.

  20. Laser-guided cervical selective nerve root block with the Dyna-CT: initial experience of three-dimensional puncture planning with an ex-vivo model.

    Directory of Open Access Journals (Sweden)

    Miriam I E Freundt

    Full Text Available BACKGROUND: Cervical selective nerve root block (CSNRB is a well-established, minimally invasive procedure to treat radicular cervical pain. However, the procedure is technically challenging and might lead to major complications. The objective of this study was to evaluate the feasibility of a three-dimensional puncture planning and two-dimensional laser-guidance system for CSNRB in an ex-vivo model. METHODS: Dyna-CT of the cervical spine of an ex-vivo lamb model was performed with the Artis Zee® Ceiling (Siemens Medical Solutions, Erlangen, Germany to acquire multiplanar reconstruction images. 15 cervical nerve root punctures were planned and conducted with the syngo iGuide® laser-guidance system. Needle tip location and contrast dye distribution were analyzed by two independent investigators. Procedural, planning, and fluoroscopic time, tract length, and dose area product (DAP were acquired for each puncture. RESULTS: All 15 punctures were rated as successful with 12 punctures on the first attempt. Total procedural time was approximately 5 minutes. Mean planning time for the puncture was 2.03 (±0.39 min. Mean puncture time was 2.16 (±0.32 min, while mean fluoroscopy time was 0.17 (±0.06 min. Mean tract length was 2.68 (±0.23 cm. Mean total DAP was 397.45 (±15.63 µGy m(2. CONCLUSION: CSNRB performed with Dyna-CT and the tested laser guidance system is feasible. 3D pre-puncture planning is easy and fast and the laser-guiding system ensures very accurate and intuitive puncture control.

  1. SEDDS of gliclazide: Preparation and characterization by in-vitro, ex-vivo and in-vivo techniques

    OpenAIRE

    Nipun, Tanzina Sharmin; Ashraful Islam, S.M.

    2013-01-01

    In the study, self emulsifying drug delivery system (SEDDS) of gliclazide, a poorly soluble drug, was developed and evaluated by in-vitro, ex-vivo and in-vivo techniques. Oil and surfactant were screened out according to their solubilizing capacity. Among the tested components Transcutol HP and Tween-80 showed good solubilizing capacity. These two components were used in different ratios to prepare gliclazide SEDDS. The SEDDS formulations were transparent and clear. Droplet size of the emulsi...

  2. Comparison between Different Methods for Biomechanical Assessment of Ex Vivo Fracture Callus Stiffness in Small Animal Bone Healing Studies

    OpenAIRE

    Malte Steiner; David Volkheimer; Nicholaus Meyers; Tim Wehner; Hans-Joachim Wilke; Lutz Claes; Anita Ignatius

    2015-01-01

    For ex vivo measurements of fracture callus stiffness in small animals, different test methods, such as torsion or bending tests, are established. Each method provides advantages and disadvantages, and it is still debated which of those is most sensitive to experimental conditions (i.e. specimen alignment, directional dependency, asymmetric behavior). The aim of this study was to experimentally compare six different testing methods regarding their robustness against experimental errors. There...

  3. Evaluation of an Indigenously Prepared Herbal Extract (EndoPam) as an Antimicrobial Endodontic Irrigant: An Ex Vivo Study

    OpenAIRE

    Mathew, Jain; Pathrose, Sonia; Kottoor, Jojo; Karaththodiyil, Ranjith; Alani, Mathew; Mathew, Joy

    2015-01-01

    Backgroundg: Root canal irrigation plays a pivotal role in endodontics. Constant increase in antibiotic resistance and side effects caused by synthetic irrigants has shifted the research toward developing herbal alternatives. The current study aims to assess the ex vivo effectiveness of an indigenously prepared herbal extract “EndoPam” and compare it with the conventional endodontic irrigants for disinfection of root canals infected with Enterococcus faecalis. Materials and Methods: As a prel...

  4. Scavenging and antioxidant properties of different grape cultivars against ionizing radiation-induced liver damage ex vivo.

    Science.gov (United States)

    Singha, Indrani; Das, Subir Kumar

    2016-04-01

    Ionizing radiation (IR) has become an integral part of the modern medicine--both for diagnosis as well as therapy. However, normal tissues or even distant cells also suffer IR-induced free radical insult. It may be more damaging in longer term than direct radiation exposure. Antioxidants provide protection against IR-induced damage. Grapes are the richest source of antioxidants. Here, we assessed the scavenging properties of four grape (Vitis vinifera) cultivars, namely Flame seedless (Black), Kishmish chorni (Black with reddish brown), Red globe (Red) and Thompson seedless mutant (Green), and also evaluated their protective action against γ-radiation-induced oxidative stress in liver tissue ex vivo. The scavenging abilities of grape seeds [2,2-diphenyl-1-picrylhydrazyl (DPPH) (IC₅₀ = 0.008 ± 0.001 mg/mL), hydrogen peroxide (IC₅₀ = 0.49 to 0.8 mg/mL), hydroxyl radicals (IC₅₀ = 0.08 ± 0.008 mg/mL), and nitric oxide (IC₅₀ = 0.8 ± 0.08 mg/mL)] were higher than that of skin or pulp. Gamma (γ) radiation exposure to sliced liver tissues ex vivo from goat, @ 6 Gy significantly (P antioxidant potential compared to skin or pulp extracts of different grape cultivars against oxidative damage by ionizing radiation (6 Gy, 10 Gy and 16 Gy) in sliced liver tissues ex vivo. Grape extracts at higher concentration (10 mg extract/g liver tissue) showed stronger antioxidant potential against lower dose (6 Gy) of ionizing radiation. Our results suggest that grape extracts could serve as a potential source of natural antioxidant against lower doses of IR-induced oxidative stress in liver extracts ex vivo. PMID:27295925

  5. In vitro-/ ex vivo-Hautmodelle zur Erfassung der Reaktion auf externe Reize sowie der Wirkung einer antiinflammatorischen Behandlung

    OpenAIRE

    Zimmermann, Esther

    2008-01-01

    Dermatoses are wide-spread. Beside physical discomforts like pain and itches, often psychosocial problems are caused and the quality of life of patients is strongly affected. In addition, the treatment of dermatoses is still unsatisfying in many cases, as only symptoms are considered and results are moderate. Researches in pathophysiology and therapeutics are therefore of great need. An important tool thereby are in vitro / ex vivo skin models. They allow to carry out experi...

  6. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    Science.gov (United States)

    Chou, Ming-Li; Burnouf, Thierry; Wang, Tsung-Jen

    2014-01-01

    Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml) and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml) neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices and regulatory

  7. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    Directory of Open Access Journals (Sweden)

    Ming-Li Chou

    Full Text Available Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices

  8. Improvements of Venous Tone with Pycnogenol in Chronic Venous Insufficiency: An Ex Vivo Study on Venous Segments

    OpenAIRE

    Belcaro, Gianni; Dugall, Mark; Luzzi, Roberta; Hosoi, M.; Corsi, Marcello

    2014-01-01

    This study evaluated the stretching and dilatation of venous segments ex vivo in subjects with primary varicose veins in comparison with comparable segments from subjects that used the supplement Pycnogenol (150 mg/d) for 3 months before surgery. Subjects with varicose veins and chronic venous insufficiency voluntarily used Pycnogenol for a period of at least 3 months. The segments of veins removed with surgery (in 30 subjects that had used Pycnogenol and in 10 comparable control subjects tha...

  9. Towards a PBMC “virogram assay” for precision medicine: concordance between ex vivo and in vivo viral infection transcriptomes

    Science.gov (United States)

    Gardeux, Vincent; Bosco, Anthony; Li, Jianrong; Halonen, Marilyn J.; Jackson, Daniel; Martinez, Fernando D.; Lussier, Yves A.

    2016-01-01

    Background Understanding individual patient host-response to viruses is key to designing optimal personalized therapy. Unsurprisingly, in vivo human experimentation to understand individualized dynamic response of the transcriptome to viruses are rarely studied because of the obviously limitations stemming from ethical considerations of the clinical risk. Objective In this rhinovirus study, we first hypothesized that ex vivo human cells response to virus can serve as proxy for otherwise controversial in vivo human experimentation. We further hypothesized that the N-of-1-pathways framework, previously validated in cancer, can be effective in understanding the more subtle individual transcriptomic response to viral infection. Method N-of-1-pathways computes a significance score for a given list of gene sets at the patient level, using merely the ‘omics profiles of two paired samples as input. We extracted the peripheral blood mononuclear cells (PBMC) of four human subjects, aliquoted in two paired samples, one subjected to ex vivo rhinovirus infection. Their dysregulated genes and pathways were then compared to those of 9 human subjects prior and after intranasal inoculation in vivo with rhinovirus. Additionally, we developed the Similarity Venn Diagram, a novel visualization method that goes beyond conventional overlap to show the similarity between two sets of qualitative measures. Results We evaluated the individual N-of-1-pathways results using two established cohort-based methods: GSEA and enrichment of differentially expressed genes. Similarity Venn Diagrams and individual patient ROC curves illustrate and quantify that the in vivo dysregulation is recapitulated ex vivo both at the gene and pathway level (p-values≤0.004). Conclusion We established the first evidence that an interpretable dynamic transcriptome metric, conducted as an ex vivo assays for a single subject, has the potential to predict individualized response to infectious disease without the

  10. Light cola drink is less erosive than the regular one: an in situ/ex vivo study

    OpenAIRE

    Rios, D.; Honório, H M; Magalhães, A C; Wiegand, A.; de Andrade Moreira Machado, M A; Buzalaf, M.A.R.

    2009-01-01

    OBJECTIVE: This in situ/ex vivo study assessed the erosive potential of a light cola drink when compared to a regular one. METHODS: During 2 experimental 14-days crossover phases, eight volunteers wore palatal devices with 2 human enamel blocks. The groups under study were: group light, erosive challenge with light cola drink and group regular, erosive challenge with regular cola drink. During 14 days, erosive challenges were performed extraorally 3X/day. In each challenge, the device was imm...

  11. Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo

    OpenAIRE

    Kalliopi Ioannidou; Anderson, Kurt I; David Strachan; Edgar, Julia M.; Barnett, Susan C.

    2012-01-01

    Background Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualize th...

  12. Fibre optic sensors for temperature and pressure monitoring in laser ablation: experiments on ex-vivo animal model

    Science.gov (United States)

    Tosi, Daniele; Saccomandi, Paola; Schena, Emiliano; Duraibabu, Dinesh B.; Poeggel, Sven; Adilzhan, Abzal; Aliakhmet, Kamilla; Silvestri, Sergio; Leen, Gabriel; Lewis, Elfed

    2016-05-01

    Optical fibre sensors have been applied to perform biophysical measurement in ex-vivo laser ablation (LA), on pancreas animal phantom. Experiments have been performed using Fibre Bragg Grating (FBG) arrays for spatially resolved temperature detection, and an all-glass Extrinsic Fabry-Perot Interferometer (EFPI) for pressure measurement. Results using a Nd:YAG laser source as ablation device, are presented and discussed.

  13. Recent advances in haploidentical hematopoietic stem cell transplantation using ex vivo T cell-depleted graft in children and adolescents.

    Science.gov (United States)

    Im, Ho Joon; Koh, Kyung-Nam; Seo, Jong Jin

    2016-03-01

    Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for children and adolescents with various malignant and non-malignant diseases. While human leukocyte antigen (HLA)-identical sibling donor is the preferred choice, matched unrelated volunteer donor is another realistic option for successful HSCT. Unfortunately, it is not always possible to find a HLA-matched donor for patients requiring HSCT, leading to a considerable number of deaths of patients without undergoing transplantation. Alternatively, allogeneic HSCT from haploidentical family members could provide donors for virtually all patients who need HSCT. Although the early attempts at allogeneic HSCT from haploidentical family donor (HFD) were disappointing, recent advances in the effective ex vivo depletion of T cells or unmanipulated in vivo regulation of T cells, better supportive care, and optimal conditioning regimens have significantly improved the outcomes of haploidentical HSCT. The ex vivo techniques used to remove T cells have evolved from the selection of CD34(+) hematopoietic stem cell progenitors to the depletion of CD3(+) cells, and more recently to the depletion of αβ(+) T cells. The recent emerging evidence for ex vivo T cell-depleted haploidentical HSCT has provided additional therapeutic options for pediatric patients with diseases curable by HSCT but has not found a suitable related or unrelated donor. This review discusses recent advances in haploidentical HSCT, focusing on transplant using ex vivo T cell-depleted grafts. In addition, our experiences with this novel approach for the treatment of pediatric patients with malignant and non-malignant diseases are described.

  14. Cryopreserved Ex Vivo-Expanded Allogeneic Myeloid Progenitor Cell Product Protects Neutropenic Mice From a Lethal Fungal Infection.

    Science.gov (United States)

    Domen, Jos; Christensen, Julie L; Gille, Daphne; Smith-Berdan, Stephanie; Fong, Timothy; Brown, Janice M Y; Sedello, Anna K

    2016-01-01

    Severe neutropenia induced by chemotherapy or conditioning for hematopoietic cell transplantation often results in morbidity and mortality due to infection by opportunistic pathogens. A system has been developed to generate ex vivo-expanded mouse myeloid progenitor cells (mMPCs) that produce functional neutrophils in vivo upon transplantation in a pathogen challenge model. It has previously been demonstrated that transplantation of large numbers of freshly isolated myeloid progenitors from a single donor provides survival benefit in radiation-induced neutropenic mice. In the present work, an ex vivo-expanded and cryopreserved mMPC product generated from an allogeneic donor pool retains protective activity in vivo in a lethal fungal infection model. Infusion of the allogeneic pooled mMPC product is effective in preventing death from invasive Aspergillus fumigatus in neutropenic animals, and protection is dose dependent. Cell progeny from the mMPC product is detected in the bone marrow, spleen, blood, and liver by flow cytometry 1 week postinfusion but is no longer evident in most animals 4 weeks posttransplant. In this model, the ex vivo-generated pooled allogeneic mMPC product (i) expands and differentiates in vivo; (ii) is functional and prevents death from invasive fungal infection; and (iii) does not permanently engraft or cause allosensitization. These data suggest that an analogous ex vivo-expanded human myeloid progenitor cell product may be an effective off-the-shelf bridging therapy for the infectious complications that develop during hematopoietic recovery following hematopoietic cell transplantation or intensive chemotherapy. PMID:25812169

  15. An Ex Vivo Model in Human Femoral Heads for Histopathological Study and Resonance Frequency Analysis of Dental Implant Primary Stability

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    Pedro Hernández-Cortés

    2014-01-01

    Full Text Available Objective. This study was designed to explore relationships of resonance frequency analysis (RFA—assessed implant stability (ISQ values with bone morphometric parameters and bone quality in an ex vivo model of dental implants placed in human femoral heads and to evaluate the usefulness of this model for dental implant studies. Material and Methods. This ex vivo study included femoral heads from 17 patients undergoing surgery for femoral neck fracture due to osteoporosis (OP (n=7 or for total prosthesis joint replacement due to severe hip osteoarthrosis (OA (n=10. Sixty 4.5×13 mm Dentsply Astra implants were placed, followed by RFA. CD44 immunohistochemical analysis for osteocytes was also carried out. Results. As expected, the analysis yielded significant effects of femoral head type (OA versus OA (P0.5 in all cases, and no significant differences in ISQ values were found as a function of the length or area of the cortical layer (both P>0.08. Conclusion. Although RFA-determined ISQ values are not correlated with morphometric parameters, they can discriminate bone quality (OP versus OA. This ex vivo model is useful for dental implant studies.

  16. In Vivo Tracking of Murine Adipose Tissue-Derived Multipotent Adult Stem Cells and Ex Vivo Cross-Validation

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    Chiara Garrovo

    2013-01-01

    Full Text Available Stem cells are characterized by the ability to renew themselves and to differentiate into specialized cell types, while stem cell therapy is believed to treat a number of different human diseases through either cell regeneration or paracrine effects. Herein, an in vivo and ex vivo near infrared time domain (NIR TD optical imaging study was undertaken to evaluate the migratory ability of murine adipose tissue-derived multipotent adult stem cells [mAT-MASC] after intramuscular injection in mice. In vivo NIR TD optical imaging data analysis showed a migration of DiD-labelled mAT-MASC in the leg opposite the injection site, which was confirmed by a fibered confocal microendoscopy system. Ex vivo NIR TD optical imaging results showed a systemic distribution of labelled cells. Considering a potential microenvironmental contamination, a cross-validation study by multimodality approaches was followed: mAT-MASC were isolated from male mice expressing constitutively eGFP, which was detectable using techniques of immunofluorescence and qPCR. Y-chromosome positive cells, injected into wild-type female recipients, were detected by FISH. Cross-validation confirmed the data obtained by in vivo/ex vivo TD optical imaging analysis. In summary, our data demonstrates the usefulness of NIR TD optical imaging in tracking delivered cells, giving insights into the migratory properties of the injected cells.

  17. Numerical and ex vivo studies of a bioprobe developed for laser-induced thermotherapy (LITT) in contact with liver tissue.

    Science.gov (United States)

    Chartier, T; Carpentier, O; Genestie, B; Hornez, J-C; Monchau, F

    2016-08-01

    This work is based on the production of a bioprobe that is compatible with magnetic resonance imaging (MRI) for laser-induced thermotherapy (LITT) in liver cancer laser therapy. This probe is made of an alumina tube (3-mm diameter) in which an optical fibre is centred and fixed. A shooting window (20mm) is created using a mechanical rectifier. The device is then consolidated by the injection of a transparent and heat-resistant resin. Through numerical modelling, the thermal power damping of the laser source is evaluated as well as the propagation of the heat in the ex vivo liver tissue according to different heating scenarios. These analyses allow for an estimation of the irradiated volume. Ex vivo tests were performed on bovine liver to confirm the adequacy of the bioprobe for LITT and of the irradiated volumes predicted by the numerical model. There was a difference of 8% between the simulations and ex vivo experiments. The pulsed mode heating scenario was the most effective under the experimental conditions. PMID:27212211

  18. Ex vivo and in silico feasibility study of monitoring electric field distribution in tissue during electroporation based treatments.

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    Matej Kranjc

    Full Text Available Magnetic resonance electrical impedance tomography (MREIT was recently proposed for determining electric field distribution during electroporation in which cell membrane permeability is temporary increased by application of an external high electric field. The method was already successfully applied for reconstruction of electric field distribution in agar phantoms. Before the next step towards in vivo experiments is taken, monitoring of electric field distribution during electroporation of ex vivo tissue ex vivo and feasibility for its use in electroporation based treatments needed to be evaluated. Sequences of high voltage pulses were applied to chicken liver tissue in order to expose it to electric field which was measured by means of MREIT. MREIT was also evaluated for its use in electroporation based treatments by calculating electric field distribution for two regions, the tumor and the tumor-liver region, in a numerical model based on data obtained from clinical study on electrochemotherapy treatment of deep-seated tumors. Electric field distribution inside tissue was successfully measured ex vivo using MREIT and significant changes of tissue electrical conductivity were observed in the region of the highest electric field. A good agreement was obtained between the electric field distribution obtained by MREIT and the actual electric field distribution in evaluated regions of a numerical model, suggesting that implementation of MREIT could thus enable efficient detection of areas with insufficient electric field coverage during electroporation based treatments, thus assuring the effectiveness of the treatment.

  19. A single epidermal stem cell strategy for safe ex vivo gene therapy.

    Science.gov (United States)

    Droz-Georget Lathion, Stéphanie; Rochat, Ariane; Knott, Graham; Recchia, Alessandra; Martinet, Danielle; Benmohammed, Sara; Grasset, Nicolas; Zaffalon, Andrea; Besuchet Schmutz, Nathalie; Savioz-Dayer, Emmanuelle; Beckmann, Jacques Samuel; Rougemont, Jacques; Mavilio, Fulvio; Barrandon, Yann

    2015-02-27

    There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self-inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non-blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole-genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long-term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by-passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene-editing technologies like zinc finger nucleases, TALENs and homologous recombination for next-generation gene therapy.

  20. An ex-vivo human intestinal model to study Entamoeba histolytica pathogenesis.

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    Devendra Bansal

    Full Text Available Amoebiasis (a human intestinal infection affecting 50 million people every year is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5, which have major roles in cell death, adhesion (to target cells or mucus and mucus degradation, respectively were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion.

  1. LANTCET: laser nanotechnology for screening and treating tumors ex vivo and in vivo

    Science.gov (United States)

    Lapotko, Dmitri O.; Lukianova-Hleb, Ekaterina Y.; Zhdanok, Sergei A.; Hafner, Jason H.; Rostro, Betty C.; Scully, Peter; Konopleva, Marina; Andreeff, Michael; Li, Chun; Hanna, Ehab Y.; Myers, Jeffrey N.; Oraevsky, Alexander A.

    2007-06-01

    LANTCET (laser-activated nano-thermolysis as cell elimination technology) was developed for selective detection and destruction of individual tumor cells through generation of photothermal bubbles around clusters of light absorbing gold nanoparticles (nanorods and nanoshells) that are selectively formed in target tumor cells. We have applied bare nanoparticles and their conjugates with cell-specific vectors such as monoclonal antibodies CD33 (specific for Acute Myeloid Leukemia) and C225 (specific for carcinoma cells that express epidermal growth factor -EGF). Clusters were formed by using vector-receptor interactions with further clusterization of nanoparticles due to endocytosis. Formation of clusters was verified directly with optical resonance scattering microscopy and microspectroscopy. LANTCET method was tested in vitro for living cell samples with: (1) model myeloid K562 cells (CD33 positive), (2) primary human bone marrow CD33-positive blast cells from patients with the diagnosis of acute myeloid leukemia, (3) monolayers of living EGF-positive carcinoma cells (Hep-2C), (4) human lymphocytes and red blood cells as normal cells. The LANTCET method was also tested in vivo using rats with experimental polymorphic sarcoma. Photothermal bubbles were generated and detected in vitro with a photothermal microscope equipped with a tunable Ti-Sa pulsed laser. We have found that cluster formation caused an almost 100-fold decrease in the bubble generation threshold of laser pulse fluence in tumor cells compared to the bubble generation threshold for normal cells. The animal tumor that was treated with a single laser pulse showed a necrotic area of diameter close to the pump laser beam diameter and a depth of 1-2 mm. Cell level selectivity of tumor damage with single laser pulse was demonstrated. Combining lightscattering imaging with bubble imaging, we introduced a new image-guided mode of the LANTCET operation for screening and treatment of tumors ex vivo and in vivo.

  2. Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions.

    Science.gov (United States)

    Karkampouna, Sofia; Kloen, Peter; Obdeijn, Miryam C; Riester, Scott M; van Wijnen, Andre J; Kruithof-de Julio, Marianna

    2015-01-01

    Organ fibrosis or "scarring" is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic. To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first "organ" culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

  3. Ex vivo perfusion of the isolated rat small intestine as a novel model of Salmonella enteritis.

    Science.gov (United States)

    Boyle, Erin C; Dombrowsky, Heike; Sarau, Jürgen; Braun, Janin; Aepfelbacher, Martin; Lautenschläger, Ingmar; Grassl, Guntram A

    2016-01-15

    Using an ex vivo perfused rat small intestinal model, we examined pathological changes to the tissue, inflammation induction, as well as dynamic changes to smooth muscle activity, metabolic competence, and luminal fluid accumulation during short-term infection with the enteropathogenic bacteria Salmonella enterica serovar Typhimurium and Yersinia enterocolitica. Although few effects were seen upon Yersinia infection, this system accurately modeled key aspects associated with Salmonella enteritis. Our results confirmed the importance of the Salmonella Pathogenicity Island 1 (SPI1)-encoded type 3 secretion system (T3SS) in pathology, tissue invasion, inflammation induction, and fluid secretion. Novel physiological consequences of Salmonella infection of the small intestine were also identified, namely, SPI-1-dependent vasoconstriction and SPI-1-independent reduction in the digestive and absorptive functions of the epithelium. Importantly, this is the first small animal model that allows for the study of Salmonella-induced fluid secretion. Another major advantage of this model is that one can specifically determine the contribution of resident cell populations. Accordingly, we can conclude that recruited cell populations were not involved in the pathological damage, inflammation induction, fluid accumulation, nutrient absorption deficiency, and vasoconstriction observed. Although fluid loss induced by Salmonella infection is hypothesized to be due to damage caused by recruited neutrophils, our data suggest that bacterial invasion and inflammation induction in resident cell populations are sufficient for fluid loss into the lumen. In summary, this model is a novel and useful tool that allows for detailed examination of the early physiopathological effects of Salmonella infection on the small intestine.

  4. Nonlinear acoustic properties of ex vivo bovine liver and the effects of temperature and denaturation.

    Science.gov (United States)

    Jackson, E J; Coussios, C-C; Cleveland, R O

    2014-06-21

    Thermal ablation by high intensity focused ultrasound (HIFU) has a great potential for the non-invasive treatment of solid tumours. Due to the high pressure amplitudes involved, nonlinear acoustic effects must be understood and the relevant medium property is the parameter of nonlinearity B/A. Here, B/A was measured in ex vivo bovine liver, over a heating/cooling cycle replicating temperatures reached during HIFU ablation, adapting a finite amplitude insertion technique, which also allowed for measurement of sound-speed and attenuation. The method measures the nonlinear progression of a plane wave through liver and B/A was chosen so that numerical simulations matched the measured waveforms. To create plane-wave conditions, sinusoidal bursts were transmitted by a 100 mm diameter 1.125 MHz unfocused transducer and measured using a 15 mm diameter 2.25 MHz broadband transducer in the near field. Attenuation and sound-speed were calculated using a reflected pulse from the smaller transducer using the larger transducer as the reflecting interface. Results showed that attenuation initially decreased with heating then increased after denaturation, the sound-speed initially increased with temperature and then decreased, and B/A showed an increase with temperature but no significant post-heating change. The B/A data disagree with other reports that show a significant change and we suggest that any nonlinear enhancement in the received ultrasound signal post-treatment is likely due to acoustic cavitation rather than changes in tissue nonlinearity.

  5. Nonlinear acoustic properties of ex vivo bovine liver and the effects of temperature and denaturation

    International Nuclear Information System (INIS)

    Thermal ablation by high intensity focused ultrasound (HIFU) has a great potential for the non-invasive treatment of solid tumours. Due to the high pressure amplitudes involved, nonlinear acoustic effects must be understood and the relevant medium property is the parameter of nonlinearity B/A. Here, B/A was measured in ex vivo bovine liver, over a heating/cooling cycle replicating temperatures reached during HIFU ablation, adapting a finite amplitude insertion technique, which also allowed for measurement of sound-speed and attenuation. The method measures the nonlinear progression of a plane wave through liver and B/A was chosen so that numerical simulations matched the measured waveforms. To create plane-wave conditions, sinusoidal bursts were transmitted by a 100 mm diameter 1.125 MHz unfocused transducer and measured using a 15 mm diameter 2.25 MHz broadband transducer in the near field. Attenuation and sound-speed were calculated using a reflected pulse from the smaller transducer using the larger transducer as the reflecting interface. Results showed that attenuation initially decreased with heating then increased after denaturation, the sound-speed initially increased with temperature and then decreased, and B/A showed an increase with temperature but no significant post-heating change. The B/A data disagree with other reports that show a significant change and we suggest that any nonlinear enhancement in the received ultrasound signal post-treatment is likely due to acoustic cavitation rather than changes in tissue nonlinearity. (paper)

  6. Structural layers of ex vivo rat hippocampus at 7T MRI.

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    Jeanine Manuella Kamsu

    Full Text Available Magnetic resonance imaging (MRI applied to the hippocampus is challenging in studies of the neurophysiology of memory and the physiopathology of numerous diseases such as epilepsy, Alzheimer's disease, ischemia, and depression. The hippocampus is a well-delineated cerebral structure with a multi-layered organization. Imaging of hippocampus layers is limited to a few studies and requires high magnetic field and gradient strength. We performed one conventional MRI sequence on a 7T MRI in order to visualize and to delineate the multi-layered hippocampal structure ex vivo in rat brains. We optimized a volumic three-dimensional T2 Rapid Acquisition Relaxation Enhancement (RARE sequence and quantified the volume of the hippocampus and one of its thinnest layers, the stratum granulare of the dentate gyrus. Additionally, we tested passive staining by gadolinium with the aim of decreasing the acquisition time and increasing image contrast. Using appropriated settings, six discrete layers were differentiated within the hippocampus in rats. In the hippocampus proper or Ammon's Horn (AH: the stratum oriens, the stratum pyramidale of, the stratum radiatum, and the stratum lacunosum moleculare of the CA1 were differentiated. In the dentate gyrus: the stratum moleculare and the stratum granulare layer were seen distinctly. Passive staining of one brain with gadolinium decreased the acquisition time by four and improved the differentiation between the layers. A conventional sequence optimized on a 7T MRI with a standard receiver surface coil will allow us to study structural layers (signal and volume of hippocampus in various rat models of neuropathology (anxiety, epilepsia, neurodegeneration.

  7. Development of an Ex Vivo Tissue Platform To Study the Human Lung Response to Coxiella burnetii.

    Science.gov (United States)

    Graham, Joseph G; Winchell, Caylin G; Kurten, Richard C; Voth, Daniel E

    2016-05-01

    Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute debilitating flu-like illness that can also present as chronic endocarditis. Disease typically occurs following inhalation of contaminated aerosols, resulting in an initial pulmonary infection. In human cells, C. burnetii generates a replication niche termed the parasitophorous vacuole (PV) by directing fusion with autophagosomes and lysosomes. C. burnetii requires this lysosomal environment for replication and uses a Dot/Icm type IV secretion system to generate the large PV. However, we do not understand how C. burnetii evades the intracellular immune surveillance that triggers an inflammatory response. We recently characterized human alveolar macrophage (hAM) infection in vitro and found that avirulent C. burnetii triggers sustained interleukin-1β (IL-1β) production. Here, we evaluated infection of ex vivo human lung tissue, defining a valuable approach for characterizing C. burnetii interactions with a human host. Within whole lung tissue, C. burnetii preferentially replicated in hAMs. Additionally, IL-1β production correlated with formation of an apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC)-dependent inflammasome in response to infection. We also assessed potential activation of a human-specific noncanonical inflammasome and found that caspase-4 and caspase-5 are processed during infection. Interestingly, although inflammasome activation is closely linked to pyroptosis, lytic cell death did not occur following C. burnetii-triggered inflammasome activation, indicating an atypical response after intracellular detection. Together, these studies provide a novel platform for studying the human innate immune response to C. burnetii. PMID:26902725

  8. Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

    Science.gov (United States)

    Wang, Ying; Ortega-Martinez, Antonio; Farinelli, Bill; Anderson, R. R.; Franco, Walfre

    2016-02-01

    Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and, hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for quantitative measurements in wound healing in vivo.

  9. Ex-Vivo Uterine Environment (EVE Therapy Induced Limited Fetal Inflammation in a Premature Lamb Model.

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    Yuichiro Miura

    Full Text Available Ex-vivo uterine environment (EVE therapy uses an artificial placenta to provide gas exchange and nutrient delivery to a fetus submerged in an amniotic fluid bath. Development of EVE may allow us to treat very premature neonates without mechanical ventilation. Meanwhile, elevations in fetal inflammation are associated with adverse neonatal outcomes. In the present study, we analysed fetal survival, inflammation and pulmonary maturation in preterm lambs maintained on EVE therapy using a parallelised umbilical circuit system with a low priming volume.Ewes underwent surgical delivery at 115 days of gestation (term is 150 days, and fetuses were transferred to EVE therapy (EVE group; n = 5. Physiological parameters were continuously monitored; fetal blood samples were intermittently obtained to assess wellbeing and targeted to reference range values for 2 days. Age-matched animals (Control group; n = 6 were surgically delivered at 117 days of gestation. Fetal blood and tissue samples were analysed and compared between the two groups.Fetal survival time in the EVE group was 27.0 ± 15.5 (group mean ± SD hours. Only one fetus completed the pre-determined study period with optimal physiological parameters, while the other 4 animals demonstrated physiological deterioration or death prior to the pre-determined study end point. Significant elevations (p0.05 in surfactant protein mRNA expression level between the two groups.In this study, we achieved limited fetal survival using EVE therapy. Despite this, EVE therapy only induced a modest fetal inflammatory response and did not promote lung maturation. These data provide additional insight into markers of treatment efficacy for the assessment of future studies.

  10. Novel Techniques for Ex Vivo Expansion of Cord Blood: Clinical Trials

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    Rohtesh S Mehta

    2015-12-01

    Full Text Available Cord blood (CB provides an excellent alternative source of hematopoietic progenitor cells (HPC for patients lacking human leukocyte antigen (HLA-matched peripheral blood or bone marrow graft for transplantation. However, due to the limited cell dose in CB graft, it is associated with prolonged time to engraftment, risk of graft rejection, infections and treatment-related mortality. To increase the cell dose, a variety of ex vivo expansion techniques have been developed. Results of traditional methods of CB expansion using cytokines alone were disappointing. Expanding CB cells with mesenchymal progenitor cells led to sizeable increase in graft content and improved engraftment. Other methods used HPC-differentiation blockers, such as nicotinamide analogs, copper chelators, inducing constitutive Notch signaling, or an aryl hydrocarbon receptor antagonist (StemReginin1. Many of these methods lead to substantial expansions of total nucleated cells and CD34+ cells, and significantly improved time to neutrophil or platelet engraftment in patients transplanted with the expanded products compared to the recipients of unmanipulated CBT. These studies differ not only in the expansion method, but also with regards to the cytokines used, patient population, conditioning regimens and transplantation practices, to name a few. Some of these methods employed expansion of a portion of CB unit in the setting of single CBT, while others in the setting of double CBT. Here, we review various procedures used for CB expansion and highlight some of the key differences. Novel methods of improving engraftment that aim at improving bone marrow homing potential of CB cells are not reviewed.

  11. Ex vivo vs. in vivo antibacterial activity of two antiseptics on oral biofilm

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    ISABEL ePRADA-LÓPEZ

    2015-07-01

    Full Text Available Aim: To compare the immediate antibacterial effect of two application methods (passive immersion and active mouthwash of two antiseptic solutions on the in situ oral biofilm.Material and Methods: A randomized observer-masked crossover study was conducted. Fifteen healthy volunteers wore a specific intraoral device for 48 hours to form a biofilm in three glass disks. One of these disks was used as a baseline; another one was immersed in a solution of 0.2% Chlorhexidine (0.2% CHX, remaining the third in the device, placed in the oral cavity, during the 0.2% CHX mouthwash application. After a two-week washout period, the protocol was repeated using a solution of Essential Oils (EO. Samples were analysed for bacterial viability with the confocal laser scanning microscope after previous staining with LIVE/DEAD® BacLight™.Results: The EO showed a better antibacterial effect compared to the 0.2% CHX after the mouthwash application (% of bacterial viability= 1.16 ± 1.00% vs. 5.08 ± 5.79%, respectively, and was more effective in all layers (p<0.05. In the immersion, both antiseptics were significantly less effective (% of bacterial viability= 26.93 ± 13.11%, EO vs. 15.17 ± 6.14%, 0.2% CHX; in the case of EO immersion, there were no significant changes in the bacterial viability of the deepest layer in comparison with the baseline. Conclusions: The method of application conditioned the antibacterial activity of the 0.2% CHX and EO solutions on the in situ oral biofilm. The in vivo active mouthwash was more effective than the ex vivo passive immersion in both antiseptic solutions. There was more penetration of the antiseptic inside the biofilm with an active mouthwash, especially with the EO.

  12. An ex vivo swine tracheal organ culture for the study of influenza infection

    Science.gov (United States)

    Nunes, Sandro F.; Murcia, Pablo R.; Tiley, Laurence S.; Brown, Ian H.; Tucker, Alexander W.; Maskell, Duncan J.; Wood, James Lionel N.

    2009-01-01

    Background  The threat posed by swine influenza viruses with potential to transmit from pig populations to other hosts, including humans, requires the development of new experimental systems to study different aspects of influenza infection. Ex vivo organ culture (EVOC) systems have been successfully used in the study of both human and animal respiratory pathogens. Objectives  We aimed to develop an air interface EVOC using pig tracheas in the study of influenza infection demonstrating that tracheal explants can be effectively maintained in organ culture and support productive influenza infection. Methods  Tracheal explants were maintained in the air interface EVOC system for 7 days. Histological characteristics were analysed with different staining protocols and co‐ordinated ciliary movement on the epithelial surface was evaluated through a bead clearance assay. Explants were infected with a swine H1N1 influenza virus. Influenza infection of epithelial cells was confirmed by immunohistochemistry and viral replication was quantified by plaque assays and real‐time RT‐PCR. Results  Histological analysis and bead clearance assay showed that the tissue architecture of the explants was maintained for up to 7 days, while ciliary movement exhibited a gradual decrease after 4 days. Challenge with swine H1N1 influenza virus showed that the EVOC tracheal system shows histological changes consistent with in vivo influenza infection and supported productive viral replication over multiple cycles of infection. Conclusion  The air interface EVOC system using pig trachea described here constitutes a useful biological tool with a wide range of applications in the study of influenza infection. PMID:20021502

  13. Improving donor lung suitability: from protective strategies to ex-vivo reconditioning.

    Science.gov (United States)

    Solidoro, Paolo; Schreiber, Annia; Boffini, Massimo; Braido, Fulvio; DI Marco, Fabiano

    2016-06-01

    Lung transplant is a therapeutic option for end stage lung diseases, but only a limited number of lung grafts is considered suitable for transplantation. It has been recently suggested an approach to improve and maximize donor lung suitability, namely ventilation strategies to prevent lung damage and preserve function before transplantation. In potential lung donor patients, the use of lung-protective ventilatory strategies may protect against and at least partially reverse some conditions, such as ventilator-induced lung injury, atelectasis and neurogenic pulmonary edema, resulting in improved donor organ procurement. The novelty recently proposed lies in the integration of ventilatory strategies of previous studies, using an algorithmic approach for the management of potential donors, based on their clinical response and PaO2/FiO2 ratio. This approach could be further improved by using lung ultrasound (LUS) which demonstrated to be more accurate than bedside chest radiography in detecting and distinguishing different degrees of aeration loss, and could be useful in the evaluation of alveolar recruitment following the application of PEEP or after performing any recruitment maneuver. Finally, the close future is the exploration of ex-vivo reconditioning which introduces the exciting concept of both a protective ventilation and a protective perfusion, reducing the risk of ventilation associated damage, and, on the other hand, washing out potential inflammatory cytokines by low volume high oncotic pressure perfusion, managing the risk of edema by capillary leakage. Addressing these challenges will allow more patients with end-stage lung disease access to a lung transplant. PMID:27424500

  14. Ethosomes for skin delivery of ropivacaine: preparation, characterization and ex vivo penetration properties.

    Science.gov (United States)

    Zhai, Yingjie; Xu, Rui; Wang, Yi; Liu, Jiyong; Wang, Zimin; Zhai, Guangxi

    2015-01-01

    Ropivacaine, a novel long-acting local anesthetic, has been proved to own superior advantage. However, Naropin® Injection, the applied form in clinic, can cause patient non-convenience. The purpose of this study was to formulate ropivacaine (RPV) in ethosomes and evaluate the potential of ethosome formulation in delivering RPV transdermally. The RPV-loaded ethosomes were prepared with thin-film dispersion technique and the formulation was characterized in terms of size, zeta potential, differential scanning calorimetry (DSC) analysis and X-ray diffraction (XRD) study. The results showed that the optimized RPV-ethosomes displayed a typical lipid bilayer structure with a narrow size distribution of 73.86 ± 2.40 nm and drug loading of 8.27 ± 0.37%, EE of 68.92 ± 0.29%. The results of DSC and XRD study indicated that RPV was in amorphous state when encapsulated into ethosomes. Furthermore, the results of ex vivo permeation study proved that RPV-ethosomes could promote the permeability in a high-efficient, rapid way (349.0 ± 11.5 μg cm(-2) at 12 h and 178.8 ± 7.1 μg cm(-2) at 0.5 h). The outcomes of histopathology study forecasted that the interaction between ethosomes and skin could loosen the tight conjugation of corneocyte layers and weaken the permeation barrier. In conclusion, RPV-ethosomes could be a promising delivery system to encapsulate RPV and deliver RPV for transdermal administration.

  15. An ex-vivo human intestinal model to study Entamoeba histolytica pathogenesis.

    Science.gov (United States)

    Bansal, Devendra; Ave, Patrick; Kerneis, Sophie; Frileux, Pascal; Boché, Olivier; Baglin, Anne Catherine; Dubost, Geneviève; Leguern, Anne-Sophie; Prevost, Marie-Christine; Bracha, Rivka; Mirelman, David; Guillén, Nancy; Labruyère, Elisabeth

    2009-11-17

    Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion.

  16. Laser welding of urinary tissues, ex vivo, using a tunable Thulium fiber laser

    Science.gov (United States)

    Ngo, Anthony K.; Sharma, Utkarsh; Kang, Jin U.; Fried, Nathaniel M.

    2006-02-01

    Recent advances in Thulium fiber laser technology have resulted in the availability of a compact, inexpensive, tunable, mid-infrared laser for potential use in laser surgery. The objective of this study was to tune the Thulium fiber laser wavelength and corresponding optical penetration depth to match the tissue thickness, and thus produce full-thickness, watertight tissue closure during microsurgical laser welding of urinary tissues. 1-cm-length incisions were made, ex vivo, in porcine ureters. Thulium fiber laser radiation with a wavelength of 1873 nm, power of 550-650 mW, and 750-μm-diameter spot was delivered to the tissue in continuous-wave mode through a 600-μm silica optical fiber. The fiber was scanned over the weld site once at a rate of 0.1 mm/s using a motion controller and linear stage controlled by a PC. Optical coherence tomography, histology, flow rates, and temperature measurements were used to optimize and evaluate laser welding success. Histologic analysis demonstrated full-thickness welding of the ureteral wall. Weld success rates ranged from 67% (8/12) at an incident laser power of 550 mW to 91% (10/11) at 650 mW. Peak flow rates greater than 200 ml/min were measured, however, mean flow rates were only about 50 ml/min. Average tissue temperatures increased with incident laser power from 59-89 °C. The tunable Thulium fiber laser may be useful for surgical applications requiring variable control of thermal coagulation depth, such as microsurgical laser tissue welding.

  17. Inhibition of human platelet function in vitro and ex vivo by acetaminophen.

    Science.gov (United States)

    Lages, B; Weiss, H J

    1989-03-15

    The effects of acetaminophen (APAP) in vitro, or ex vivo following APAP ingestion, on human platelet aggregation, 14C-5HT secretion, and thromboxane B2 (TxB2) formation were assessed. APAP added in vitro to citrated platelet-rich plasma (PRP) inhibited aggregation, secretion, and TxB2 formation induced by collagen, epinephrine, arachidonate, and the ionophore A23187, but had no effect on the responses induced by the endoperoxide analog U44069. Arachidonate-induced responses were inhibited by lower concentrations of APAP than were the responses to the other agonists. In PRP obtained 1 hour after ingestion of 650 mg or 1000 mg APAP, arachidonate-induced TxB2 formation was inhibited by 40-99% in five subjects tested, whereas inhibition of collagen- or epinephrine-induced TxB2 formation was less consistent. Aggregation and secretion responses were not altered by APAP ingestion in 4 of the 5 subjects, but were inhibited in the remaining subject, who had the highest plasma APAP levels. In contrast to aspirin and indomethacin, APAP-induced inhibition of collagen-stimulated TxB2 formation could be partially overcome with increasing collagen concentrations. No such partial correction occurred with epinephrine, however. In washed platelet suspensions labeled with 3H-arachidonate, both APAP and aspirin inhibited the formation of labeled PGD2 and PGE2, as well as TxB2. These results suggest that APAP acts in human platelets as a reversible inhibitor of cyclo-oxygenase, as found previously in other tissues, and that recent APAP ingestion can, on occasion, produce inhibition of platelet functional responses measured in vitro. PMID:2499947

  18. Ex vivo stimulation of whole blood as a means to determine glucocorticoid sensitivity

    Directory of Open Access Journals (Sweden)

    Burnsides C

    2012-08-01

    Full Text Available Christopher Burnsides,1,* Jacqueline Corry,1,* Jacob Alexander,1 Catherine Balint,1 David Cosmar,1 Gary Phillips,2 Jeanette I Webster Marketon1,31Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Department of Internal Medicine, 2Center for Biostatistics, 3Institute for Behavioral Medicine Research, Wexner Medical Center at The Ohio State University, Columbus, OH, USA*JC and CB have equally contributed to this workPurpose: Glucocorticoids are commonly prescribed to treat a number of diseases including the majority of inflammatory diseases. Despite considerable interpersonal variability in response to glucocorticoids, an insensitivity rate of about 30%, and the risk of adverse side effects of glucocorticoid therapy, currently no assay is performed to determine sensitivity.Patients and methods: Here we propose a whole blood ex vivo stimulation assay to interrogate known glucocorticoid receptor (GR up- and downregulated genes to indicate glucocorticoid sensitivity. We have chosen to employ real-time PCR in order to provide a relatively fast and inexpensive assay.Results: We show that the GR-regulated genes, GILZ and FKBP51, are upregulated in whole blood by treatment with dexamethasone and that LPS-induction of cytokines (IL-6 and TNFα are repressed by dexamethasone in a dose responsive manner. There is considerable interpersonal variability in the maximum induction of these genes but little variation in the EC50 and IC50 concentrations. The regulation of the GR-induced genes differs throughout the day whereas the suppression of LPS-induced cytokines is not as sensitive to time of day.Conclusion: In all, this assay would provide a method to determine glucocorticoid receptor responsiveness in whole blood.Keywords: glucocorticoid responsiveness, gene regulation, nuclear receptor, GILZ, FKBP51, cytokines

  19. Ethosomes for skin delivery of ropivacaine: preparation, characterization and ex vivo penetration properties.

    Science.gov (United States)

    Zhai, Yingjie; Xu, Rui; Wang, Yi; Liu, Jiyong; Wang, Zimin; Zhai, Guangxi

    2015-01-01

    Ropivacaine, a novel long-acting local anesthetic, has been proved to own superior advantage. However, Naropin® Injection, the applied form in clinic, can cause patient non-convenience. The purpose of this study was to formulate ropivacaine (RPV) in ethosomes and evaluate the potential of ethosome formulation in delivering RPV transdermally. The RPV-loaded ethosomes were prepared with thin-film dispersion technique and the formulation was characterized in terms of size, zeta potential, differential scanning calorimetry (DSC) analysis and X-ray diffraction (XRD) study. The results showed that the optimized RPV-ethosomes displayed a typical lipid bilayer structure with a narrow size distribution of 73.86 ± 2.40 nm and drug loading of 8.27 ± 0.37%, EE of 68.92 ± 0.29%. The results of DSC and XRD study indicated that RPV was in amorphous state when encapsulated into ethosomes. Furthermore, the results of ex vivo permeation study proved that RPV-ethosomes could promote the permeability in a high-efficient, rapid way (349.0 ± 11.5 μg cm(-2) at 12 h and 178.8 ± 7.1 μg cm(-2) at 0.5 h). The outcomes of histopathology study forecasted that the interaction between ethosomes and skin could loosen the tight conjugation of corneocyte layers and weaken the permeation barrier. In conclusion, RPV-ethosomes could be a promising delivery system to encapsulate RPV and deliver RPV for transdermal administration. PMID:25625544

  20. Nuove strategie manipolative ex vivo per il purging delle unità di cellule staminali ematopoietiche periferiche destinate al trapianto autologo di midollo

    OpenAIRE

    Veronesi, Arianna

    2015-01-01

    NEW MANIPULATIVE STRATEGIES EX VIVO FOR PURGING OF PHERIPHERAL HAEMATOPOIETICS STEM CELLS UNITS AIMED FOR AUTOLOGOUS BONE MARROW TRANSPLANTATION This thesis aims to evaluate the possibility of using the Gemtuzumab ozogamicin in ex vivo purging of autologous pheripheral blood mononuclear cells, in order to eliminate possible contaminants leukaemia cells. The use of this drug is subject to verification that the depletion of malignant cells does not cause cytotoxic effects on normal stem cel...

  1. Comparison of xenobiotic metabolizing enzyme activities in ex vivo human skin and reconstructed human skin models from SkinEthic.

    Science.gov (United States)

    Eilstein, Joan; Léreaux, Guillaume; Budimir, Natali; Hussler, Georges; Wilkinson, Simon; Duché, Daniel

    2014-09-01

    Skin function is not limited to a physical barrier. According to its total surface area, it is also considered as an extra-hepatic metabolizing organ. In vitro engineered human skins have been developed to replace limited ex vivo normal human skin samples (NHS). Thus, assessing and comparing skin models from SkinEthic [Episkin™, RHE™ and the full thickness model (FTM)] with NHS in terms of metabolic capability are essential. The apparent activities of main cutaneous isoforms of cytochrome P450-dependent monooxygenases (CYP1A1/1B1, 2B6/2C18/2E1, 3A5/3A7), esterase, glutathione-S-[(GST), A, M, P, T], N-acetyl-(NAT1), uridinyl-diphosphate glucuronyl-(UDPGT 1A family) and sulfo-(SULT1A1) transferases were determined using probe substrates. Mean activities indicative of CYP1A1/1B1 (expressed as pmol/mg protein/6 h) in RHE™ (2.8) and FTM (2.6) were very similar to NHS (3.0) while Episkin™ showed a higher activity (9.1). Activities of CYP3A5/3A7 in FTM (3.3) and Episkin™ (3.6) were similar to NHS (3.8) while activity in RHE™ (13.3) was higher. CYP2B6/2C18/2E1 activity was below LOQ (0.5) in all skin models and NHS. Comparable intrinsic metabolic clearances were measured between NHS and skin models for esterase, UDPGT, GST and NAT1 activities. SULT1A1 activity toward probe substrates was not detected in skin models and observed at the limit of detection in NHS. Weak cytochrome P450-dependent monooxygenases, high esterase and transferase activities suggested that NHS and skin models exhibited limited functionalization and much greater detoxification (hydrolytic and conjugating) capacities. These results demonstrate that skin models are similar to NHS in terms of metabolic functionality toward xenobiotics investigated and useful tools to assess both the local efficiency and safety of cosmetics. PMID:24658324

  2. Lyophilized phytosomal nanocarriers as platforms for enhanced diosmin delivery: optimization and ex vivo permeation

    Directory of Open Access Journals (Sweden)

    Freag MS

    2013-07-01

    Full Text Available May S Freag, Yosra SR Elnaggar, Ossama Y AbdallahDepartment of Pharmaceutics, Faculty of Pharmacy, Alexandria University, Alexandria, EgyptAbstract: Diosmin (DSN is an outstanding phlebotonic flavonoid with a tolerable potential for the treatment of colon and hepatocellular carcinoma. Being highly insoluble, DSN bioavailability suffers from high inter-subject variation due to variable degrees of permeation. This work endeavored to develop novel DSN loaded phytosomes in order to improve drug dissolution and intestinal permeability. Three preparation methods (solvent evaporation, salting out, and lyophilization were compared. Nanocarrier optimization encompassed different soybean phospholipid (SPC types, different solvents, and different DSN:SPC molar ratios (1:1, 1:2, and 1:4. In vitro appraisal encompassed differential scanning calorimetry, infrared spectroscopy, particle size, zeta potential, polydispersity index, transmission electron microscopy, drug content, and in vitro stability. Comparative dissolution studies were performed under sink versus non-sink conditions. Ex vivo intestinal permeation studies were performed on rats utilizing noneverted sac technique and high-performance liquid chromatography analysis. The results revealed lyophilization as the optimum preparation technique using SPC and solvent mixture (Dimethyl sulphoxide:t-butylalchol in a 1:2 ratio. Complex formation was contended by differential scanning calorimetry and infrared data. Optimal lyophilized phytosomal nanocarriers (LPNs exhibited the lowest particle size (316 nm, adequate zeta-potential (−27 mV, and good in vitro stability. Well formed, discrete vesicles were revealed by transmission electron microscopy, drug content, and in vitro stability. Comparative dissolution studies were performed. LPNs demonstrated significant enhancement in DSN dissolution compared to crude drug, physical mixture, and generic and brand DSN products. Permeation studies revealed 80% DSN

  3. Manganese ferrite-based nanoparticles induce ex vivo, but not in vivo, cardiovascular effects

    Directory of Open Access Journals (Sweden)

    Nunes ADC

    2014-07-01

    rate or arterial blood pressure in conscious rats. In summary, although the MNPs were able to induce effects ex vivo, no significant changes were observed in vivo. Thus, given the proper dosages, these MNPs should be considered for possible therapeutic applications. Keywords: cardiac function, isolated heart, magnetic fluids, magnetic nanoparticles, nanomedicine

  4. Ex vivo and in vivo evaluation of an ultrasonic device for precise dissection, coagulation, and transection

    Directory of Open Access Journals (Sweden)

    Bertke BD

    2014-12-01

    Full Text Available Brian D Bertke,1 Patrick J Scoggins,1 Alissa L Welling,1 Tamara V Widenhouse,1 Chaoyang Chen,2 Srinivasu Kallakuri,2 John M Cavanaugh,2 Jeffrey W Clymer,1 Joseph F Amaral1 1Ethicon, Inc., Cincinnati, OH, USA; 2Spine Research Laboratory, Bioengineering Center, Wayne State University, Detroit, MI, USA Background: A new ultrasonic device, Harmonic Focus®+, has been developed that is smaller and delivers energy more efficiently than its predecessor via the inclusion of Adaptive Tissue Technology. This study was undertaken to compare its dissection capabilities to an advanced bipolar electrosurgery device in benchtop and preclinical evaluations. Methods: In ex vivo testing, Focus+ and LigaSure™ Small Jaw were evaluated for physical dimensions, device and tissue temperature after repeated applications to porcine jejunum, and burst pressure of vessel seals, transection time, and tissue sticking in 3–5 mm porcine carotid arteries. In in vivo testing, the devices were tested on intact porcine carotid arteries for thermal damage via collagen denaturation and in muscle incisions near rat sciatic nerve for acute inflammation via hematoxylin and eosin and for impaired axonal transport via β-APP. Results: Focus+ was smaller than the Small Jaw in width and height, yet it had a longer active blade and larger jaw aperture. Device temperatures were not different after application, but thermal spread (tissue temperature above 50°C was 78% greater for Small Jaw (9.6 mm than for Focus+ (5.4 mm. Burst pressures of sealed vessels were not significantly different between the devices: 900 (±466 mmHg for Focus+ versus 974 (±500 mmHg for Small Jaw. Small Jaw had a shorter individual transection time (5.0 seconds compared to 6.3 seconds for Focus+, whereas Focus+ had 70% less tissue sticking. Thermal damage, neural inflammation, and impaired axonal transport were all significantly lower for Focus+ compared to Small Jaw, by 19%, 57%, and 50%, respectively

  5. A novel human ex vivo model for the analysis of molecular events during lung cancer chemotherapy

    Directory of Open Access Journals (Sweden)

    Lang Dagmar S

    2007-06-01

    Full Text Available Abstract Background Non-small cell lung cancer (NSCLC causes most of cancer related deaths in humans and is characterized by poor prognosis regarding efficiency of chemotherapeutical treatment and long-term survival of the patients. The purpose of the present study was the development of a human ex vivo tissue culture model and the analysis of the effects of conventional chemotherapy, which then can serve as a tool to test new chemotherapeutical regimens in NSCLC. Methods In a short-term tissue culture model designated STST (Short-Term Stimulation of Tissues in combination with the novel *HOPE-fixation and paraffin embedding method we examined the responsiveness of 41 human NSCLC tissue specimens to the individual cytotoxic drugs carboplatin, vinorelbine or gemcitabine. Viability was analyzed by LIFE/DEAD assay, TUNEL-staining and colorimetric MTT assay. Expression of Ki-67 protein and of BrdU (bromodeoxyuridine uptake as markers for proliferation and of cleaved (activated effector caspase-3 as indicator of late phase apoptosis were assessed by immunohistochemistry. Transcription of caspase-3 was analyzed by RT-PCR. Flow cytometry was utilized to determine caspase-3 in human cancer cell lines. Results Viability, proliferation and apoptosis of the tissues were moderately affected by cultivation. In human breast cancer, small-cell lung cancer (SCLC and human cell lines (CPC-N, HEK proliferative capacity was clearly reduced by all 3 chemotherapeutic agents in a very similar manner. Cleavage of caspase-3 was induced in the chemo-sensitive types of cancer (breast cancer, SCLC. Drug-induced effects in human NSCLC tissues were less evident than in the chemo-sensitive tumors with more pronounced effects in adenocarcinomas as compared to squamous cell carcinomas. Conclusion Although there was high heterogeneity among the individual tumor tissue responses as expected, we clearly demonstrate specific multiple drug-induced effects simultaneously. Thus, STST

  6. Effect of anti-CD52 antibody alemtuzumab on ex-vivo culture of umbilical cord blood stem cells

    Directory of Open Access Journals (Sweden)

    Law Ping

    2008-10-01

    Full Text Available Abstract Background Excessive maturation of hematopoietic cells leads to a reduction of long-term proliferative capability during cord blood (CB expansion. In this study, we report the effects of anit-CD52 (Alemtuzumab, Campath on both short- and long-term ex vivo expansion of CB hematopoietic stem cells (HSC by evaluating the potential role of Alemtuzumab in preserving the repopulating capability in CB HSC and nonlymphoid progenitors. Methods Ex vivo expansion experiments were carried out using freshly purified CB CD34+ cells in StemSpan™ SFEM medium in the presence of stem cell factor, Flt3-Ligand and thrombopoietin at 50 ng/ml. Alemtuzumab (10 μg/ml was used to deplete CD52+ cells during the cultures. Flow cytometry was used to monitor CB HSC and their differentiation. Colony forming unit (CFU assays and long term culture-initiating cell (LTC-IC assays were performed on cells obtained from day 0 (before culture and day 14 after cultures. Secondary cultures was performed using CD34+ cells isolated at 35 days from primary cultures and further cultured in StemSpan™ SFEM medium for another 14 days to confirm the long term effect of alemtuzumab in liquid cultures. Results Compared to cytokines alone, addition of alemtuzumab resulted in a significant increase in total nucleated cells, absolute CD34+ cells, myeloid and megakaryocytic progenitors, multi-lineage and myeloid CFU and LTC-IC. Conclusion The results from current study suggested that the use of alemtuzumab for ex vivo expansion of CBHSC maybe advantageous. Our findings may improve current technologies for CBHSC expansion and increase the availability of CB units for transplantation. However, in vivo studies using animal models are likely needed in further studies to test the hematopoietic effects using such expanded CB products.

  7. CT Fluoroscopy-Guided Lung Biopsy with Novel Steerable Biopsy Canula: Ex-Vivo Evaluation in Ventilated Porcine Lung Explants

    International Nuclear Information System (INIS)

    The purpose was to evaluate ex-vivo a prototype of a novel biopsy canula under CT fluoroscopy-guidance in ventilated porcine lung explants in respiratory motion simulations. Using an established chest phantom for porcine lung explants, n = 24 artificial lesions consisting of a fat-wax-Lipiodol mixture (approx. 70HU) were placed adjacent to sensible structures such as aorta, pericardium, diaphragm, bronchus and pulmonary artery. A piston pump connected to a reservoir beneath a flexible silicone reconstruction of a diaphragm simulated respiratory motion by rhythmic inflation and deflation of 1.5 L water. As biopsy device an 18-gauge prototype biopsy canula with a lancet-like, helically bended cutting edge was used. The artificial lesions were punctured under CT fluoroscopy-guidance (SOMATOM Sensation 64, Siemens, Erlangen, Germany; 30mAs/120 kV/5 mm slice thickness) implementing a dedicated protocol for CT fluoroscopy-guided lung biopsy. The mean-diameter of the artificial lesions was 8.3 ± 2.6 mm, and the mean-distance of the phantom wall to the lesions was 54.1 ± 13.5 mm. The mean-displacement of the lesions by respiratory motion was 14.1 ± 4.0 mm. The mean-duration of CT fluoroscopy was 9.6 ± 5.1 s. On a 4-point scale (1 = central; 2 = peripheral; 3 = marginal; 4 = off target), the mean-targeted precision was 1.9 ± 0.9. No misplacement of the biopsy canula affecting adjacent structures could be detected. The novel steerable biopsy canula proved to be efficient in the ex-vivo set-up. The chest phantom enabling respiratory motion and the steerable biopsy canula offer a feasible ex-vivo system for evaluating and training CT fluoroscopy-guided lung biopsy adapted to respiratory motion.

  8. Fundamental analysis and ex vivo validation of thermal lesion mapping using harmonic motion imaging for focused ultrasound (HMIFU)

    Science.gov (United States)

    Hou, Gary Y.; Luo, Jianwen; Maleke, Caroline; Vappou, Jonathan; Marquet, Fabrice; Konofagou, Elisa E.

    2012-10-01

    Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a novel high-intensity focused ultrasound (HIFU) therapy monitoring method with feasibilities demonstrated in vitro, ex vivo and in vivo. Its principle is based on Amplitude-modulated (AM) - Harmonic Motion Imaging (HMI), an oscillatory radiation force used for imaging the tissue mechanical response during thermal ablation. In this study, a theoretical framework of HMIFU is presented, comprising a customized nonlinear wave propagation model, a finite-element (FE) analysis module, and an image-formation model. The objective of this study is to develop such a framework in order to 1) assess the fundamental performance of HMIFU in detecting HIFU lesions based on the change in tissue apparent elasticity, i.e., the increasing Young's modulus, and the HIFU lesion size with respect to the HIFU exposure time and 2) validate the simulation findings ex vivo. The same HMI and HMIFU parameters as in the experimental studies were used, i.e., 4.5-MHz HIFU frequency and 25-Hz AM frequency. For a lesion-to-background Young's modulus ratio of 3, 6, and 9, the estimated HMI displacement ratios were equal to 1.65, 3.19, 4.59, respectively. In experiments, the HMI displacement followed a similar increasing trend of 1.19, 1.28, 1.78 at 10-s, 20-s, and 30-s HIFU exposure, respectively. In addition, moderate agreement in lesion size growth was also found in both simulations (16.2, 73.1 and 334.7 mm2) and experiments (26.2, 94.2 and 206.2 mm2). Therefore, the feasibility of HMIFU for HIFU lesion detection based on the underlying tissue elasticity changes was verified through the developed theoretical framework, i.e., validation of the fundamental performance of the HMIFU system for lesion detection, localization and quantification, was demonstrated both theoretically and ex vivo.

  9. Influence of ultrasonic scattering in the calculation of thermal dose in ex-vivo bovine muscular tissues.

    Science.gov (United States)

    Cortela, Guillermo A; von Krüger, Marco A; Negreira, Carlos A; Pereira, Wagner C A

    2016-02-01

    This study explores the effect of ultrasound scattering on the temperature increase in phantoms and in samples of ex-vivo biological tissue through the calculation of the thermal dose (TD). Phantoms with different weight percentages of graphite powder (0-1%w/w, different scattering mean free paths, ℓS) and ex-vivo bovine muscle tissue were isonified by therapeutic ultrasound (1 MHz). The TD values were calculated from the first 4 min of experimental temperature curves obtained at several depths and were compared with those acquired from the numerical solution of the bio-heat transfer equation (simulated with 1 MHz and 0.5-2.0 W cm(-2)). The temperature curves suggested that scattering had an important role because the temperature increments were found to be higher for higher percentages of graphite powder (lower ℓS). For example, at a 30-mm depth and a 4-min therapeutic ultrasound application (0.5 W cm(-2)), the TDs (in equivalent minutes at 43 °C) were 7.2, 17.8, and 58.3 for the phantom with ℓS of 4.35, 3.85, and 3.03 mm, respectively. In tissue, the inclusion of only absorption or full attenuation in the bio-heat transfer equation (BHTE) heat source term of the simulation leads to under- or overestimation of the TD, respectively, as compared to the TD calculated from experimental data. The experiments with phantoms (with different scatterer concentrations) and ex-vivo samples show that the high values of TD were caused by the increase of energy absorption due to the lengthening of the propagation path caused by the changing in the propagation regime. PMID:26522957

  10. TGF-b Downregulation by RNAi Technique in ex Vivo-Expanded HSCs on 3D DBM Scaffold

    Directory of Open Access Journals (Sweden)

    N Amirizadeh

    2012-05-01

    Full Text Available Background: Bone Marrow Transplantations (BMT are limited by low CD34+ cell counts in umbilical cord blood (UCB and these cells need to be expanded for success in such procedures. To achieve this goal, ex vivo expansion of hematopoietic stem cells (HSCs by enhancing their self-renewal activity on demineralized bone matrix (DBM scaffold coated with mesenchymal progenitor cells (MPCs and unrestricted somatic stem cells (USSCs was recommended. TGF-b pathway is a key inhibitory factor for HSCs self-renewal. In this study ex vivo expansion and downregulation of TGF-b pathway were simultaneously performed. Methods: USSC cells were isolated from UCB and then coated on DBM scaffold as a feeder layer. UCB CD34+ cells were isolated from UCB by magnetic activated cell sorting (MACS method and were transfected by siRNA against TGFbR2 in two-dimensional (2D and three-dimensional (3D cultures by co-cultivation with USSC. TGFbR2 expression levels were evaluated by quantitative real-time PCR. Cell count and flow cytometry were performed and clonogenic activity was evaluated. Results: Ex vivo expansion of CD34+ cells was significantly enhanced (41±0.7 folds by TGFbR2 downregulation, especially in 2D than 3D cultures. Finally, 2D culture showed less TGFbR2 expression levels and higher increase in the percentage of CD34 markers by flow cytometry assay. Conclusion: The 3D siRNA delivery system would be of lower efficiency in contrast to 2D settings where the cells have less freedom and are in more contact with the feeder layer.

  11. Time-lapse imaging of the dynamics of CNS glial-axonal interactions in vitro and ex vivo.

    Directory of Open Access Journals (Sweden)

    Kalliopi Ioannidou

    Full Text Available BACKGROUND: Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualize the various stages of myelination. METHODOLOGY/PRINCIPAL FINDINGS: We took two approaches to follow myelination over time: i time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii ex vivo imaging of the spinal cord of shiverer (Mbp mutant mice, transplanted with GFP-labelled murine neurospheres. We demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, we provide evidence from time-lapse fluorescence microscopy and confocal microscopy that the oligodendrocytes' cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs, which then extend longitudinally along the axons. We predict from this model that these spiral cuffs must extend over each other first before extending to form internodes of myelin. CONCLUSION: These experiments show the feasibility of visualizing the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualization of an entire internodal length of myelin and the ex vivo approach validating the in vitro data.

  12. High-resolution ex vivo imaging of coronary artery stents using 64-slice computed tomography - initial experience

    Energy Technology Data Exchange (ETDEWEB)

    Rist, Carsten; Nikolaou, Konstantin; Wintersperger, Bernd J.; Reiser, Maximilian F.; Becker, Christoph R. [Ludwig-Maximilians University, Department of Clinical Radiology, Munich (Germany); Flohr, Thomas [Siemens Medical Solutions, CT Division, Forchheim (Germany)

    2006-07-15

    The aim of the study was to evaluate the potential of new-generation multi-slice computed tomography (CT) scanner technology for the delineation of coronary artery stents in an ex vivo setting. Nine stents of various diameters (seven stents 3 mm, two stents 2.5 mm) were implanted into the coronary arteries of ex vivo porcine hearts and filled with a mixture of an iodine-containing contrast agent. Specimens were scanned with a 16-slice CT (16SCT) machine; (Somatom Sensation 16, Siemens Medical Solutions), slice thickness 0.75 mm, and a 64-slice CT (64SCT, Somatom Sensation 64), slice-thickness 0.6 mm. Stent diameters as well as contrast densities were measured, on both the 16SCT and 64SCT images. No significant differences of CT densities were observed between the 16SCT and 64SCT images outside the stent lumen: 265{+-}25HU and 254{+-}16HU (P=0.33), respectively. CT densities derived from the 64SCT images and 16SCT images within the stent lumen were 367{+-}36HU versus 402{+-}28HU, P<0.05, respectively. Inner and outer stent diameters as measured from 16SCT and 64SCT images were 2.68{+-}0.08 mm versus 2.81{+-}0.07 mm and 3.29{+-}0.06 mm versus 3.18{+-}0.07 mm (P<0.05), respectively. The new 64SCT scanner proved to be superior in the ex vivo assessment of coronary artery stents to the conventional 16SCT machine. Increased spatial resolution allows for improved assessment of the coronary artery stent lumen. (orig.)

  13. Ex Vivo Smooth Muscle Pharmacological Effects of a Novel Bradykinin-Related Peptide, and Its Analogue, from Chinese Large Odorous Frog, Odorrana livida Skin Secretions

    Science.gov (United States)

    Xiang, Jie; Wang, Hui; Ma, Chengbang; Zhou, Mei; Wu, Yuxin; Wang, Lei; Guo, Shaodong; Chen, Tianbao; Shaw, Chris

    2016-01-01

    Bradykinin-related peptides (BRPs) are one of the most extensively studied frog secretions-derived peptide families identified from many amphibian species. The diverse primary structures of BRPs have been proven essential for providing valuable information in understanding basic mechanisms associated with drug modification. Here, we isolated, identified and characterized a dodeca-BRP (RAP-L1, T6-BK), with primary structure RAPLPPGFTPFR, from the skin secretions of Chinese large odorous frogs, Odorrana livida. This novel peptide exhibited a dose-dependent contractile property on rat bladder and rat ileum, and increased the contraction frequency on rat uterus ex vivo smooth muscle preparations; it also showed vasorelaxant activity on rat tail artery smooth muscle. In addition, the analogue RAP-L1, T6, L8-BK completely abolished these effects on selected rat smooth muscle tissues, whilst it showed inhibition effect on bradykinin-induced rat tail artery relaxation. By using canonical antagonist for bradykinin B1 or B2 type receptors, we found that RAP-L1, T6-BK -induced relaxation of the arterial smooth muscle was very likely to be modulated by B2 receptors. The analogue RAP-L1, T6, L8-BK further enhanced the bradykinin inhibitory activity only under the condition of co-administration with HOE140 on rat tail artery, suggesting a synergistic inhibition mechanism by which targeting B2 type receptors. PMID:27690099

  14. Human tumor-derived genomic DNA transduced into a recipient cell induces tumor-specific immune responses ex vivo

    OpenAIRE

    Whiteside, Theresa L.; Gambotto, Andrea; Albers, Andreas; Stanson, Joanna; Cohen, Edward P.

    2002-01-01

    This article describes a DNA-based vaccination strategy evaluated ex vivo with human cells. The vaccine was prepared by transferring tumor-derived genomic DNA to PCI-13 cells, a highly immunogenic tumor cell line (“recipient cell”), which had been genetically modified to secrete IL-2 (PCI-13/IL-2). PCI-13 cells expressed class I MHC determinants (HLA-A2) shared with the tumor from which the DNA was obtained as well as allogeneic determinants. DNA from a gp100+ melanoma ce...

  15. Promotion of wound healing by Plantago major L. leaf extracts--ex-vivo experiments confirm experiences from traditional medicine.

    Science.gov (United States)

    Zubair, Muhammad; Nybom, Hilde; Lindholm, Christina; Brandner, Johanna M; Rumpunen, Kimmo

    2016-01-01

    The wound-healing properties of Plantago major L. (plantain) were evaluated using an ex-vivo porcine wound-healing model. Ethanol- and water-based extracts were prepared from greenhouse-grown and freeze-dried leaves of P. major. Both types of extracts stimulated wound healing in porcine skin, but the ethanol-based extracts had a somewhat stronger effect. A concentration of 1.0 mg/mL (on dry weight basis) produced the best results for both types of extracts.

  16. Promotion of wound healing by Plantago major L. leaf extracts--ex-vivo experiments confirm experiences from traditional medicine.

    Science.gov (United States)

    Zubair, Muhammad; Nybom, Hilde; Lindholm, Christina; Brandner, Johanna M; Rumpunen, Kimmo

    2016-01-01

    The wound-healing properties of Plantago major L. (plantain) were evaluated using an ex-vivo porcine wound-healing model. Ethanol- and water-based extracts were prepared from greenhouse-grown and freeze-dried leaves of P. major. Both types of extracts stimulated wound healing in porcine skin, but the ethanol-based extracts had a somewhat stronger effect. A concentration of 1.0 mg/mL (on dry weight basis) produced the best results for both types of extracts. PMID:25898918

  17. Inhibitory action of two zinc oxide sources on the ex vivo growth of porcine small intestine bacteria.

    Science.gov (United States)

    Vahjen, W; Zentek, J; Durosoy, S

    2012-12-01

    Pharmacological dosage of zinc oxide in piglet weaning diets is a common practice to improve growth performance and gut health. However, high zinc excretion in animal wastes poses environmental challenges. Alternatives to current practice are studied. In this study, the inhibitory action of 2 zinc oxide sources on the ex vivo growth of small intestinal bacteria from weaned piglets was studied. Lag time was higher (P < 0.05) in media supplemented with a new zinc oxide preparation in stomach samples, but not in jejunum samples. Bacterial growth reduction (P < 0.05) was more drastic and more rapid in media supplemented with the new zinc oxide preparation.

  18. Multi-feature-based plaque characterization in ex vivo MRI trained by registration to 3D histology

    DEFF Research Database (Denmark)

    Engelen, Arna van; Niessen, Wiro J.; Klein, Stefan;

    2012-01-01

    We present a new method for automated characterization of atherosclerotic plaque composition in ex vivo MRI. It uses MRI intensities as well as four other types of features: smoothed, gradient magnitude and Laplacian images at several scales, and the distances to the lumen and outer vessel wall....... The ground truth for fibrous, necrotic and calcified tissue was provided by histology and micro-CT in 12 carotid plaque specimens. Semi-automatic registration of a 3D stack of histological slices and micro-CT images to MRI allowed for 3D rotations and inplane deformations of histology. By basing voxelwise...

  19. A Short Period of Ventilation without Perfusion Seems to Reduce Atelectasis without Harming the Lungs during Ex Vivo Lung Perfusion

    OpenAIRE

    Sandra Lindstedt; Leif Pierre; Richard Ingemansson

    2013-01-01

    To evaluate the lung function of donors after circulatory deaths (DCDs), ex vivo lung perfusion (EVLP) has been shown to be a valuable method. We present modified EVLP where lung atelectasis is removed, while the lung perfusion is temporarily shut down. Twelve pigs were randomized into two groups: modified EVLP and conventional EVLP. When the lungs had reached 37°C in the EVLP circuit, lung perfusion was temporarily shut down in the modified EVLP group, and positive end-expiratory pressure (P...

  20. GONAD: A Novel CRISPR/Cas9 Genome Editing Method that Does Not Require Ex Vivo Handling of Embryos.

    Science.gov (United States)

    Gurumurthy, Channabasavaiah B; Takahashi, Gou; Wada, Kenta; Miura, Hiromi; Sato, Masahiro; Ohtsuka, Masato

    2016-01-01

    Transgenic technologies used for creating a desired genomic change in animals involve three critical steps: isolation of fertilized eggs, microinjection of transgenic DNA into them and their subsequent transfer to recipient females. These ex vivo steps have been widely used for over 3 decades and they were also readily adapted for the latest genome editing technologies such as ZFNs, TALENs, and CRISPR/Cas9 systems. We recently developed a method called GONAD (Genome editing via Oviductal Nucleic Acids Delivery) that does not require all the three critical steps of transgenesis and therefore relieves the bottlenecks of widely used animal transgenic technologies. Here we provide protocols for the GONAD system.

  1. Buttressing staples with cholecyst-derived extracellular matrix (CEM) reinforces staple lines in an ex vivo peristaltic inflation model.

    LENUS (Irish Health Repository)

    Burugapalli, Krishna

    2008-11-01

    Staple line leakage and bleeding are the most common problems associated with the use of surgical staplers for gastrointestinal resection and anastomotic procedures. These complications can be reduced by reinforcing the staple lines with buttressing materials. The current study reports the potential use of cholecyst-derived extracellular matrix (CEM) in non-crosslinked (NCEM) and crosslinked (XCEM) forms, and compares their mechanical performance with clinically available buttress materials [small intestinal submucosa (SIS) and bovine pericardium (BP)] in an ex vivo small intestine model.

  2. NEURO-PROTECTION AND NEURO-THERAPY EFFECTS OF Acalypha indica Linn. WATER EXTRACT EX VIVO ON Musculus gastrocnemius Frog

    OpenAIRE

    Arjo Tedjo; Hamdani Zain; Nurhadi Ibrahim; Ernie H. Purwaningsih

    2008-01-01

    The studies of neuro-protection and neuro-therapy effects of Acalypha indica Linn. water extract ex vivo on Musculus gastrocnemius frog have already done at three Departments in Faculty of Medicine, University of Indonesia. The experimental studies were done on 2 groups of frog for neuro-protection and neuro-therapy effects. Each group of frog was divided into 7 subgroups of application, 4 samples each. There were 5 subgroups of doses: 5; 10; 15; 20; 25 mg and 2 subgroups as control. Pancuro...

  3. Development and clinical translation of OTIS: a wide-field OCT imaging device for ex-vivo tissue characterization

    Science.gov (United States)

    Munro, Elizabeth A.; Rempel, David; Danner, Christine; Atchia, Yaaseen; Valic, Michael S.; Berkeley, Andrew; Davoudi, Bahar; Magnin, Paul A.; Akens, Margarete; Done, Susan J.; Kulkarni, Supriya; Leong, Wey-Liang; Wilson, Brian C.

    2016-03-01

    We have developed an automated, wide-field optical coherence tomography (OCT)-based imaging device (OTISTM Perimeter Medical Imaging) for peri-operative, ex-vivo tissue imaging. This device features automated image acquisition, enabling rapid capture of high-resolution (15 μm) OCT images from samples up to 10 cm in diameter. We report on the iterative progression of device development from phantom and pre-clinical (tumor xenograft) models through to initial clinical results. We discuss the challenges associated with proving a novel imaging technology against the clinical "gold standard" of conventional post-operative pathology.

  4. Radiosynthesis and ex vivo evaluation of (R)-(-)-2-chloro-N-[1-11C-propyl]n-propylnorapomorphine

    DEFF Research Database (Denmark)

    Palner, Mikael; McCormick, Patrick; Gillings, Nicolas;

    2010-01-01

    Several dopamine D(2) agonist radioligands have been used with positron emission tomography (PET), including [(11)C-]-(-)-MNPA, [(11)C-]-(-)-NPA and [(11)C]-(+)-PHNO. These radioligands are considered particularly powerful for detection of endogenous dopamine release, but they either provide PET...... brain images with limited contrast or have affinity for both D(2) and D(3) receptors. We here present the carbon-11 radiolabeling and ex vivo evaluation of 2-Cl-(-)-NPA, a novel PET-tracer candidate with high in vitro D(2)/D(3) selectivity....

  5. A new electromagnetic shock-wave generator "SLX-F2" with user-selectable dual focus size: ex vivo evaluation of renal injury.

    Science.gov (United States)

    Leistner, Rasmus; Wendt-Nordahl, Gunnar; Grobholz, Rainer; Michel, Maurice Stephan; Marlinghaus, Ernst; Köhrmann, Kai Uwe; Alken, Peter; Häcker, Axel

    2007-08-01

    Storz Medical AG (Kreutzlingen/Switzerland) has developed a new electromagnetic shockwave (SW) generator, the "SLX-F2", which allows the user to choose between a small-focus, high-pressure treatment regime or a wide-focus, low-pressure option. The aim of this study was to investigate, under standardized conditions, the impact of these two different treatment regimes on SW-induced renal injury. SW-induced renal injury was investigated by using the standardized model of the perfused ex vivo kidney. SWs were applied under ultrasound control in the parenchyma of a kidney pole. Different SW numbers (20, 50, 125, 250, 500, 1,000) were applied in three groups: group A was treated with a wider focus (80 MPa), groups B (60 MPa) and C (120 MPa) with a smaller focus (each parameter setting was repeated ten-fold). Disintegration capacity (measured by crater volume in cubes of plaster of Paris) was the same in groups A and C. After SW exposure, barium sulphate suspension was perfused through the renal artery. The maximum diameter (mm) of the extravasation in the cortex, representing the extent of vascular injury, was measured on X-ray mammography films. H&E staining was performed. In all three groups (A, B, C) a higher number of SWs caused the diameter of the extravasate to increase, with statistical significance appearing at 1,000 shots versus 20 shots (p < 0.05). Vascular injury was not influenced by the focal size and positive peak pressure at identical SW numbers applied. Histology of the focal area showed gap-like defects. Our ex vivo data show that renal vascular injury is independent of the focal diameter of the SW generator at the same peak positive pressure and disintegration power. This confirms the in vivo findings that show renal injury caused by SW as being related to the number of SWs administered. Clinical studies are needed to investigate whether there is any advantage to offering both treatment regimes in one SW machine-for example, by using the "wide

  6. 3D high spectral and spatial resolution imaging of ex vivo mouse brain

    Energy Technology Data Exchange (ETDEWEB)

    Foxley, Sean, E-mail: sean.foxley@ndcn.ox.ac.uk; Karczmar, Gregory S. [Department of Radiology, University of Chicago, Chicago, Illinois 60637 (United States); Domowicz, Miriam [Department of Pediatrics, University of Chicago, Chicago, Illinois 60637 (United States); Schwartz, Nancy [Department of Pediatrics, Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637 (United States)

    2015-03-15

    Purpose: Widely used MRI methods show brain morphology both in vivo and ex vivo at very high resolution. Many of these methods (e.g., T{sub 2}{sup *}-weighted imaging, phase-sensitive imaging, or susceptibility-weighted imaging) are sensitive to local magnetic susceptibility gradients produced by subtle variations in tissue composition. However, the spectral resolution of commonly used methods is limited to maintain reasonable run-time combined with very high spatial resolution. Here, the authors report on data acquisition at increased spectral resolution, with 3-dimensional high spectral and spatial resolution MRI, in order to analyze subtle variations in water proton resonance frequency and lineshape that reflect local anatomy. The resulting information compliments previous studies based on T{sub 2}{sup *} and resonance frequency. Methods: The proton free induction decay was sampled at high resolution and Fourier transformed to produce a high-resolution water spectrum for each image voxel in a 3D volume. Data were acquired using a multigradient echo pulse sequence (i.e., echo-planar spectroscopic imaging) with a spatial resolution of 50 × 50 × 70 μm{sup 3} and spectral resolution of 3.5 Hz. Data were analyzed in the spectral domain, and images were produced from the various Fourier components of the water resonance. This allowed precise measurement of local variations in water resonance frequency and lineshape, at the expense of significantly increased run time (16–24 h). Results: High contrast T{sub 2}{sup *}-weighted images were produced from the peak of the water resonance (peak height image), revealing a high degree of anatomical detail, specifically in the hippocampus and cerebellum. In images produced from Fourier components of the water resonance at −7.0 Hz from the peak, the contrast between deep white matter tracts and the surrounding tissue is the reverse of the contrast in water peak height images. This indicates the presence of a shoulder in

  7. Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock’s testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex

  8. Ex-vivo Potential of Cadaveric and Fresh Limbal Tissues to Regenerate Cultured Epithelium

    Directory of Open Access Journals (Sweden)

    Vemuganti Geeta

    2004-01-01

    Full Text Available Purpose: To evaluate and compare the ex-vivo growth potential and formation of cultured corneal epithelium from residual corneo-limbal rings obtained from the operating room after penetrating keratoplasty, and fresh limbal tissues from patients undergoing routine cataract surgery. Methods: With the approval of the Institutional Review Board and informed consent from patients, 1-2mm of limbal tissues from 15 patients and 31 tissues from the cadaveric limbal ring preserved in MK medium (16 tissues and Optisol (15 tissues were used for the study. Donor data included age, time lapse between death and collection, collection and preservation and preservation and culture. Tiny bits of the limbal tissue were explanted on the de-epithelialised human amniotic membrane prepared following standard guidelines, and cultured using Human Corneal Epithelial cell medium. Radial growth from the explant was observed and measured by phase contrast microscopy over 2-4 weeks. After adequate confluent growth, whole mount preparation of the membrane was made and stained with haematoxylin and eosin. Part of the membrane was fixed in formalin and processed for routine histologic examination. The sections were stained with haematoxylin and eosin. Results: Forty-six tissues were evaluated from 42 eyes (15 from patients, 31 from cadaveric eyes with a mean age of 55.3 years ± 21.23 years (range 18 years - 110 years. The growth pattern observed was similar in all the positive cases with clusters of cells budding from the explant over 24- 72 hours, and subsequent formation of a monolayer over the next 2-3 weeks. The stained whole mount preparation showed a radial growth of cells around explants with diameter ranging from 5 to 16mm. Histologic evaluation of the membrane confirmed the growth of 2-3 cell-layered epithelium over the amniotic membrane. Cultivated epithelium around explant cell cultures was observed in 100% (15/15 of limbal tissue obtained from patients, as against

  9. Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection

    Institute of Scientific and Technical Information of China (English)

    LI BiChun; YU Fei; WANG KeHua; CHEN GuoHong; SUN GuoBo; SUN HuaiChang; XU Qi; GAO Bo; ZHOU GuanYue; ZHAO WenMing; WU XinSheng; BAO WenBin

    2008-01-01

    The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (ITSSCs). For the TMGT approach, four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection, the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, head, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex

  10. Ex vivo corneal epithelial wound healing following exposure to ophthalmic nonsteroidal anti-inflammatory drugs

    Directory of Open Access Journals (Sweden)

    Keping Xu

    2011-02-01

    bromfenac 0.09%.Conclusion: Corneas treated with ketorolac 0.45% healed as rapidly as those treated with MEM, likely secondary to addition of CMC and removal of BAK. In the ex vivo corneal organ culture model, ketorolac 0.45% had statistically less impact on corneal re-epithelialization than prior ketorolac formulations (0.4% and 0.5%, bromfenac 0.09%, and nepafenac 0.01%.Keywords: bromfenac 0.09%, corneal epithelial wound healing, epithelial toxicity, ketorolac 0.45%, nepafenac 0.1%, ocular surgery

  11. The effect of a hydrogen sulfide releasing molecule (Na2S) on the cold storage of livers from cardiac dead donor rats. A study in an ex vivo model.

    Science.gov (United States)

    Balaban, Cecilia Lucía; Rodríguez, Joaquín Valentín; Tiribelli, Claudio; Guibert, Edgardo Elvio

    2015-08-01

    Liver transplantation is currently the preferred treatment option for end-stage liver disease. Donation after cardiac death was a common practice in the early years of organ donation before brain death criteria were established. Those organs were subjected to variable periods of warm ischemia that might intensify cold ischemia/reperfusion injuries. In the present, shortage of brain dead donors has led to the reassessment of organ donation after cardiac death. Since many cytoprotective roles have been describe for H2S during ischemia/reperfusion on a variety of tissues, we hypothesized that graft exposure to this bioactive gas might improve preservation of non-heart beating donated organs. Therefore, to establish a rat model of donation post-cardiac arrest and using this approach to judge H2S delivery effects on graft hypothermic preservation, were the main objectives of this investigation. Cardiopulmonary arrest was induced in sedated rats by overload of potassium (K(+)). Livers were surgically removed and subsequently stored in HTK Solution (Histidine-tryptophan-ketoglutarate) at 0-4°C. After 24 h of hypothermic preservation, livers were rewarmed in an ex vivo model. Three experimental groups were established as follows: I--Livers procured before cardiac death and cold stored 24 h in HTK (BCD); II--Livers procured after cardiac death (45 min) and cold stored 24 h in HTK (ACD); III--Livers procured after cardiac death (45 min) and cold stored 24 h in HTK+10 μM Sodium Sulfide (Na2S) (ACD-SS). Data suggest that after 45 min of warm ischemia, viability parameters assessed during reperfusion in the ex vivo model were significantly impaired. Real time PCR revealed that after ex vivo reperfusion there is an increased expression of HO-1 and TNF-α and a modest drop in Bcl-2 mRNA, which could be interpreted as the cellular response to the hypoxic insult sustained during warm ischemia. On the other hand, warm ischemic livers exposed to H2S during cold storage, improved

  12. Somatostatin receptor expression in the human spleen - Answer to an enigma by ex-vivo and in-vitro autoradiography after 177Lu-DOTA-octreotate administration

    International Nuclear Information System (INIS)

    Full text of publication follows. Aim: radiolabelled somatostatin analogues are being used for diagnostic and therapeutic (PRRT) purposes in patients with somatostatin receptor (SSTR) expressing tumours. During PRRT a significant spleen uptake may lead to radiation doses of > 20 Gy. Yet, the threshold dose for spleen radiation induced toxicity is currently unknown. Based on previous 68Ga-DOTATOC PET/CT studies, demonstrating higher uptake in spleen than in splenosis, white pulp (WP) localization of radioactivity was suggested. This hypothesis was investigated in the current pilot study using the longer lived 177Lu-DOTA-octreotate. Methods: a patient diagnosed with neuroendocrine neoplasm of the pancreatic tail (SUVmax on 68Ga-DOTATOC PET/CT 100.4) with liver metastasis (SUV 47.3, normal liver SUV 12.5) and uptake in the spleen (SUV 41.0) received 1 GBq 177Lu-DOTA-octreotate. 2 h after administration whole-body planar scintigraphy and SPECT/CT of the upper abdomen was performed, followed by laparoscopic resection of the pancreatic tumour and splenectomy the next day. After spleen transport from Bad Berka to Rotterdam ex-vivo micro-SPECT of the removed spleen was acquired for 73 min using 2.5 mm diameter pinholes. Spleen fragments (∼10 * 10 * 5 mm) were either snap-frozen in liquid nitrogen or fixed in 10% formalin and paraffin embedded. Ex-vivo autoradiography of 10 μm cryo-sections was performed and serial sections were used for 111In-DOTA-octreotate in-vitro autoradiography after decay of 177Lu. FFPE sections were used for HE- and immunostaining for SSTR2A and cell subsets CD4 (Th-cell), CD8 (Ts-cell), CD20 (B-cell) and CD68 (macrophage). Results: 177Lu-DOTA-octreotate scintigraphy and SPECT/CT demonstrated high uptake in the pancreatic tumor, hepatic metastasis and homogeneously in the normal spleen. High resolution micro-SPECT imaging of the isolated spleen also revealed a relatively homogeneous uptake (calculated rest activity 60 MBq 177Lu). The vast

  13. In vitro and ex vivo methods predict the enhanced lung residence time of liposomal ciprofloxacin formulations for nebulisation.

    Science.gov (United States)

    Ong, Hui Xin; Benaouda, Faiza; Traini, Daniela; Cipolla, David; Gonda, Igor; Bebawy, Mary; Forbes, Ben; Young, Paul M

    2014-01-01

    Liposomal ciprofloxacin formulations have been developed with the aim of enhancing lung residence time, thereby reducing the burden of inhaled antimicrobial therapy which requires multiple daily administration due to rapid absorptive clearance of antibiotics from the lungs. However, there is a lack of a predictive methodology available to assess controlled release inhalation delivery systems and their effect on drug disposition. In this study, three ciprofloxacin formulations were evaluated: a liposomal formulation, a solution formulation and a 1:1 combination of the two (mixture formulation). Different methodologies were utilised to study the release profiles of ciprofloxacin from these formulations: (i) membrane diffusion, (ii) air interface Calu-3 cells and (iii) isolated perfused rat lungs. The data from these models were compared to the performance of the formulations in vivo. The solution formulation provided the highest rate of absorptive transport followed by the mixture formulation, with the liposomal formulation providing substantially slower drug release. The rank order of drug release/transport from the different formulations was consistent across the in vitro and ex vivo methods, and this was predictive of the profiles in vivo. The use of complimentary in vitro and ex vivo methodologies provided a robust analysis of formulation behaviour, including mechanistic insights, and predicted in vivo pharmacokinetics.

  14. Ex vivo evaluation of the serotonin 1A receptor partial agonist [³H]CUMI-101 in awake rats

    DEFF Research Database (Denmark)

    Palner, Mikael; Underwood, Mark D; Kumar, Dileep J S;

    2011-01-01

    -DL-phenylalanine, a serotonin synthesis inhibitor, did not show any effect on the standardized uptake values (SUVs) in any region. Citalopram did alter SBR, but this was due to changes in cerebellar SUVs. Our results indicate that [³H]CUMI-101 is a good radioligand for imaging 5-HT(1A) high-density regions in rats; however...... different challenge paradigms. [³H]CUMI-101 shows good uptake and good specific binding ratio (SBR) in frontal cortex 5.18 and in hippocampus 3.18. Binding was inhibited in a one-binding-site fashion by WAY100635 and unlabeled CUMI-101. The ex vivo B(max) of [³H]CUMI-101 in frontal cortex (98.7 fmol....../mg) and hippocampus (131 fmol/kg) agree with the ex vivo B(max) of [³H]MPPF in frontal cortex (147.1 fmol/mg) and hippocampus (72.1 fmol/mg) and with in vitro values reported with 8-OH-DPAT. Challenges with citalopram, a selective serotonin reuptake inhibitor, fenfluramine, a serotonin releaser, and 4-chloro...

  15. SEDDS of gliclazide: Preparation and characterization by in-vitro, ex-vivo and in-vivo techniques.

    Science.gov (United States)

    Nipun, Tanzina Sharmin; Ashraful Islam, S M

    2014-09-01

    In the study, self emulsifying drug delivery system (SEDDS) of gliclazide, a poorly soluble drug, was developed and evaluated by in-vitro, ex-vivo and in-vivo techniques. Oil and surfactant were screened out according to their solubilizing capacity. Among the tested components Transcutol HP and Tween-80 showed good solubilizing capacity. These two components were used in different ratios to prepare gliclazide SEDDS. The SEDDS formulations were transparent and clear. Droplet size of the emulsion was determined by Laser Diffraction Technology of Malvern. Formulation F1 containing 1:1 (m/m) mixture of Transcutol HP/Tween-80 showed minimum mean droplet size (50.959 μm). In-vitro drug release from F1 was higher (99% within 20 min) than other formulations. The developed SEDDS was also evaluated for ex-vivo permeability profile by using chicken intestinal sac. Formulation F1 showed optimal drug diffusion. In-vivo performance of SEDDS was evaluated in albino mice using plasma glucose level as a pharmacodynamic marker parameter. The test formulation (F1) showed significant reduction in plasma glucose level, after oral administration. So SEDDS may be an alternative technique for the oral administration of gliclazide. PMID:25161379

  16. Design and validation of a system to simulate coronary flexure dynamics on arterial segments perfused ex vivo.

    Science.gov (United States)

    VanEpps, J Scott; Londono, Ricardo; Nieponice, Alejandro; Vorp, David A

    2009-02-01

    Cyclic flexure of the coronary arteries can lead to spatially varying fluid and solid stress patterns. These patterns may explain the heterogenous distribution of atherosclerotic lesions. Here we describe the design and validation of an experimental system to simulate coronary-like flexure dynamics on intact arterial segments ex vivo. Our previously described ex vivo perfusion system was modified with a polymer flexure membrane controlled by a custom data acquisition/motion control system. The system was validated by perfusing arterial segments with pulsatile hemodynamics with or without cyclic flexure. Digital images were obtained to quantify dynamic vessel curvature and arc length. Tissue integrity was assessed by histology. The device generated physiologic curvatures (0-1.8 cm(-1)) at 1 Hz with a physiologic phase relationship with the pressure waveform. Additionally, the in vivo longitudinal extension ratio (40%) was maintained within 2.3% during the flexure cycle. Twelve hours of cyclic contact with the membrane did not compromise arterial segment integrity. This device provides a novel method to examine how the local biomechanical milieu could impact atherosclerotic lesion localization. PMID:18297319

  17. THE CELLS WITH MYCOBACTERIA IN GRANULOMATOUS AGGREGATES FROM MICE WITH LATENT TUBERCULOUS INFECTION IN EX VIVO CULTURE

    Directory of Open Access Journals (Sweden)

    E. G. Ufimtseva

    2013-01-01

    Full Text Available Abstract. The aim of this study was to obtain ex vivo monolayer culture cells migrated from individual granulomas isolated from the spleens of the Balb/c line mice through 1–2 months after BCG vaccine infection. The second goal was to evaluate influence of different types of cells in the development of granulomatic inflammation and analysis of BCG bacteria content in these cells in the latent stage of tuberculosis. Granulomas were presented by macrophages in general. The number of granulomas was varied as in one mouse as between mice. Granulomas contained also dendritic cells (in average 10% from macrophages of granulomas and lymphocytes. In some granulomas fibroblasts, neutrophils, eosiniphils, multinuclear cells of Pirogov–Langhans, megacariocytes and platelets were observed in all stages of infection. The number of these cells was also varied between granulomas. The acid staining BCG bacteria were only detected in macrophages, dendritic cells and Pirogov–Langhans cells of mice granulomas. Mice were different as by number of cells with BCG bacteria in granulomas as by number of granulomas with BCG-containing cells. The proposed model of granuloma cells of mice in ex vivo culture can be used to study interaction between host cells and mycobacteria to find new ways and methods of influence to intracellular pathogens in latent stage of tuberculosis. 

  18. Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites

    Directory of Open Access Journals (Sweden)

    Thompson Joanne

    2007-05-01

    Full Text Available Abstract Background Sensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites. Methods After surface labelling with biotin in the presence of the inhibitor furosemide, detergent extraction and osmotic lysis methods of enriching for the membrane fractions were compared to determine the efficiency of purification and recovery. Biotin-labelled proteins were identified on silver-stained SDS-polyacrylamide gels. Results Detergent extraction and osmotic lysis were compared for their capacity to purify biotin-labelled Plasmodium falciparum and Plasmodium chabaudi erythrocyte surface antigens. The pellet fraction formed after osmotic lysis of P. falciparum-infected erythrocytes is notably enriched in suface antigens, including PfEMP1, when compared to detergent extraction. There is also reduced co-extraction of host proteins such as spectrin and Band 3. Conclusion Biotinylation and osmotic lysis provides an improved method to label and purify parasitised erythrocyte surface antigen extracts from both in vitro and ex vivo Plasmodium parasite preparations.

  19. Microemulsion System for Topical Delivery of Thai Mango Seed Kernel Extract: Development, Physicochemical Characterisation and Ex Vivo Skin Permeation Studies

    Directory of Open Access Journals (Sweden)

    Jiraporn Leanpolchareanchai

    2014-10-01

    Full Text Available A microemulsion system containing Thai mango seed kernel extract (MSKE, cultivar “Fahlun” was developed and characterised for the purpose of topical skin delivery. The MSKE-loaded microemulsions were prepared by using the spontaneous emulsification method. Isopropyl myristate (IPM was selected as the oil phase. A polyoxyethylene sorbitan monooleate and sorbitan monododecanoate (1:1, w/w system was used as the surfactant phase; an aqueous mixture of different cosurfactants (absolute ethanol, 96.3% v/v ethanol, 1-propanol, 2-propanol or 1,2-propanediol at a weight ratio of 1:1 was used as the aqueous phase. Among the cosurfactants studied, the 1-propanol aqueous mixture had the largest microemulsion region (48.93% in the pseudo-ternary phase diagram. Microemulsions containing 1% MSKE demonstrated good physicochemical stability during a six-month study period at 25 ± 2 °C/60% ± 5% RH. The ex vivo skin permeation study demonstrated that the microemulsions exhibited a potent skin enhancement effect allowing MSKE to penetrate skin layers up to 60-fold higher compared with the control. Neither skin irritation nor skin corrosion was observed in ex vivo studies. The present study revealed that IPM-based microemulsion systems may be promising carriers to enhance skin penetration and delivering MSKE for topical treatment.

  20. Effects of Maté Tea Intake on ex Vivo LDL Peroxidation Induced by Three Different Pathways

    Directory of Open Access Journals (Sweden)

    Ruth Lobato T. Matsumoto

    2009-06-01

    Full Text Available Yerba maté (Ilex paraguariensis is a native South America plant widely consumed as different beverages. Yerba maté leaves contains high concentrations of polyphenols that are responsible for its high in vitro and in vivo antioxidant activity. The in vivo antioxidant properties vis a vis LDL particles has not yet been studied for maté tea, the roasted yerba maté product. The aim of this study was to evaluate the antioxidant activity of maté tea ingestion ex vivo on human LDL. Fasting peripheral venous blood samples of healthy women were taken in three different times: before drinking the tea, one hour later and after one week (7 days of daily consumption of maté tea. The isolated LDL was oxidized by three different pathways [copper (CuSO4, lipoxygenase and peroxynitrite (SIN-1]. Conjugated dienes and structural modifications on LDL were evaluated. Ingestion of maté tea increased LDL resistance towards ex vivo copper oxidation, but did not alter the peroxidation pattern when SIN-1 or lipoxygenase were used as oxidants

  1. Ex-vivo gene therapy restores LEKTI activity and corrects the architecture of Netherton syndrome-derived skin grafts.

    Science.gov (United States)

    Di, Wei-Li; Larcher, Fernado; Semenova, Ekaterina; Talbot, Gill E; Harper, John I; Del Rio, Marcela; Thrasher, Adrian J; Qasim, Waseem

    2011-02-01

    Netherton syndrome (NS) is a debilitating congenital skin disorder caused by mutations in the SPINK5 gene encoding the lymphoepithelial Kazal-type-related inhibitor (LEKTI). It is characterized by defective keratinization, recurrent infections, and hypernatraemic dehydration with a mortality rate of about 10% in the first year of life. Currently, there are no curative treatments for NS. We have developed a HIV-1 based, self-inactivating lentiviral vector to express SPINK5 in keratinocytes as part of an ex-vivo gene therapy strategy for NS. High transduction efficiency was achieved in NS keratinocytes and reconstitution of LEKTI expression was confirmed in previously deficient cells. These genetically corrected keratinocytes were further tested in an in vitro organotypic culture (OTC) system and in vivo mouse/human skin engraftment model. Results showed correction of epidermal architecture in both OTCs and regenerated skin grafts. Importantly, the results from corrected skin grafts indicated that even where detectable LEKTI expression was restored to a limited numbers of cells, a wider bystander benefit occurred around these small populations. As LEKTI is a secreted protein, the genetically modified graft may provide not only an immediate local protective barrier, but also act as a source of secreted LEKTI providing a generalized benefit following ex-vivo gene therapy.

  2. 3D thoracoscopic ultrasound volume measurement validation in an ex vivo and in vivo porcine model of lung tumours

    Science.gov (United States)

    Hornblower, V. D. M.; Yu, E.; Fenster, A.; Battista, J. J.; Malthaner, R. A.

    2007-01-01

    The purpose of this study was to validate the accuracy and reliability of volume measurements obtained using three-dimensional (3D) thoracoscopic ultrasound (US) imaging. Artificial 'tumours' were created by injecting a liquid agar mixture into spherical moulds of known volume. Once solidified, the 'tumours' were implanted into the lung tissue in both a porcine lung sample ex vivo and a surgical porcine model in vivo. 3D US images were created by mechanically rotating the thoracoscopic ultrasound probe about its long axis while the transducer was maintained in close contact with the tissue. Volume measurements were made by one observer using the ultrasound images and a manual-radial segmentation technique and these were compared with the known volumes of the agar. In vitro measurements had average accuracy and precision of 4.76% and 1.77%, respectively; in vivo measurements had average accuracy and precision of 8.18% and 1.75%, respectively. The 3D thoracoscopic ultrasound can be used to accurately and reproducibly measure 'tumour' volumes both in vivo and ex vivo.

  3. Fluorescent probes concentration estimation in vitro and ex vivo as a model for early detection of Alzheimer's disease

    Science.gov (United States)

    Harbater, Osnat; Gannot, Israel

    2014-12-01

    The pathogenic process of Alzheimer's disease (AD) begins years before clinical diagnosis. Here, we suggest a method that may detect AD several years earlier than current exams. The method is based on previous reports that relate the concentration ratio of biomarkers (amyloid-beta and tau) in the cerebrospinal fluid (CSF) to the development of AD. Our method replaces the lumbar puncture process required for CSF drawing by using fluorescence measurements. The system uses an optical fiber coupled to a laser source and a detector. The laser radiation excites two fluorescent probes which may bond to the CSF biomarkers. Their concentration ratio is extracted from the fluorescence intensities and can be used for future AD detection. First, we present a theoretical model for fluorescence concentration ratio estimation. The method's feasibility was validated using Monte Carlo simulations. Its accuracy was then tested using multilayered tissue phantoms simulating the epidural fat, CSF, and bone. These phantoms have various optical properties, thicknesses, and fluorescence concentrations in order to simulate human anatomy variations and different fiber locations. The method was further tested using ex vivo chicken tissue. The average errors of the estimated concentration ratios were low both in vitro (4.4%) and ex vivo (10.9%), demonstrating high accuracy.

  4. Microemulsion system for topical delivery of thai mango seed kernel extract: development, physicochemical characterisation and ex vivo skin permeation studies.

    Science.gov (United States)

    Leanpolchareanchai, Jiraporn; Padois, Karine; Falson, Françoise; Bavovada, Rapepol; Pithayanukul, Pimolpan

    2014-01-01

    A microemulsion system containing Thai mango seed kernel extract (MSKE, cultivar "Fahlun") was developed and characterised for the purpose of topical skin delivery. The MSKE-loaded microemulsions were prepared by using the spontaneous emulsification method. Isopropyl myristate (IPM) was selected as the oil phase. A polyoxyethylene sorbitan monooleate and sorbitan monododecanoate (1:1, w/w) system was used as the surfactant phase; an aqueous mixture of different cosurfactants (absolute ethanol, 96.3% v/v ethanol, 1-propanol, 2-propanol or 1,2-propanediol) at a weight ratio of 1:1 was used as the aqueous phase. Among the cosurfactants studied, the 1-propanol aqueous mixture had the largest microemulsion region (48.93%) in the pseudo-ternary phase diagram. Microemulsions containing 1% MSKE demonstrated good physicochemical stability during a six-month study period at 25 ± 2 °C/60% ± 5% RH. The ex vivo skin permeation study demonstrated that the microemulsions exhibited a potent skin enhancement effect allowing MSKE to penetrate skin layers up to 60-fold higher compared with the control. Neither skin irritation nor skin corrosion was observed in ex vivo studies. The present study revealed that IPM-based microemulsion systems may be promising carriers to enhance skin penetration and delivering MSKE for topical treatment. PMID:25347456

  5. Effect of anticonvulsant drugs on (/sup 35/S)t-butylbicyclophosphorothionate binding in vitro and ex vivo

    Energy Technology Data Exchange (ETDEWEB)

    Pitkaenen, A.; Riekkinen, P.J.; Saano, V.; Tuomisto, L.

    1987-01-01

    Using several concentrations of eight anticonvulsant drugs in clinical use (carbamazepine, clonazepam, phenytoin, phenobarbital, ethosuximide, primidone, sodium valproate, and D,L-..gamma..-vinyl GABA), we studied their abilities in vitro to displace (/sup 35/S)t-butylbicyclophosphorothionate (/sup 35/S-TBPS) from its binding site in a homogenate of rat brain. Thereafter ethosuximide (150 mg/kg), phenobarbital (30 mg/kg), clonazepam (0.3 mg/kg), or phenytoin (100 mg/kg) was injected intraperitoneally into rats for 16-20 days; and the effect of drug administration on /sup 35/S-TBPS binding was studied in the cortex and hippocampus ex vivo. Phenobarbital (100 ..mu..M, P<0.001), ethosuximide (500 ..mu..M, P<0.001), and phenytoin (40 ..mu..M, P<0.001) decreased the specific /sup 35/S-TBPS binding in vitro by 10-16%. After drug administration of phenobarbital (concentration in plasma 168 ..mu..M), the number of binding sites decreased and the binding affinity (p<0.05) in the cortex increased. Other anticonvulsants did not modulate /sup 35/S-TBPS binding in vitro at the concentration analogous to therapeutic plasma levels or ex vivo at the dose used. These results suggest that the use of phenobarbital may modulate the TBPS binding site, but the role of the present findings in the anticonvulsant action of phenobarbital needs to be further studied.

  6. Fast glomerular quantification of whole ex vivo mouse kidneys using Magnetic Resonance Imaging at 9.4 Tesla

    Energy Technology Data Exchange (ETDEWEB)

    Chacon-Caldera, Jorge; Kraemer, Philipp; Schad, Lothar R. [Heidelberg Univ., Mannheim (Germany). Computer Assisted Clinical Medicine; Geraci, Stefania; Gretz, Norbert [Heidelberg Univ., Mannheim (Germany). Medical Research Centre; Cullen-McEwen, Luise; Bertram, John F. [Monash Univ., Melbourne, VIC (Australia). Development and Stem Cells Program and Dept. of Anatomy and Developmental Biology

    2016-05-01

    A method to measure total glomerular number (N{sub glom}) in whole mouse kidneys using MRI is presented. The method relies on efficient acquisition times. A 9.4 T preclinical MRI system with a surface cryogenic coil and a 3D gradient echo sequence were used to image nine whole ex vivo BALB/c mouse kidneys labelled with cationized-ferritin (CF). A novel method to segment the glomeruli was developed. The quantification of glomeruli was achieved by identifying and fitting the probability distribution of glomeruli thus reducing variations due to noise. For validation, N{sub glom} of the same kidneys were also obtained using the gold standard: design-based stereology. Excellent agreement was found between the MRI and stereological measurements of N{sub glom}, with values differing by less than 4%: (mean ± SD) MRI = 15 606 ± 1 178; stereology = 16 273 ± 1 523. Using a robust segmentation method and a reliable quantification method, it was possible to acquire N{sub glom} with a scanning time of 33 minutes and 20 seconds. This was more than 8 times faster than previously presented MRI-based methods. Thus, an efficient approach to measure N{sub glom} ex vivo in health and disease is provided.

  7. Fast glomerular quantification of whole ex vivo mouse kidneys using Magnetic Resonance Imaging at 9.4 Tesla.

    Science.gov (United States)

    Chacon-Caldera, Jorge; Geraci, Stefania; Krämer, Philipp; Cullen-McEwen, Luise; Bertram, John F; Gretz, Norbert; Schad, Lothar R

    2016-03-01

    A method to measure total glomerular number (Nglom) in whole mouse kidneys using MRI is presented. The method relies on efficient acquisition times. A 9.4 T preclinical MRI system with a surface cryogenic coil and a 3D gradient echo sequence were used to image nine whole ex vivo BALB/c mouse kidneys labelled with cationized-ferritin (CF). A novel method to segment the glomeruli was developed. The quantification of glomeruli was achieved by identifying and fitting the probability distribution of glomeruli thus reducing variations due to noise. For validation, Nglom of the same kidneys were also obtained using the gold standard: design-based stereology. Excellent agreement was found between the MRI and stereological measurements of Nglom, with values differing by less than 4%: (mean ± SD) MRI = 15 606±1 178; stereology = 16 273±1 523. Using a robust segmentation method and a reliable quantification method, it was possible to acquire Nglom with a scanning time of 33minutes and 20seconds. This was more than 8 times faster than previously presented MRI-based methods. Thus, an efficient approach to measure Nglom ex vivo in health and disease is provided. PMID:26777317

  8. Rapid Stereomicroscopic Imaging of HER2 Overexpression in Ex Vivo Breast Tissue Using Topically Applied Silica-Based Gold Nanoshells

    Directory of Open Access Journals (Sweden)

    Lissett R. Bickford

    2012-01-01

    Full Text Available Tumor margin detection for patients undergoing breast conservation surgery primarily occurs postoperatively. Previously, we demonstrated that gold nanoshells rapidly enhance contrast of HER2 overexpression in ex vivo tissue sections. Our ultimate objective, however, is to discern HER2 overexpressing tissue from normal tissue in whole, nonsectioned, specimens to facilitate rapid diagnoses. Here, we use targeted nanoshells to quickly and effectively visualize HER2 receptor expression in intact ex vivo human breast tissue specimens. Punch biopsies of human breast tissue were analyzed after a brief 5-minute incubation with and without HER2-targeted silica-gold nanoshells using two-photon microscopy and stereomicroscopy. Labeling was subsequently verified using reflectance confocal microscopy, darkfield hyperspectral imaging, and immunohistochemistry to confirm levels of HER2 expression. Our results suggest that anti-HER2 nanoshells used in tandem with a near-infrared reflectance confocal microscope and a standard stereomicroscope may potentially be used to discern HER2-overexpressing cancerous tissue from normal tissue in near real time and offer a rapid supplement to current diagnostic techniques.

  9. Acceleration of Enterococcus faecalis biofilm formation by aggregation substance expression in an ex vivo model of cardiac valve colonization.

    Directory of Open Access Journals (Sweden)

    Olivia N Chuang-Smith

    Full Text Available Infectious endocarditis involves formation of a microbial biofilm in vivo. Enterococcus faecalis Aggregation Substance (Asc10 protein enhances the severity of experimental endocarditis, where it has been implicated in formation of large vegetations and in microbial persistence during infection. In the current study, we developed an ex vivo porcine heart valve adherence model to study the initial interactions between Asc10(+ and Asc10(-E. faecalis and valve tissue, and to examine formation of E. faecalis biofilms on a relevant tissue surface. Scanning electron microscopy of the infected valve tissue provided evidence for biofilm formation, including growing masses of bacterial cells and the increasing presence of exopolymeric matrix over time; accumulation of adherent biofilm populations on the cardiac valve surfaces during the first 2-4 h of incubation was over 10-fold higher than was observed on abiotic membranes incubated in the same culture medium. Asc10 expression accelerated biofilm formation via aggregation between E. faecalis cells; the results also suggested that in vivo adherence to host tissue and biofilm development by E. faecalis can proceed by Asc10-dependent or Asc10-independent pathways. Mutations in either of two Asc10 subdomains previously implicated in endocarditis virulence reduced levels of adherent bacterial populations in the ex vivo system. Interference with the molecular interactions involved in adherence and initiation of biofilm development in vivo with specific inhibitory compounds could lead to more effective treatment of infectious endocarditis.

  10. Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34+ Cells

    Directory of Open Access Journals (Sweden)

    Hui Xie

    2016-01-01

    Full Text Available Mesenchymal stem cells (MSCs are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+ cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

  11. An improved cryopreservation method for porcine buccal mucosa in ex vivo drug permeation studies using Franz diffusion cells.

    Science.gov (United States)

    Amores, Sonia; Domenech, José; Colom, Helena; Calpena, Ana C; Clares, Beatriz; Gimeno, Álvaro; Lauroba, Jacinto

    2014-08-18

    The use of isolated animal models to assess percutaneous absorption of molecules is frequently reported. The porcine buccal mucosa has been proposed as a substitute for the buccal mucosa barrier on ex vivo permeability studies avoiding unnecessary sacrifice of animals. But it is not always easy to obtain fresh buccal mucosa. Consequently, human and porcine buccal mucosa is sometimes frozen and stored in liquid nitrogen, but this procedure is not always feasible. One cheaper and simpler alternative is to freeze the buccal mucosa of freshly slaughtered pigs in a mechanical freezer, using DMSO and albumin as cryoprotective agents. This study compared the ex vivo permeability parameters of propranolol hydrochloride through porcine buccal mucosa using a Franz diffusion cell system and HPLC as detection method. The freezing effects on drug permeability parameters were evaluated. Equally histological studies were performed. Furthermore, the use of the parameter transmucosal water loss (TMWL) as an indicator of the buccal mucosa integrity was evaluated just as transepidermal water loss (TEWL) is utilized for skin integrity. The results showed no difference between fresh and frozen mucosal flux, permeability coefficient or lag time of propranolol. However, statistical significant difference in TMWL between fresh and frozen mucosa was observed. PMID:24813111

  12. Uncovering effects of ex vivo protease activity during proteomics and peptidomics sample extraction in rat brain tissue by oxygen-18 labeling.

    Science.gov (United States)

    Stingl, Christoph; Söderquist, Marcus; Karlsson, Oskar; Borén, Mats; Luider, Theo M

    2014-06-01

    In biological samples, proteins and peptides are altered by proteolytic activity. The actual ex vivo form of the peptidome or proteome analyzed, therefore, does not always reflect the natural in vivo state. Sample stabilization and sample treatment are thereby decisive for how far these two states diverge. To assess ex vivo formation of peptides, we used enzymatic incorporation of oxygen-18 water during proteolysis (PALeO approach) to label ex-vivo-formed peptides in rodent brain tissue. Rates of ex-vivo-formed peptides were determined in 25 samples that were stabilized and treated by six different protocols, whereby samples were subjected to different conditions such as temperature, urea concentration, and duration of treatment. Samples were measured by nano LC-Orbitrap-MS, and incorporation of oxygen-18 was determined by MS/MS database search and analysis of the precursor isotope pattern. Extent of ex vivo degradations was affected relevantly by the sample treatment protocol applied and stopped almost completely by heat stabilization. Determination of the formation state by oxygen-18 incorporation by MS/MS database search correlated well to more elaborate analysis of the MS isotope pattern. Overall, oxygen-18 labeling in combination with shotgun data-acquisition and MS/MS database search offers an adjuvant and easily applicable tool to monitor sample quality and fidelity in peptide and neuropeptide sample preparations. PMID:24738752

  13. Ex vivo expansion of regulatory T cells for clinical applications against graft-versus-host disease in allogeneic hematopoietic stem cell transplantation

    Institute of Scientific and Technical Information of China (English)

    ZHANG Lan-fang; XIA Chang-qing

    2013-01-01

    Objective To review the characteristics of regulatory T cells (Tregs) and ex vivo expansion of Tregs for treatment of graftversus-host disease (GVHD).Data sources The data used in this review were retrieved from PubMed (1970-2013).The terms "ex vivo expansion","regulatory T cell",and "graft-versus-host disease" were used for literature search.Study selection The publications about the characteristics of Tregs,ex vivo expansion of Tregs and clinical applications of Tregs against GVHD were identified,retrieved and reviewed.Results Tregs can be classified as natural Tregs (nTregs) and induced Tregs (iTregs).Both subsets share most Treg features.Given their immunosuppressive property,Tregs have been tested for their capability of preventing GVHD.The bottleneck of Treg therapy is the limited numbers of naturally existing Tregs.To solve this problem,ex vivo expansion of nTregs or iTregs has been executed.The initial data indicate Treg therapy is effective in reducing GVHD without compromising graft-versus-leukemia (GVL).Conclusion Ex vivo expansion of Tregs is a reliable way to prepare sufficient number of Tregs for management of GVHD.

  14. In vitro and ex vivo models indicate that the molecular clock in fast skeletal muscle of Atlantic cod is not autonomous.

    Science.gov (United States)

    Lazado, Carlo C; Kumaratunga, Hiruni P S; Nagasawa, Kazue; Babiak, Igor; Caipang, Christopher Marlowe A; Fernandes, Jorge M O

    2014-10-01

    The notion that the circadian rhythm is exclusively regulated by a central clock has been challenged by the discovery of peripheral oscillators. These peripheral clocks are known to have a direct influence on the biological processes in a tissue or cell. In fish, several peripheral clocks respond directly to light, thus raising the hypothesis of autonomous regulation. Several clock genes are expressed with daily rhythmicity in Atlantic cod (Gadus morhua) fast skeletal muscle. In the present study, myosatellite cell culture and short-term cultured fast skeletal muscle explant models were developed and characterized, in order to investigate the autonomy of the clock system in skeletal muscle of Atlantic cod. Myosatellite cells proliferated and differentiated in vitro, as shown by the changes in cellular and myogenic gene markers. The high expression of myogenic differentiation 1 during the early days post-isolation implied the commitment to myogenic lineage and the increasing mRNA levels of proliferating cell nuclear antigen (pcna) indicated the proliferation of the cells in vitro. Transcript levels of myogenic marker genes such as pcna and myogenin increased during 5 days in culture of skeletal muscle explants, indicating that the muscle cells were proliferating and differentiating under ex vivo conditions. Transcript levels of the clock gene aryl hydrocarbon receptor nuclear translocator-like 2 (arntl2) in myosatellite cells showed no daily oscillation regardless of photoperiod manipulation. On the other hand, mRNA levels of the clock gene circadian locomotor output cycles kaput (clock) showed circadian rhythmicity in 5-day-old skeletal muscle explant under different photoperiod regimes. The expression of arntl2, cryptochrome2 (cry2), period 2a (per2a) and nuclear receptor subfamily 1, group D, member 1 was not rhythmic in muscle explants but photoperiod manipulation had a significant effect on mRNA levels of cry2 and per2a. Taken together, the lack of rhythmicity

  15. A comparison of the forces required to produce tooth movement ex vivo through three types of pre-adjusted brackets when subjected to determined tip or torque values.

    Science.gov (United States)

    Sims, A P; Waters, N E; Birnie, D J

    1994-11-01

    Friction in fixed appliance systems has received considerable attention in recent literature, although that attributable to varying second order (tip) and third order (torque) adjustments in either the bracket or the archwire has not been fully investigated. The ex vivo study of 0.022 x 0.028-inch slot Minitwin, Activa, and Standard Straight Wire brackets investigates friction when known values of tip or torque were applied to 0.018 x 0.025-inch stainless steel wires. The resistance to sliding of the wire through the ligated brackets was measured on a vertically-mounted Instron testing machine. The results showed that the self-ligating Activa brackets consistently produced less friction than the other conventionally tied brackets. Minitwin brackets were slightly more resistant to movement than the Standard brackets during torquing, but the converse was found when tip was applied. Increasing tip and torque (ranges tested 0-6 degrees and 0-25 degrees, respectively) produced almost linear increases in friction for all brackets, although increasing tip had the more profound effect on friction, particularly in Activa brackets. PMID:7857896

  16. Controlled nail delivery of a novel lipophilic antifungal agent using various modern drug carrier systems as well as in vitro and ex vivo model systems.

    Science.gov (United States)

    Naumann, Sandy; Meyer, Jean-Philippe; Kiesow, Andreas; Mrestani, Yahya; Wohlrab, Johannes; Neubert, Reinhard H H

    2014-04-28

    The penetration behavior into human nails and animal hoof membranes of a novel antifungal agent (EV-086K) for the treatment of onychomycosis was investigated in this study. The new drug provides a high lipophilicity which is adverse for penetration into nails. Therefore, four different formulations were developed, with particular focus on a colloidal carrier system (CCS) due to its penetration enhancing properties. On the one hand, ex vivo penetration experiments on human nails were performed. Afterwards the human nail plates were cut by cryomicrotome in order to quantify the drug concentration in the dorsal, intermediate and ventral nail layer using high-performance liquid chromatography (HPLC) with UV detection. On the other hand, equine and bovine hoof membranes were used to determine the in vitro penetration of the drug into the acceptor compartment of an online diffusion cell coupled with Fourier transform infrared attenuated total reflectance (FTIR-ATR) spectroscopy. In combination, both results should exhibit a correlation between the EV-086K penetration behavior in human nail plates and animal hoof membranes. The investigations showed that the developed CCS could increase drug delivery through the human nail most compared to other formulations (nail lacquer, solution and hydrogel). Using animal hooves in the online diffusion cell, we were able to calculate pharmacokinetic data of the penetration process, especially diffusion and permeability coefficients. Finally, a qualitative correlation between the penetration results of human nails and equine hooves was established. PMID:24560884

  17. Controlled nail delivery of a novel lipophilic antifungal agent using various modern drug carrier systems as well as in vitro and ex vivo model systems.

    Science.gov (United States)

    Naumann, Sandy; Meyer, Jean-Philippe; Kiesow, Andreas; Mrestani, Yahya; Wohlrab, Johannes; Neubert, Reinhard H H

    2014-04-28

    The penetration behavior into human nails and animal hoof membranes of a novel antifungal agent (EV-086K) for the treatment of onychomycosis was investigated in this study. The new drug provides a high lipophilicity which is adverse for penetration into nails. Therefore, four different formulations were developed, with particular focus on a colloidal carrier system (CCS) due to its penetration enhancing properties. On the one hand, ex vivo penetration experiments on human nails were performed. Afterwards the human nail plates were cut by cryomicrotome in order to quantify the drug concentration in the dorsal, intermediate and ventral nail layer using high-performance liquid chromatography (HPLC) with UV detection. On the other hand, equine and bovine hoof membranes were used to determine the in vitro penetration of the drug into the acceptor compartment of an online diffusion cell coupled with Fourier transform infrared attenuated total reflectance (FTIR-ATR) spectroscopy. In combination, both results should exhibit a correlation between the EV-086K penetration behavior in human nail plates and animal hoof membranes. The investigations showed that the developed CCS could increase drug delivery through the human nail most compared to other formulations (nail lacquer, solution and hydrogel). Using animal hooves in the online diffusion cell, we were able to calculate pharmacokinetic data of the penetration process, especially diffusion and permeability coefficients. Finally, a qualitative correlation between the penetration results of human nails and equine hooves was established.

  18. The Hepatoprotection Provided by Taurine and Glycine against Antineoplastic Drugs Induced Liver Injury in an Ex Vivo Model of Normothermic Recirculating Isolated Perfused Rat Liver

    Directory of Open Access Journals (Sweden)

    Reza Heidari

    2016-03-01

    Full Text Available Taurine (2-aminoethane sulfonic acid is a non-protein amino acid found in high concentration in different tissues. Glycine (Amino acetic acid is the simplest amino acid incorporated in the structure of proteins. Several investigations indicate the hepatoprotective properties of these amino acids. On the other hand, antineoplastic agents-induced serum transaminase elevation and liver injury is a clinical complication. The current investigation was designed to screen the possible hepatoprotective properties of taurine and glycine against antineoplastic drugs-induced hepatic injury in an ex vivo model of isolated perfused rat liver. Rat liver was perfused with different concentration (10 μM, 100 μM and 1000 μM of antineoplastic drugs (Mitoxantrone, Cyclophosphamide, Cisplatin, 5 Fluorouracil, Doxorubicin and Dacarbazine via portal vein. Taurine and glycine were administered to drug-treated livers and liver perfusate samples were collected for biochemical measurements (ALT, LDH, AST, and K+. Markers of oxidative stress (reactive oxygen species formation, lipid peroxidation, total antioxidant capacity and glutathione were also assessed in liver tissue. Antineoplastic drugs caused significant pathological changes in perfusate biochemistry. Furthermore, markers of oxidative stress were significantly elevated in drug treated livers. It was found that taurine (5 and 10 mM and glycine (5 and 10 mM administration significantly mitigated the biomarkers of liver injury and attenuated drug induced oxidative stress. Our data indicate that taurine and glycine supplementation might help as potential therapeutic options to encounter anticancer drugs-induced liver injury.

  19. Ex Vivo Gene Therapy Using Human Mesenchymal Stem Cells to Deliver Growth Factors in the Skeletal Muscle of a Familial ALS Rat Model.

    Science.gov (United States)

    Suzuki, Masatoshi; Svendsen, Clive N

    2016-01-01

    Therapeutic protein and molecule delivery to target sites by transplanted human stem cells holds great promise for ex vivo gene therapy. Our group has demonstrated the therapeutic benefits of ex vivo gene therapy targeting the skeletal muscles in a transgenic rat model of familial amyotrophic lateral sclerosis (ALS). We used human mesenchymal stem cells (hMSCs) and genetically modified them to release neuroprotective growth factors such as glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF). Intramuscular growth factor delivery via hMSCs can enhance neuromuscular innervation and motor neuron survival in a rat model of ALS (SOD1(G93A) transgenic rats). Here, we describe the protocol of ex vivo delivery of growth factors via lentiviral vector-mediated genetic modification of hMSCs and hMSC transplantation into the skeletal muscle of a familial ALS rat model.

  20. Measurements of optical parameters of phantom solution and bulk animal tissues ex vivo at 650 nm

    Science.gov (United States)

    Sun, Ping; Wang, Yu; Liu, Jian

    2008-12-01

    Optical parameters of biological tissues, including absorption coefficient (μa), reduced scattering coefficient (μs') or scattering coefficient (μs), anisotropy factor (g) and refractive index (n) are investigated extensively and systemically at wavelength of 650 nm. Intralipid solution was selected to be the tissue phantom in order to test the validity of measurements. Considering the factors of fiber orientation and haemoglobin content, we chose some fresh bulk animal tissues in vitro which were bovine adipose, bovine muscle, porcine adipose, porcine muscle, porcine kidney, porcine liver, mutton and chicken breast. The basic assumption is that in vitro samples are a reasonable representation of the in vivo situation. We have gained numbers of experimental data of Intralipid and some tissues. Particularly, we have set up the close relationships among six optical parameters involving μa, μs', μs, g, n and μt. The experimental results show that for animal tissues, μa, μs' or μs and n rely deeply on muscle fiber orientations. Both of μs and μt range from 10mm-1 to 20mm-1. μa ranges from 10-2 mm-1 to 10-3 mm-1 and g from 0.95 to 0.99. The results of this study will be helpful in further understanding of optical properties of tissues.

  1. Correlation of in vivo and ex vivo 1H-MRI with histology in two severities of mouse spinal cord injury

    Science.gov (United States)

    Noristani, Harun N.; Lonjon, Nicolas; Cardoso, Maïda; Le Corre, Marine; Chan-Seng, Emilie; Captier, Guillaume; Privat, Alain; Coillot, Christophe; Goze-Bac, Christophe; Perrin, Florence E.

    2015-01-01

    Spinal cord injury (SCI) is a debilitating neuropathology with no effective treatment. Magnetic resonance imaging (MRI) technology is the only method used to assess the impact of an injury on the structure and function of the human spinal cord. Moreover, in pre-clinical SCI research, MRI is a non-invasive method with great translational potential since it provides relevant longitudinal assessment of anatomical and structural alterations induced by an injury. It is only recently that MRI techniques have been effectively used for the follow-up of SCI in rodents. However, the vast majority of these studies have been carried out on rats and when conducted in mice, the contusion injury model was predominantly chosen. Due to the remarkable potential of transgenic mice for studying the pathophysiology of SCI, we examined the use of both in and ex vivo 1H-MRI (9.4 T) in two severities of the mouse SCI (hemisection and over-hemisection) and documented their correlation with histological assessments. We demonstrated that a clear distinction between the two injury severities is possible using in and ex vivo 1H-MRI and that ex vivo MR images closely correlate with histology. Moreover, tissue modifications at a remote location from the lesion epicenter were identified by conventional ex vivo MRI analysis. Therefore, in vivo MRI has the potential to accurately identify in mice the progression of tissue alterations induced by SCI and is successfully implemented by ex vivo MRI examination. This combination of in and ex vivo MRI follow-up associated with histopathological assessment provides a valuable approach for further studies intended to evaluate therapeutic strategies on SCI. PMID:25798092

  2. Improved visualization of breast cancer features in multifocal carcinoma using phase-contrast and dark-field mammography: an ex vivo study

    Energy Technology Data Exchange (ETDEWEB)

    Grandl, Susanne; Sztrokay-Gaul, Aniko; Auweter, Sigrid D.; Hellerhoff, Karin [Ludwig-Maximilians-University Hospital Munich, Institute of Clinical Radiology, Munich (Germany); Scherer, Kai; Birnbacher, Lorenz; Willer, Konstantin; Chabior, Michael; Herzen, Julia; Pfeiffer, Franz [Technische Universitaet Muenchen, Department of Physics and Institute of Medical Engineering, Garching (Germany); Mayr, Doris [Ludwig-Maximilians-Universitaet Muenchen, Institute of Pathology, Munich (Germany); Bamberg, Fabian [University Hospital Tuebingen, Department of Diagnostic and Interventional Radiology, Tuebingen (Germany)

    2015-12-15

    Conventional X-ray attenuation-based contrast is inherently low for the soft-tissue components of the female breast. To overcome this limitation, we investigate the diagnostic merits arising from dark-field mammography by means of certain tumour structures enclosed within freshly dissected mastectomy samples. We performed grating-based absorption, absolute phase and dark-field mammography of three freshly dissected mastectomy samples containing bi- and multifocal carcinoma using a compact, laboratory Talbot-Lau interferometer. Preoperative in vivo imaging (digital mammography, ultrasound, MRI), postoperative histopathological analysis and ex vivo digital mammograms of all samples were acquired for the diagnostic verification of our results. In the diagnosis of multifocal tumour growth, dark-field mammography seems superior to standard breast imaging modalities, providing a better resolution of small, calcified tumour nodules, demarcation of tumour boundaries with desmoplastic stromal response and spiculated soft-tissue strands extending from an invasive ductal breast cancer. On the basis of selected cases, we demonstrate that dark-field mammography is capable of outperforming conventional mammographic imaging of tumour features in both calcified and non-calcified tumours. Presuming dose optimization, our results encourage further studies on larger patient cohorts to identify those patients that will benefit the most from this promising additional imaging modality. (orig.)

  3. Ufasomes nano-vesicles-based lyophilized platforms for intranasal delivery of cinnarizine: preparation, optimization, ex-vivo histopathological safety assessment and mucosal confocal imaging.

    Science.gov (United States)

    Salama, Alaa Hamed; Aburahma, Mona Hassan

    2016-09-01

    To circumvent the low and erratic absorption of orally administrated cinnarizine (CN), intranasal lyophilized gels containing unsaturated fatty acid liposomes (ufasomes) and encapsulating CN were prepared from oleic acid using a simple assembling strategy. The effects of varying drug concentration and cholesterol percentage on ufasomes size, polydispersity index and entrapment efficiency were investigated using 3(1)4(1) full factorial design. The optimized ufasomes that contained 14% cholesterol relative to oleic acid displayed spherical morphology with average size of 788 nm and entrapment efficiency of 80.49%. To overcome the colloidal instability of CN-loaded ufasomes dispersions and their short residence time in the nasal cavity, the ufasomes were incorporated into mucoadhesive hydrogels that were lyophilized into unit dosage forms for accurate dosing. Scanning electron micrographs of the lyophilized gel revealed that the included ufasomes were intact, non-aggregating and maintained their spherical morphology. Rheological characterization of reconstituted ufasomal lyophilized gel ensured ease of application. Furthermore, the gel induced minor histopathological alterations in sheeps' nasal mucosa. Ex-vivo confocal laser imaging confirmed the ability of ufasomes to penetrate deep through nasal mucosa layers. The results highlighted in the current work confirm the feasibility of using CN-loaded ufasomal gels for intranasal drug delivery.

  4. Update on donor assessment, resuscitation, and acceptance criteria, including novel techniques--non-heart-beating donor lung retrieval and ex vivo donor lung perfusion.

    Science.gov (United States)

    Yeung, Jonathan C; Cypel, Marcelo; Waddell, Thomas K; van Raemdonck, Dirk; Keshavjee, Shaf

    2009-05-01

    The shortage of adequate organ donors remains a great challenge in clinical lung transplantation. With increasing experience in the medical management and surgical technique of lung transplantation, gradual expansion of the criteria for lung donor selection has occurred with beneficial effects on the donor pool. Interest in donation after cardiac death also is increasing as the gap increases between donors and the needs of listed patients. Successful use of these new sources of lungs depends on the accurate assessment and prediction of transplanted lung function. Promising techniques for lung assessment and diagnostics include investigating key genes associated with graft failure or good graft performance using molecular approaches, and ex vivo evaluation. Further studies are needed to answer remaining questions about the best technique and solution to reperfuse human lungs for several hours without edema formation. As the predictive ability to discern good from injured donor lungs improves, strategies to repair donor lungs become increasingly important. Prolonged normothermic EVLP seems to be a platform on which many reparative strategies can be realized. With these new methods for assessing and resuscitating lungs accurately, it is hoped that inroads will be made toward providing every listed patient a chance for successful lung transplantation. PMID:19662970

  5. Improved visualization of breast cancer features in multifocal carcinoma using phase-contrast and dark-field mammography: an ex vivo study

    International Nuclear Information System (INIS)

    Conventional X-ray attenuation-based contrast is inherently low for the soft-tissue components of the female breast. To overcome this limitation, we investigate the diagnostic merits arising from dark-field mammography by means of certain tumour structures enclosed within freshly dissected mastectomy samples. We performed grating-based absorption, absolute phase and dark-field mammography of three freshly dissected mastectomy samples containing bi- and multifocal carcinoma using a compact, laboratory Talbot-Lau interferometer. Preoperative in vivo imaging (digital mammography, ultrasound, MRI), postoperative histopathological analysis and ex vivo digital mammograms of all samples were acquired for the diagnostic verification of our results. In the diagnosis of multifocal tumour growth, dark-field mammography seems superior to standard breast imaging modalities, providing a better resolution of small, calcified tumour nodules, demarcation of tumour boundaries with desmoplastic stromal response and spiculated soft-tissue strands extending from an invasive ductal breast cancer. On the basis of selected cases, we demonstrate that dark-field mammography is capable of outperforming conventional mammographic imaging of tumour features in both calcified and non-calcified tumours. Presuming dose optimization, our results encourage further studies on larger patient cohorts to identify those patients that will benefit the most from this promising additional imaging modality. (orig.)

  6. Ufasomes nano-vesicles-based lyophilized platforms for intranasal delivery of cinnarizine: preparation, optimization, ex-vivo histopathological safety assessment and mucosal confocal imaging.

    Science.gov (United States)

    Salama, Alaa Hamed; Aburahma, Mona Hassan

    2016-09-01

    To circumvent the low and erratic absorption of orally administrated cinnarizine (CN), intranasal lyophilized gels containing unsaturated fatty acid liposomes (ufasomes) and encapsulating CN were prepared from oleic acid using a simple assembling strategy. The effects of varying drug concentration and cholesterol percentage on ufasomes size, polydispersity index and entrapment efficiency were investigated using 3(1)4(1) full factorial design. The optimized ufasomes that contained 14% cholesterol relative to oleic acid displayed spherical morphology with average size of 788 nm and entrapment efficiency of 80.49%. To overcome the colloidal instability of CN-loaded ufasomes dispersions and their short residence time in the nasal cavity, the ufasomes were incorporated into mucoadhesive hydrogels that were lyophilized into unit dosage forms for accurate dosing. Scanning electron micrographs of the lyophilized gel revealed that the included ufasomes were intact, non-aggregating and maintained their spherical morphology. Rheological characterization of reconstituted ufasomal lyophilized gel ensured ease of application. Furthermore, the gel induced minor histopathological alterations in sheeps' nasal mucosa. Ex-vivo confocal laser imaging confirmed the ability of ufasomes to penetrate deep through nasal mucosa layers. The results highlighted in the current work confirm the feasibility of using CN-loaded ufasomal gels for intranasal drug delivery. PMID:25996631

  7. Quantitative structural markers of colorectal dysplasia in a cross sectional study of ex vivo murine tissue using label-free multiphoton microscopy

    Science.gov (United States)

    Prieto, Sandra P.; Greening, Gage J.; Lai, Keith K.; Muldoon, Timothy J.

    2016-03-01

    Two-photon excitation of label-free tissue is of increasing interest, as advances have been made in endoscopic clinical application of multiphoton microscopy, such as second harmonic generation (SHG) scanning endoscopy used to monitor cervical collagen in mice1. We used C57BL mice as a model to investigate the progression of gastrointestinal structures, specifically glandular area and circularity. We used multiphoton microscopy to image ex-vivo label-free murine colon, focusing on the collagen structure changes over time, in mice ranging from 10 to 20 weeks of age. Series of images were acquired within the colonic and intestinal tissue at depth intervals of 20 microns from muscularis to the epithelium, up to a maximum depth of 180 microns. The imaging system comprised a two-photon laser tuned to 800nm wavelength excitation, and the SHG emission was filtered with a 400/40 bandpass filter before reaching the photomultiplier tube. Images were acquired at 15 frames per second, for 200 to 300 cumulative frames, with a field of view of 261um by 261um, and 40mW at sample. Image series were compared to histopathology H&E slides taken from adjacent locations. Quantitative metrics for determining differences between murine glandular structures were applied, specifically glandular area and circularity.

  8. Pulsed ultrasound enhances the delivery of nitric oxide from bubble liposomes to ex vivo porcine carotid tissue

    Directory of Open Access Journals (Sweden)

    Sutton JT

    2014-10-01

    Full Text Available JT Sutton,1 JL Raymond,1 MC Verleye,2 GJ Pyne-Geithman,3 CK Holland4 1University of Cincinnati, Biomedical Engineering Program, Cincinnati, OH, 2University of Notre Dame Department of Chemical Engineering, Notre Dame, IN, 3University of Cincinnati, College of Medicine, Department of Neurosurgery and the University of Cincinnati Neuroscience Institute, and Mayfield Clinic, Cincinnati, OH, 4University of Cincinnati, College of Medicine, Internal Medicine, Division of Cardiovascular Diseases, Cincinnati, OH, USA Abstract: Ultrasound-mediated drug delivery is a novel technique for enhancing the penetration of drugs into diseased tissue beds noninvasively. By encapsulating drugs into microsized and nanosized liposomes, the therapeutic can be shielded from degradation within the vasculature until delivery to a target site by ultrasound exposure. Traditional in vitro or ex vivo techniques to quantify this delivery profile include optical approaches, cell culture, and electrophysiology. Here, we demonstrate an approach to characterize the degree of nitric oxide (NO delivery to porcine carotid tissue by direct measurement of ex vivo vascular tone. An ex vivo perfusion model was adapted to assess ultrasound-mediated delivery of NO. This potent vasodilator was coencapsulated with inert octafluoropropane gas to produce acoustically active bubble liposomes. Porcine carotid arteries were excised post mortem and mounted in a physiologic buffer solution. Vascular tone was assessed in real time by coupling the artery to an isometric force transducer. NO-loaded bubble liposomes were infused into the lumen of the artery, which was exposed to 1 MHz pulsed ultrasound at a peak-to-peak acoustic pressure amplitude of 0.34 MPa. Acoustic cavitation emissions were monitored passively. Changes in vascular tone were measured and compared with control and sham NO bubble liposome exposures. Our results demonstrate that ultrasound-triggered NO release from bubble liposomes

  9. Ex vivo susceptibility of Plasmodium falciparum isolates from Dakar, Senegal, to seven standard anti-malarial drugs

    Directory of Open Access Journals (Sweden)

    Pradines Bruno

    2011-10-01

    Full Text Available Abstract Background As a result of widespread chloroquine and sulphadoxine-pyrimethamine resistance, artemisinin-based combination therapy (ACT (which includes artemether-lumefantrine and artesunate-amodiaquine has been recommended as a first-line anti-malarial regimen in Senegal since 2006. Since then, there have been very few reports on the ex vivo susceptibility of Plasmodium falciparum to anti-malarial drugs. To examine whether parasite susceptibility has been affected by the widespread use of ACT, the ex vivo susceptibility of local isolates was assessed at the military hospital of Dakar. Methods The ex vivo susceptibility of 93 P. falciparum isolates from Dakar was successfully determined using the Plasmodium lactate dehydrogenase (pLDH ELISA for the following drugs: chloroquine (CQ, quinine (QN, mefloquine (MQ, monodesethylamodiaquine (MDAQ, lumefantrine (LMF, dihydroartemisinin (DHA and doxycycline (DOX. Results After transformation of the isolate IC50 in ratio of IC50 according to the susceptibility of the 3D7 reference strain (isolate IC50/3D7 IC50, the prevalence of the in vitro resistant isolates with reduced susceptibility was 50% for MQ, 22% for CQ, 12% for DOX, 6% for both QN and MDAQ and 1% for the drugs LMF and DHA. The highest significant positive correlations were shown between responses to CQ and MDAQ (r = 0.569; P r = 0.511; P r = 0.428; P = 0.0001, LMF and MQ (r = 0.413; P = 0.0002, QN and DHA (r = 0.402; P = 0.0003 and QN and MQ (r = 0.421; P = 0.0001. Conclusions The introduction of ACT in 2002 has not induced a decrease in P. falciparum susceptibility to the drugs DHA, MDAQ and LMF, which are common ACT components. However, the prevalence of P. falciparum isolates with reduced susceptibility has increased for both MQ and DOX. Taken together, these data suggest that intensive surveillance of the P. falciparum in vitro susceptibility to anti-malarial drugs in Senegal is required.

  10. Effect of different concentrations of fluoride in dentifrices on dentin erosion subjected or not to abrasion in situ/ex vivo

    OpenAIRE

    Magalhães, A C; Rios, D.; Moino, A L; Wiegand, A.; Attin, T.; Buzalaf, M.A.R.

    2008-01-01

    This in situ/ex vivo study assessed the effect of different concentrations of fluoride in dentifrices on dentin subjected to erosion or to erosion plus abrasion. Ten volunteers took part in this crossover and double-blind study performed in 3 phases (7 days). They wore acrylic palatal appliances containing 4 bovine dentin blocks divided in two rows: erosion and erosion plus abrasion. The blocks were subjected to erosion by immersion ex vivo in a cola drink (60 s, pH 2.6) 4 times daily. During...

  11. Cord Blood-Derived Hematopoietic Stem/Progenitor Cells: Current Challenges in Engraftment, Infection, and Ex Vivo Expansion

    Directory of Open Access Journals (Sweden)

    Katsuhiro Kita

    2011-01-01

    Full Text Available Umbilical cord blood has served as an alternative to bone marrow for hematopoietic transplantation since the late 1980s. Numerous clinical studies have proven the efficacy of umbilical cord blood. Moreover, the possible immaturity of cells in umbilical cord blood gives more options to recipients with HLA mismatch and allows for the use of umbilical cord blood from unrelated donors. However, morbidity and mortality rates associated with hematopoietic malignancies still remain relatively high, even after cord blood transplantation. Infections and relapse are the major causes of death after cord blood transplantation in patients with hematopoietic diseases. Recently, new strategies have been introduced to improve these major problems. Establishing better protocols for simple isolation of primitive cells and ex vivo expansion will also be very important. In this short review, we discuss several recent promising findings related to the technical improvement of cord blood transplantation.

  12. Cord Blood-Derived Hematopoietic Stem/Progenitor Cells: Current Challenges in Engraftment, Infection, and Ex Vivo Expansion

    Science.gov (United States)

    Kita, Katsuhiro; Lee, Jong O.; Finnerty, Celeste C.; Herndon, David N.

    2011-01-01

    Umbilical cord blood has served as an alternative to bone marrow for hematopoietic transplantation since the late 1980s. Numerous clinical studies have proven the efficacy of umbilical cord blood. Moreover, the possible immaturity of cells in umbilical cord blood gives more options to recipients with HLA mismatch and allows for the use of umbilical cord blood from unrelated donors. However, morbidity and mortality rates associated with hematopoietic malignancies still remain relatively high, even after cord blood transplantation. Infections and relapse are the major causes of death after cord blood transplantation in patients with hematopoietic diseases. Recently, new strategies have been introduced to improve these major problems. Establishing better protocols for simple isolation of primitive cells and ex vivo expansion will also be very important. In this short review, we discuss several recent promising findings related to the technical improvement of cord blood transplantation. PMID:21603139

  13. Tissue differentiation by diffuse reflectance spectroscopy for automated oral and maxillofacial laser surgery: ex vivo pilot study

    Science.gov (United States)

    Zam, Azhar; Stelzle, Florian; Tangermann-Gerk, Katja; Adler, Werner; Nkenke, Emeka; Schmidt, Michael; Douplik, Alexandre

    2010-02-01

    Remote laser surgery lacks of haptic feedback during the laser ablation of tissue. Hence, there is a risk of iatrogenic damage or destruction of anatomical structures like nerves or salivary glands. Diffuse reflectance spectroscopy provides a straightforward and simple approach for optical tissue differentiation. We measured diffuse reflectance from seven various tissue types ex vivo. We applied Linear Discriminant Analysis (LDA) to differentiate the seven tissue types and computed the area under the ROC curve (AUC). Special emphasis was taken on the identification of nerves and salivary glands as the most crucial tissue for maxillofacial surgery. The results show a promise for differentiating tissues as guidance for oral and maxillofacial laser surgery by means of diffuse reflectance.

  14. Modelling and characterization of photothermal effects assisted with gold nanorods in ex vivo samples and in a murine model

    Science.gov (United States)

    Lamela Rivera, Horacio; Rodríguez Jara, Félix; Cunningham, Vincent

    2011-03-01

    We discuss in this article the implementation of a laser-tissue interaction and bioheat-transfer 2-D finite-element model for Photothermal Therapy assisted with Gold Nanorods. We have selected Gold Nanorods as absorbing nanostructures in order to improve the efficiency of using compact diode lasers because of their high opto-thermal conversion efficiency at 808 and 850 nm. The goal is to model the distribution of the optical energy among the tissue including the skin absorption effects and the tissue thermal response, with and without the presence of Gold Nanorods. The heat generation due to the optical energy absorption and the thermal propagation will be computationally modeled and optimized. The model has been evaluated and compared with experimental ex-vivo data in fresh chicken muscle samples and in-vivo BALB/c mice animal model.

  15. An ex vivo rat eye model to aid development of high-resolution retina imaging devices for rodents

    Science.gov (United States)

    van Oterendorp, Christian; Martin, Keith R.; Zhong, Jiang Jian; Diaz-Santana, Luis

    2010-09-01

    High resolution in vivo retinal imaging in rodents is becoming increasingly important in eye research. Development of suitable imaging devices currently requires many lengthy animal procedures. We present an ex vivo rat model eye with fluorescently labelled retinal ganglion cells (RGC) and nerve fibre bundles that reduces the need for animal procedures while preserving key properties of the living rat eye. Optical aberrations and scattering of four model eyes and eight live rat eyes were quantified using a Shack-Hartmann sensor. Fluorescent images from RGCs were obtained using a prototype scanning laser ophthalmoscope. The wavefront aberration root mean square value without defocus did not significantly differ between model and living eyes. Higher order aberrations were slightly higher but RGC image quality was comparable to published in vivo work. Overall, the model allows a large reduction in number and duration of animal procedures required to develop new in vivo retinal imaging devices.

  16. Effect of Coffea canephora aqueous extract on microbial counts in ex vivo oral biofilms: a case study.

    Science.gov (United States)

    Antonio, Andréa Gonçalves; Iorio, Natália Lopes Pontes; Farah, Adriana; Netto dos Santos, Kátia Regina; Maia, Lucianne Cople

    2012-05-01

    In the present study, the ex vivo antimicrobial effect of brewed coffee was tested on oral biofilms. For this, unsweetened and sweetened (10 % sucrose) brewed light-roasted Coffea canephora at 20 % was used in biofilms formed by non-stimulated saliva from three volunteers. After 30 min contact with unsweetened and sweetened brews, the average microorganism count in the biofilms reduced by 15.2 % and 12.4 %, respectively, with no statistical difference among them. We also observed a drop of microorganisms in the biofilms after treatment with sucrose solution at 5 % compared to control (saline) and to sucrose at 1 % and 3 %. In conclusion, Coffea canephora extract reduces the microbial count in oral biofilm, and our data suggest that sucrose concentration in coffee brew can influence its antimicrobial property against the referred biofilm.

  17. Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins during ex vivo Osteoblast Differentiation of Human Stromal Stem Cells

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Chen, Li; Nielsen, Maria Overbeck;

    2012-01-01

    labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited...... more than 2-fold up or down-regulation. The pulsed SILAC method revealed a strong correlation between the fraction of isotope labeling and the subset of proteins known to be secreted and involved in OB differentiation. We verified SILAC data using qRT-PCR analysis of 9 identified potential novel......It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process...

  18. Assessing ex vivo dental biofilms and in vivo composite restorations using cross-polarization optical coherence tomography

    Science.gov (United States)

    Jones, R.; Aparicio, C.; Chityala, R.; Chen, R.; Fok, A.; Rudney, J.

    2012-01-01

    A cross-polarization 1310-nm optical coherence tomography system (CP-OCT), using a beam splitter based design, was used to assess ex vivo growth of complex multi-species dental biofilms. These biofilm microcosms were derived from plaque samples along the interface of composite or amalgam restoration in children with a history of early childhood caries. This paper presents a method of measuring the mean biofilm height of mature biofilms using CP-OCT. For our in vivo application, the novel swept source based CP-OCT intraoral probe (Santec Co. Komaki, Japan) dimensions and system image acquisition speed (20 image frames/second) allowed imaging pediatric subjects as young as 4 years old. The subsurface enamel under the interface of composite resin restorations of pediatric subjects were imaged using CP-OCT. Cavitated secondary caries is clearly evident from sound resin composite restorations.

  19. Biomimetic aggrecan reduces cartilage extracellular matrix from degradation and lowers catabolic activity in ex vivo and in vivo models.

    Science.gov (United States)

    Sharma, Shaili; Lee, Aeju; Choi, Kuiwon; Kim, Kwangmeyung; Youn, Inchan; Trippel, Stephen B; Panitch, Alyssa

    2013-09-01

    Aggrecan, a major macromolecule in cartilage, protects the extracellular matrix (ECM) from degradation during the progression of osteoarthritis (OA). However, aggrecan itself is also susceptible to proteolytic cleavage. Here, the use of a biomimetic proteoglycan (mAGC) is presented, which functionally mimics aggrecan but lacks the known cleavage sites, protecting the molecule from proteolytic degradation. The objective of this study is to test the efficacy of this molecule in ex vivo (human OA synovial fluid) and in vivo (Sprague-Dawley rats) osteoarthritic models. These results indicate that mAGC's may protect articular cartilage against the loss of key ECM components, and lower catabolic protein and gene expression in both models. This suppression of matrix degradation has the potential to provide a healthy environment for tissue repair.

  20. Radiosynthesis and ex vivo evaluation of [11C-carbonyl]carbamate- and urea-based monoacylglycerol lipase inhibitors

    International Nuclear Information System (INIS)

    Introduction: Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are the two primary enzymes that regulate the tone of endocannabinoid signaling. Although new PET radiotracers have been discovered for imaging FAAH in vivo, no such radiotracer exists for imaging MAGL. Here we report the radiosynthesis of five candidate MAGL radiotracers and their ex vivo evaluations in mice and rats. Methods: Candidate carbamate and urea MAGL inhibitors were radiolabeled at the carbonyl position by [11C]CO2 fixation. Radiotracers were administered (tail-vein injection) to rodents and brain uptake of radioactivity measured at early and late time points ex vivo. Specificity of uptake was explored by pretreatment with unlabeled inhibitors (2 mg/kg, ip) 30 min prior to radiotracer administration. Results: All five candidate MAGL radiotracers were prepared in high specific activity (> 65 GBq/μmol) and radiochemical purity (> 98%). Moderate brain uptake (0.2–0.8 SUV) was observed for each candidate while pretreatment did not reduce uptake for four of the five tested. For two candidates ([11C]12 and [11C]14), high retention of radioactivity was observed in the blood (ca. 10 and 4 SUV at 40 min) which was blocked by pretreatment with unlabeled inhibitors. The most promising candidate, [11C]18, demonstrated moderate brain uptake (ca. 0.8 SUV) which showed circa 50% blockade by pretreatment with unlabeled 18. Conclusion: One putative and four reported potent and selective MAGL inhibitors have been radiolabeled via [11C]CO2 fixation as radiotracers for this enzyme. Despite the promising in vitro pharmacological profile, none of the five candidate radiotracers exhibited in vivo behavior suitable for PET neuroimaging

  1. Preserved ex vivo inflammatory status in decidual cells from women with preterm labor and subclinical intrauterine infection.

    Directory of Open Access Journals (Sweden)

    Violeta Castro-Leyva

    Full Text Available OBJECTIVE: To compare the inflammatory response preserved ex vivo by decidual cells isolated from women who experienced preterm labor with and without subclinical intrauterine infection. METHODS: Fetal membranes were obtained after cesarean section from 35 women who delivered before 37 weeks of gestation following spontaneous preterm labor, with no clinical evidence of intrauterine infection. Decidua was microbiologically tested and cultured. Concentrations of anti-inflammatory cytokines (IL-2, IL-4, IL-10, pro-inflammatory cytokines (IL-6, IL-8, IL-1β and TNF-α, and matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9 were measured in the supernatants using Bio-Plex, and prostaglandin E(2 (PGE(2 was measured by enzyme immunoassay. RESULTS: Subclinical infection was confirmed in 10 women (28.5%. Microorganisms isolated were Ureaplasma urealyticum (4, group B streptococci (3, Gardnerella vaginalis (1, and Escherichia coli (2. We found a significant increase of pro-inflammatory cytokines and a significant decrease of anti-inflammatory cytokines in supernatants from decidual cells obtained from women with preterm labor and subclinical intrauterine infection compared to women without infection. Secretion of MMP-1, MMP-8, MMP-9 and PGE(2 was significantly higher in infected women. Secretion of IL-8 by decidual cells from infected women persisted upon repeated in vitro culture passages. CONCLUSIONS: Almost 30% of idiopathic preterm labor cases were associated with subclinical intrauterine infection, and decidual cells isolated from these cases preserved an ex vivo inflammatory status after in vivo bacterial exposure.

  2. Downregulation of glutamine synthetase via GLAST suppression induces retinal axonal swelling in a rat ex vivo hydrostatic pressure model.

    Science.gov (United States)

    Ishikawa, Makoto; Yoshitomi, Takeshi; Zorumski, Charles F; Izumi, Yukitoshi

    2011-08-01

    PURPOSE. High levels of glutamate can be toxic to retinal GCs. Thus, effective buffering of extracellular glutamate is important in preserving retinal structure and function. GLAST, a major glutamate transporter in the retina, and glutamine synthetase (GS) regulate extracellular glutamate accumulation and prevent excitotoxicity. This study was an examination of changes in function and expression of GLAST and GS in ex vivo rat retinas exposed to acute increases in ambient pressure. METHODS. Ex vivo rat retinas were exposed to elevated hydrostatic pressure for 24 hours. The expression of GLAST and GS were examined using immunochemistry and real-time PCR analysis. Also examined were the effects of (2S,3S)-3-[3-[4-(trifluoromethyl) benzoylamino] benzyloxy] aspartate (TFB-TBOA), an inhibitor of glutamate transporters, and l-methionine-S-sulfoximine (MSO), an inhibitor of GS. RESULTS. In this acute model, Western blot and real-time RT-PCR analyses revealed that substantially (75 mm Hg), but not moderately (35 mm Hg), elevated pressure depressed GLAST expression, diminished GS activity, and induced axonal swelling between the GC layer and the inner limiting membrane. However, at the moderately elevated pressure (35 mm Hg), administration of either TFB-TBOA or MSO also induced axonal swelling and excitotoxic neuronal damage. MSO did not depress GLAST expression but TFB-TBOA significantly suppressed GS, suggesting that downregulation of GS during pressure loading may result from impaired GLAST expression. CONCLUSIONS. The retina is at risk during acute intraocular pressure elevation due to downregulation of GS activity resulting from depressed GLAST expression. PMID:21775659

  3. Bidirectional Transfer Study of Polystyrene Nanoparticles across the Placental Barrier in an ex Vivo Human Placental Perfusion Model

    Science.gov (United States)

    Grafmueller, Stefanie; Manser, Pius; Diener, Liliane; Diener, Pierre-André; Maeder-Althaus, Xenia; Maurizi, Lionel; Jochum, Wolfram; Krug, Harald F.; Buerki-Thurnherr, Tina; von Mandach, Ursula

    2015-01-01

    Background Nanoparticle exposure in utero might not be a major concern yet, but it could become more important with the increasing application of nanomaterials in consumer and medical products. Several epidemiologic and in vitro studies have shown that nanoparticles can have potential toxic effects. However, nanoparticles also offer the opportunity to develop new therapeutic strategies to treat specifically either the pregnant mother or the fetus. Previous studies mainly addressed whether nanoparticles are able to cross the placental barrier. However, the transport mechanisms underlying nanoparticle translocation across the placenta are still unknown. Objectives In this study we examined which transport mechanisms underlie the placental transfer of nanoparticles. Methods We used the ex vivo human placental perfusion model to analyze the bidirectional transfer of plain and carboxylate modified polystyrene particles in a size range between 50 and 300 nm. Results We observed that the transport of polystyrene particles in the fetal to maternal direction was significantly higher than for the maternal to fetal direction. Regardless of their ability to cross the placental barrier and the direction of perfusion, all polystyrene particles accumulated in the syncytiotrophoblast of the placental tissue. Conclusions Our results indicate that the syncytiotrophoblast is the key player in regulating nanoparticle transport across the human placenta. The main mechanism underlying this translocation is not based on passive diffusion, but is likely to involve an active, energy-dependent transport pathway. These findings will be important for reproductive toxicology as well as for pharmaceutical engineering of new drug carriers. Citation Grafmueller S, Manser P, Diener L, Diener PA, Maeder-Althaus X, Maurizi L, Jochum W, Krug HF, Buerki-Thurnherr T, von Mandach U, Wick P. 2015. Bidirectional transfer study of polystyrene nanoparticles across the placental barrier in an ex vivo human

  4. Ex Vivo and In Vivo Mice Models to Study Blastocystis spp. Adhesion, Colonization and Pathology: Closer to Proving Koch's Postulates.

    Science.gov (United States)

    Ajjampur, Sitara S R; Png, Chin Wen; Chia, Wan Ni; Zhang, Yongliang; Tan, Kevin S W

    2016-01-01

    Blastocystis spp. are widely prevalent extra cellular, non-motile anerobic protists that inhabit the gastrointestinal tract. Although Blastocystis spp. have been associated with gastrointestinal symptoms, irritable bowel syndrome and urticaria, their clinical significance has remained controversial. We established an ex vivo mouse explant model to characterize adhesion in the context of tissue architecture and presence of the mucin layer. Using confocal microscopy with tissue whole mounts and two axenic isolates of Blastocystis spp., subtype 7 with notable differences in adhesion to intestinal epithelial cells (IEC), isolate B (ST7-B) and isolate H (more adhesive, ST7-H), we showed that adhesion is both isolate dependent and tissue trophic. The more adhesive isolate, ST7-H was found to bind preferentially to the colon tissue than caecum and terminal ileum. Both isolates were also found to have mucinolytic effects. We then adapted a DSS colitis mouse model as a susceptible model to study colonization and acute infection by intra-caecal inoculation of trophic Blastocystis spp.cells. We found that the more adhesive isolate ST7-H was also a better colonizer with more mice shedding parasites and for a longer duration than ST7-B. Adhesion and colonization was also associated with increased virulence as ST7-H infected mice showed greater tissue damage than ST7-B. Both the ex vivo and in vivo models used in this study showed that Blastocystis spp. remain luminal and predominantly associated with mucin. This was further confirmed using colonic loop experiments. We were also successfully able to re-infect a second batch of mice with ST7-H isolates obtained from fecal cultures and demonstrated similar histopathological findings and tissue damage thereby coming closer to proving Koch's postulates for this parasite. PMID:27508942

  5. An ex vivo laser-induced spinal cord injury model to assess mechanisms of axonal degeneration in real-time.

    Science.gov (United States)

    Okada, Starlyn L M; Stivers, Nicole S; Stys, Peter K; Stirling, David P

    2014-01-01

    Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular

  6. Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in Ex vivo Organ Culture

    Directory of Open Access Journals (Sweden)

    Li-Guo Zhu

    2016-01-01

    Methods: IVD motion segments were harvested from rabbit lumbar spines and cultured in no-loading 6-well plates (control conditions or custom-made apparatuses under a constant, compressive load (3 kg, 0.5 MPa for up to 14 days. Tissue integrity, matrix synthesis, and the matrix gene expression profile were assessed after 3, 7, and 14 days of culturing and compared with those of fresh tissues. Results: The results showed that ex vivo culturing of motion segments preserved tissue integrity under no-loading conditions for 14 days whereas the static load gradually destroyed the morphology after 3 days. Proteoglycan contents were decreased under both conditions, with a more obvious decrease under static load, and proteoglycan gene expression was also downregulated. However, under static load, immunohistochemical staining intensity and collagen Type II alpha 1 (COL2A1 gene expression were significantly enhanced (61.54 ± 5.91, P = 0.035 and upregulated (1.195 ± 0.040, P = 0.000, respectively, compared with those in the controls (P < 0.05. In contrast, under constant compression, these trends were reversed. Our initial results indicated that short-term static load stimulated the synthesis of collagen Type II alpha 1; however, sustained constant compression led to progressive degeneration and specifically to a decreased proteoglycan content. Conclusions: A loading and organ culture system for ex vivo rabbit IVD motion segments was developed. Using this system, we were able to study the effects of mechanical stimulation on the biology of IVDs, as well as the pathomechanics of IVD degeneration.

  7. Preliminary in vivo and ex vivo evaluation of the 5-HT2A imaging probe [18F]MH.MZ

    International Nuclear Information System (INIS)

    Introduction: The 5-HT2A receptor is one of the most interesting targets within the serotonergic system because it is involved in a number of important physiological processes and diseases. Methods: [18F]MH.MZ, a 5-HT2A antagonistic receptor ligand, is labeled by 18F-fluoroalkylation of the corresponding desmethyl analogue MDL 105725 with 2-[18F]fluoroethyltosylate ([18F]FETos). In vitro binding experiments were performed to test selectivity toward a broad spectrum of neuroreceptors by radioligand binding assays. Moreover, first micro-positron emission tomography (μPET) experiments, ex vivo organ biodistribution, blood cell and protein binding and brain metabolism studies of [18F]MH.MZ were carried out in rats. Results: [18F]MH.MZ showed a Ki of 3 nM toward the 5-HT2A receptor and no appreciable affinity for a variety of receptors and transporters. Ex vivo biodistribution as well as μPET showed highest brain uptake at ∼5 min p.i. and steady state after ∼30 min p.i. While [18F]MH.MZ undergoes extensive first-pass metabolism which significantly reduces its bioavailability, it is insignificantly metabolized within the brain. The binding potential in the rat frontal cortex is 1.45, whereas the cortex to cerebellum ratio was determined to be 2.7 after ∼30 min. Conclusion: Results from μPET measurements of [18F]MH.MZ are in no way inferior to data known for [11C]MDL 100907 at least in rats. [18F]MH.MZ appears to be a highly potent and selective serotonergic PET ligand in small animals.

  8. Plasmodium falciparum Polymorphisms associated with ex vivo drug susceptibility and clinical effectiveness of artemisinin-based combination therapies in Benin.

    Science.gov (United States)

    Dahlström, Sabina; Aubouy, Agnès; Maïga-Ascofaré, Oumou; Faucher, Jean-François; Wakpo, Abel; Ezinmègnon, Sèm; Massougbodji, Achille; Houzé, Pascal; Kendjo, Eric; Deloron, Philippe; Le Bras, Jacques; Houzé, Sandrine

    2014-01-01

    Artemisinin-based combination therapies (ACTs) are the main option to treat malaria, and their efficacy and susceptibility must be closely monitored to avoid resistance. We assessed the association of Plasmodium falciparum polymorphisms and ex vivo drug susceptibility with clinical effectiveness. Patients enrolled in an effectiveness trial comparing artemether-lumefantrine (n = 96), fixed-dose artesunate-amodiaquine (n = 96), and sulfadoxine-pyrimethamine (n = 48) for the treatment of uncomplicated malaria 2007 in Benin were assessed. pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps polymorphisms were analyzed pretreatment and in recurrent infections. Drug susceptibility was determined in fresh baseline isolates by Plasmodium lactate dehydrogenase enzyme-linked immunosorbent assay (ELISA). A majority had 50% inhibitory concentration (IC50) estimates (the concentration required for 50% growth inhibition) lower than those of the 3D7 reference clone for desethylamodiaquine, lumefantrine, mefloquine, and quinine and was considered to be susceptible, while dihydroartemisinin and pyrimethamine IC50s were higher. No association was found between susceptibility to the ACT compounds and treatment outcome. Selection was observed for the pfmdr1 N86 allele in artemether-lumefantrine recrudescences (recurring infections) (4/7 [57.1%] versus 36/195 [18.5%]), and of the opposite allele, 86Y, in artesunate-amodiaquine reinfections (new infections) (20/22 [90.9%] versus 137/195 [70.3%]) compared to baseline infections. The importance of pfmdr1 N86 in lumefantrine tolerance was emphasized by its association with elevated lumefantrine IC50s. Genetic linkage between N86 and Y184 was observed, which together with the low frequency of 1246Y may explain regional differences in selection of pfmdr1 loci. Selection of opposite alleles in artemether-lumefantrine and artesunate-amodiaquine recurrent infections supports the strategy of multiple first-line treatment. Surveillance based on clinical, ex

  9. Ex Vivo and In Vivo Mice Models to Study Blastocystis spp. Adhesion, Colonization and Pathology: Closer to Proving Koch's Postulates

    Science.gov (United States)

    Ajjampur, Sitara S. R.; Png, Chin Wen; Chia, Wan Ni; Zhang, Yongliang; Tan, Kevin S. W.

    2016-01-01

    Blastocystis spp. are widely prevalent extra cellular, non-motile anerobic protists that inhabit the gastrointestinal tract. Although Blastocystis spp. have been associated with gastrointestinal symptoms, irritable bowel syndrome and urticaria, their clinical significance has remained controversial. We established an ex vivo mouse explant model to characterize adhesion in the context of tissue architecture and presence of the mucin layer. Using confocal microscopy with tissue whole mounts and two axenic isolates of Blastocystis spp., subtype 7 with notable differences in adhesion to intestinal epithelial cells (IEC), isolate B (ST7-B) and isolate H (more adhesive, ST7-H), we showed that adhesion is both isolate dependent and tissue trophic. The more adhesive isolate, ST7-H was found to bind preferentially to the colon tissue than caecum and terminal ileum. Both isolates were also found to have mucinolytic effects. We then adapted a DSS colitis mouse model as a susceptible model to study colonization and acute infection by intra-caecal inoculation of trophic Blastocystis spp.cells. We found that the more adhesive isolate ST7-H was also a better colonizer with more mice shedding parasites and for a longer duration than ST7-B. Adhesion and colonization was also associated with increased virulence as ST7-H infected mice showed greater tissue damage than ST7-B. Both the ex vivo and in vivo models used in this study showed that Blastocystis spp. remain luminal and predominantly associated with mucin. This was further confirmed using colonic loop experiments. We were also successfully able to re-infect a second batch of mice with ST7-H isolates obtained from fecal cultures and demonstrated similar histopathological findings and tissue damage thereby coming closer to proving Koch’s postulates for this parasite. PMID:27508942

  10. Preparation, quality criteria, and properties of human blood platelet lysate supplements for ex vivo stem cell expansion.

    Science.gov (United States)

    Shih, Daniel Tzu-Bi; Burnouf, Thierry

    2015-01-25

    Most clinical applications of human multipotent mesenchymal stromal cells (MSCs) for cell therapy, tissue engineering, regenerative medicine, and treatment of immune and inflammatory diseases require a phase of isolation and ex vivo expansion allowing a clinically meaningful cell number to be reached. Conditions used for cell isolation and expansion should meet strict quality and safety requirements. This is particularly true for the growth medium used for MSC isolation and expansion. Basal growth media used for MSC expansion are supplemented with multiple nutrients and growth factors. Fetal bovine serum (FBS) has long been the gold standard medium supplement for laboratory-scale MSC culture. However, FBS has a poorly characterized composition and poses risk factors, as it may be a source of xenogenic antigens and zoonotic infections. FBS has therefore become undesirable as a growth medium supplement for isolating and expanding MSCs for human therapy protocols. In recent years, human blood materials, and most particularly lysates and releasates of platelet concentrates have emerged as efficient medium supplements for isolating and expanding MSCs from various origins. This review analyzes the advantages and limits of using human platelet materials as medium supplements for MSC isolation and expansion. We present the modes of production of allogeneic and autologous platelet concentrates, measures taken to ensure optimal pathogen safety profiles, and methods of preparing PLs for MSC expansion. We also discuss the supply of such blood preparations. Produced under optimal conditions of standardization and safety, human platelet materials can become the future 'gold standard' supplement for ex vivo production of MSCs for translational medicine and cell therapy applications.

  11. Gene expression patterns related to osteogenic differentiation of bone marrow-derived mesenchymal stem cells during ex vivo expansion.

    Science.gov (United States)

    Granchi, Donatella; Ochoa, Gorka; Leonardi, Elisa; Devescovi, Valentina; Baglìo, Serena Rubina; Osaba, Lourdes; Baldini, Nicola; Ciapetti, Gabriela

    2010-06-01

    Bone marrow is commonly used as a source of adult multipotent mesenchymal stem cells (MSCs), defined for their ability to differentiate in vitro into multiple lineages. The ex vivo-expanded MSCs are currently being evaluated as a strategy for the restoration of function in damaged skeletal tissue, both in cell therapy and tissue engineering applications. The aim of this study was to define gene expression patterns underlying the differentiation of MSCs into mature osteoblasts during the expansion in vitro, and to explore a variety of cell functions that cannot be easily evaluated using morphological, cytochemical, and biochemical assays. Cell cultures were obtained from bone marrow samples of six individuals undergoing total hip replacement, and a large-scale transcriptome analysis, using Affymetrix HG-U133A Plus 2.0 array (Affymetrix((R)), Santa Clara, CA), was performed at the occurrence of specific events, including the appearance of MSC surface markers, formation of colonies, and deposition of mineral nodules. We focused our attention on 213 differentially upregulated genes, some belonging to well-known pathways and some having one or more Gene Ontology annotations related to bone cell biology, including angiogenesis, bone-related genes, cell communication, development and morphogenesis, transforming growth factor-beta signaling, and Wnt signaling. Twenty-nine genes, whose role in bone cell pathophysiology has not been described yet, were found. In conclusion, gene expression patterns that characterize the early, intermediate, and late phases of the osteogenic differentiation process of ex vivo-expanded MSCs were defined. These signatures represent a useful tool to monitor the osteogenic process, and to analyze a broad spectrum of functions of MSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration.

  12. Transdermal glimepiride delivery system based on optimized ethosomal nano-vesicles: Preparation, characterization, in vitro, ex vivo and clinical evaluation.

    Science.gov (United States)

    Ahmed, Tarek A; El-Say, Khalid M; Aljaeid, Bader M; Fahmy, Usama A; Abd-Allah, Fathy I

    2016-03-16

    This work aimed to develop an optimized ethosomal formulation of glimepiride then loading into transdermal films to offer lower drug side effect, extended release behavior and avoid first pass effect. Four formulation factors were optimized for their effects on vesicle size (Y1), entrapment efficiency (Y2) and vesicle flexibility (Y3). Optimum desirability was identified and, an optimized formulation was prepared, characterized and loaded into transdermal films. Ex-vivo permeation study for the prepared films was conducted and, the permeation parameters and drug permeation mechanism were identified. Penetration through rat skin was studied using confocal laser microscope. In-vivo study was performed following transdermal application on human volunteers. The percent of alcohol was significantly affecting all the studied responses while the other factors and their interaction effects were varied on their effects on each response. The optimized ethosomal formulation showed observed values for Y1, Y2 and Y3 of 61 nm, 97.12% and 54.03, respectively. Ex-vivo permeation of films loaded with optimized ethosomal formulation was superior to that of the corresponding pure drug transdermal films and this finding was also confirmed after confocal laser microscope study. Permeation of glimepiride from the prepared films was in favor of Higushi-diffusion model and exhibited non-Fickian or anomalous release mechanism. In-vivo study revealed extended drug release behavior and lower maximum drug plasma level from transdermal films loaded with drug ethosomal formulation. So, the ethosomal formulation could be considered a suitable drug delivery system especially when loaded into transdermal vehicle with possible reduction in side effects and controlling the drug release. PMID:26775063

  13. Development of lovastatin-loaded poly(lactic acid microspheres for sustained oral delivery: in vitro and ex vivo evaluation

    Directory of Open Access Journals (Sweden)

    Guan QG

    2015-02-01

    Full Text Available Qigang Guan,1 Wei Chen,2 Xianming Hu2 1Department of Cardiology, The First Affiliated Hospital of China Medical University, Shenyang, People’s Republic of China; 2Department of Pharmaceutical, Shenyang Institute of Pharmaceutical Industry, Shenyang, People’s Republic of China Background: A novel lovastatin (LVT-loaded poly(lactic acid microsphere suitable for oral administration was developed in this study, and in vitro and in vivo characteristics were evaluated. Methods: The designed microspheres were obtained by an improved emulsion-solvent evaporation method. The morphological examination, particle size, encapsulation ratio, drug loading, and in vitro release were characterized. Pharmacokinetics studies were used to show that microspheres possess more advantages than the conventional formulations. Results: By using the emulsion-solvent evaporation method, it was simple to prepare microspheres and easy to scale up production. The morphology of formed microspheres showed a spherical shape with a smooth surface, without any particle aggregation. Mean size of the microspheres was 2.65±0.69 µm; the encapsulation efficiency was 92.5%±3.6%, and drug loading was 16.7%±2.1%. In vitro release indicated that the LVT microspheres had a well-sustained release efficacy, and ex vivo studies showed that after LVT was loaded to microspheres, the area under the plasma concentration-time curve from zero to the last measurable plasma concentration point and the extrapolation to time infinity increased significantly, which represented 2.63-fold and 2.49-fold increases, respectively, compared to suspensions. The rate of ex vivo clearance was significantly reduced. Conclusion: This research proved that poly(lactic acid microspheres can significantly prolong the drug circulation time in vivo and can also significantly increase the relative bioavailability of the drug. Keywords: lovastatin, microspheres, PLA, in vitro release, pharmacokinetics 

  14. Root canals decontamination by coherent photons initiated photoacustic streaming (PIPS) of irrigants: an ex-vivo study

    Science.gov (United States)

    Pedullà, E.; Genovese, C.; Scolaro, C.; Cutroneo, M.; Tempera, G.; Rapisarda, E.; Torrisi, L.

    2014-04-01

    The aim of this ex vivo study was to assess the antibacterial effectiveness of coherent photon initiated photoacoustic streaming (PIPS) of irrigants using an Er:YAG laser equipped with a newly designed, stripped and tapered, tip in extracted teeth with infected root canals. One hundred-forty-eight single-rooted extracted teeth were prepared using a rotary abrasive instrument providing a root channel with a suitable size. The samples were sterilized and all teeth except ten (negative control group) were inoculated with Enterococcus faecalis and incubated in a CO2 chamber at 37°C for 15 days in Eppendorff tubes filled with trypticase soy broth medium changed every 2 days. Infected teeth were then randomly divided into 4 test groups (n=32 for each): pulsed erbium:YAG laser at non-ablative settings for 30 seconds with sterile bi-distilled water (Group A) or 5% sodium hypochlorite (NaOCl) (Group B); without laser activated sterile bi-distilled water irrigation for 30 seconds (Group C) or 5% NaOCl irrigation for 30 seconds (Group D); the positive control group received no treatment in infected teeth (n=10). Colony-forming units (CFUs) were counted from bacteriologic samples taken before (S1) and after treatment (S2). Data were analyzed by Kruskal-Wallis and post hoc Dunn's multiple comparison tests. CFU counts were significantly lower in groups B and D than in group C (P0.05). None of the four groups predictably generated negative samples. Under the conditions of this ex vivo study, statistically significant difference wasn't found in planktonic bacteria reduction between the laser and NaOCl or NaOCl alone groups.

  15. Ex vivo pretreatment with melatonin improves survival, proangiogenic/mitogenic activity, and efficiency of mesenchymal stem cells injected into ischemic kidney.

    Science.gov (United States)

    Mias, Céline; Trouche, Elodie; Seguelas, Marie-Hélène; Calcagno, Fabien; Dignat-George, Françoise; Sabatier, Florence; Piercecchi-Marti, Marie-Dominique; Daniel, Laurent; Bianchi, Pascale; Calise, Denis; Bourin, Philippe; Parini, Angelo; Cussac, Daniel

    2008-07-01

    Bone marrow mesenchymal stem cells (MSCs) have shown great potential in cell therapy of solid organs. Approaches to improving the ability of grafted MSCs to survive and secrete paracrine factors represent one of the challenges for the further development of this novel therapy. In the present study, we designed a strategy of ex vivo pretreatment with the pineal hormone melatonin to improve survival, paracrine activity, and efficiency of MSCs. Using a rat model of acute renal failure, we showed that melatonin pretreatment strongly increased survival of MSCs after intraparenchymal injection. This effect was concomitant with overstimulation of angiogenesis, proliferation of renal cells, and accelerated recovery of renal function. To gain insight into the mechanisms involved in the effects observed in vivo, melatonin was tested in vitro on cultured MSCs. Our results show that through stimulation of specific melatonin receptors, melatonin induced an overexpression of the antioxidant enzyme catalase and superoxide dismutase-1 and increased the resistance of MSCs to hydrogen peroxide-dependent apoptosis. Compared with untreated cells, MSCs incubated with melatonin displayed a higher expression of basic fibroblast growth factor and hepatocyte growth factor. In addition, conditioned culture media from melatonin-treated MSCs stimulated tube formation by endothelial progenitor cells and proliferation of proximal tubule cells in culture. In conclusion, our results show that melatonin behaves as a preconditioning agent increasing survival, paracrine activity, and efficiency of MSCs. The use of this molecule for pretreatment of stem cells may represent a novel and safe approach to improving the beneficial effects of cell therapy of solid organs.

  16. EFFECT OF OIL COMBUSTION PARTICLE BIOAVAILABLE CONSTITUENTS ON EX VIVO VASCULAR FUNCTION OF AORTAS RECOVERED FROM NORMAL AND TYPE 2 DIABETIC RATS

    Science.gov (United States)

    Effect of Oil Combustion Particle Bioavailable Constituents on Ex Vivo Vascular Function of Aortae Recovered from Healthy and Early Type 2 Diabetic RatsKL Dreher1, SE Kelly2, SD Proctor2, and JC Russell2. 1National Health and Environmental Effects Laboratory, US EPA, RTP, NC;...

  17. Normothermic ex vivo lung perfusion of non-heart-beating donor lungs in pigs : from pretransplant function analysis towards a 6-h machine preservation

    NARCIS (Netherlands)

    Erasmus, ME; Fernhout, MH; Elstrodt, JM; Rakhorst, G

    2006-01-01

    Donor shortage urges optimal use of all lungs available. Ex vivo lung perfusion (EVLP) is a method to evaluate lung function before implantation. EVLP was performed in pigs to evaluate lung function, using two different clinical non-heart-beating (NHS) donor protocols: flush perfusion and topical co

  18. Ex vivo intracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants.

    Science.gov (United States)

    Raissadati, Alireza; Jokinen, Janne J; Syrjälä, Simo O; Keränen, Mikko A I; Krebs, Rainer; Tuuminen, Raimo; Arnaudova, Ralica; Rouvinen, Eeva; Anisimov, Andrey; Soronen, Jarkko; Pajusola, Katri; Alitalo, Kari; Nykänen, Antti I; Lemström, Karl

    2013-11-01

    Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies.

  19. Astaxanthin, a Carotenoid, Stimulates Immune Responses by Enhancing IFN-γ and IL-2 Secretion in Primary Cultured Lymphocytes in Vitro and ex Vivo

    Directory of Open Access Journals (Sweden)

    Kuan-Hung Lin

    2015-12-01

    Full Text Available Astaxanthin, a potent antioxidant carotenoid, plays a major role in modulating the immune response. In this study, we examined the immunomodulatory effects of astaxanthin on cytokine production in primary cultured lymphocytes both in vitro and ex vivo. Direct administration of astaxanthin (70–300 nM did not produce cytotoxicity in lipopolysaccharide (LPS, 100 µg/ mL- or concanavalin A (Con A, 10 µg/ mL-activated lymphocytes, whereas astaxanthin alone at 300 nM induced proliferation of splenic lymphocytes (p < 0.05 in vitro. Although astaxanthin, alone or with Con A, had no apparent effect on interferon (INF-γ and interleukin (IL-2 production in primary cultured lymphocytes, it enhanced LPS-induced INF-γ production. In an ex vivo experiment, oral administration of astaxanthin (0.28, 1.4 and 7 mg/kg/day for 14 days did not cause alterations in the body or spleen weights of mice and also was not toxic to lymphocyte cells derived from the mice. Moreover, treatment with astaxanthin significantly increased LPS-induced lymphocyte proliferation ex vivo but not Con A-stimulated lymphocyte proliferation ex vivo. Enzyme linked immunosorbent assay (ELISA analysis revealed that administration of astaxanthin significantly enhanced INF-γ production in response to both LPS and Con A stimulation, whereas IL-2 production increased only in response to Con A stimulation. Also, astaxanthin treatment alone significantly increased IL-2 production in lymphocytes derived from mice, but did not significantly change production of INF-γ. These findings suggest that astaxanthin modulates lymphocytic immune responses in vitro, and that it partly exerts its ex vivo immunomodulatory effects by increasing INF-γ and IL-2 production without inducing cytotoxicity.

  20. Cell enrichment-free massive ex-vivo expansion of peripheral CD20⁺ B cells via CD40-CD40L signals in non-human primates.

    Science.gov (United States)

    Kim, Jung-Sik; Byun, Nari; Chung, Hyunwoo; Kim, Hyun-Je; Kim, Jong-Min; Chun, Taehoon; Lee, Won-Woo; Park, Chung-Gyu

    2016-04-22

    Non-human primates (NHPs) are valuable as preclinical resources that bridge the gap between basic science and clinical application. B cells from NHPs have been utilized for the development of B-cell targeted drugs and cell-based therapeutic modalities; however, few studies on the ex-vivo expansion of monkey B cells have been reported. In this study, we developed a highly efficient ex-vivo expansion protocol for monkey B cells resulting in 99% purity without the requirement for prior cell-enrichment procedures. To this end, monkey peripheral blood mononuclear cells (PBMCs) were stimulated for 12 days with cells constitutively expressing monkey CD40L in expansion medium optimized for specific and massive expansion of B cells. The B cells expansion rates obtained were 2-5 times higher than those previously reported in humans, with rates ranging from 7.9 to 16.6 fold increase. Moreover, expanded B cells sustained high expression of co-stimulatory molecules including CD83 and CD86 until day 12 of culture, and the simple application of a brief centrifugation resulted in a CD20(+) B cell purity rate of greater than 99%. Furthermore, small amounts of CD3(+)CD20(+)BT-like cells were generated and CD16 was expressed at moderate levels on expanded B cells. Thus, the establishment of this protocol provides a method to produce quantities of homogeneous, mature B cells in numbers sufficient for the in vitro study of B cell immunity as well as for the development of B cell-diagnostic tools and cell-based therapeutic modalities.

  1. Integrating dimension reduction and out-of-sample extension in automated classification of ex vivo human patellar cartilage on phase contrast X-ray computed tomography.

    Directory of Open Access Journals (Sweden)

    Mahesh B Nagarajan

    Full Text Available Phase contrast X-ray computed tomography (PCI-CT has been demonstrated as a novel imaging technique that can visualize human cartilage with high spatial resolution and soft tissue contrast. Different textural approaches have been previously investigated for characterizing chondrocyte organization on PCI-CT to enable classification of healthy and osteoarthritic cartilage. However, the large size of feature sets extracted in such studies motivates an investigation into algorithmic feature reduction for computing efficient feature representations without compromising their discriminatory power. For this purpose, geometrical feature sets derived from the scaling index method (SIM were extracted from 1392 volumes of interest (VOI annotated on PCI-CT images of ex vivo human patellar cartilage specimens. The extracted feature sets were subject to linear and non-linear dimension reduction techniques as well as feature selection based on evaluation of mutual information criteria. The reduced feature set was subsequently used in a machine learning task with support vector regression to classify VOIs as healthy or osteoarthritic; classification performance was evaluated using the area under the receiver-operating characteristic (ROC curve (AUC. Our results show that the classification performance achieved by 9-D SIM-derived geometric feature sets (AUC: 0.96 ± 0.02 can be maintained with 2-D representations computed from both dimension reduction and feature selection (AUC values as high as 0.97 ± 0.02. Thus, such feature reduction techniques can offer a high degree of compaction to large feature sets extracted from PCI-CT images while maintaining their ability to characterize the underlying chondrocyte patterns.

  2. Radiosynthesis and ex vivo evaluation of (R)-(-)-2-chloro-N-[1-{sup 11}C-propyl]n-propylnorapomorphine

    Energy Technology Data Exchange (ETDEWEB)

    Palner, Mikael [Neurobiology Research Unit, Rigshospitalet, University of Copenhagen, DK-2300, Copenhagen (Denmark); Center for Integrated Molecular Brain Imaging, Copenhagen (Denmark)], E-mail: mikael.palner@nru.dk; McCormick, Patrick [PET Centre, Centre for Addiction and Mental Health, M5T 1RB, Toronto, ON (Canada); Gillings, Nic [Center for Integrated Molecular Brain Imaging, Copenhagen (Denmark); PET and Cyclotron Unit, Rigshospitalet, DK-2300, Copenhagen (Denmark); Begtrup, Mikael [Center for Integrated Molecular Brain Imaging, Copenhagen (Denmark); Institute for Medicinal Chemistry, Pharmaceutical Faculty, University of Copenhagen, DK-2300, Copenhagen (Denmark); Wilson, Alan A. [PET Centre, Centre for Addiction and Mental Health, M5T 1RB, Toronto, ON (Canada); Knudsen, Gitte M. [Neurobiology Research Unit, Rigshospitalet, University of Copenhagen, DK-2300, Copenhagen (Denmark); Center for Integrated Molecular Brain Imaging, Copenhagen (Denmark)

    2010-01-15

    Introduction: Several dopamine D{sub 2} agonist radioligands have been used with positron emission tomography (PET), including [{sup 11}C-]-(-)-MNPA, [{sup 11}C-]-(-)-NPA and [{sup 11}C]-(+)-PHNO. These radioligands are considered particularly powerful for detection of endogenous dopamine release, but they either provide PET brain images with limited contrast or have affinity for both D{sub 2} and D{sub 3} receptors. We here present the carbon-11 radiolabeling and ex vivo evaluation of 2-Cl-(-)-NPA, a novel PET-tracer candidate with high in vitro D{sub 2}/D{sub 3} selectivity. Methods: 2-Cl-[{sup 11}C]-(-)-NPA and [{sup 11}C]-(-)-NPA were synthesized by a two step N-acylation-reduction process using [{sup 11}C]-propionyl chloride. Awake rats were injected with either tracer, via the tail vein. The rats were decapitated at various times, the brains were removed and quickly dissected, and plasma metabolites were measured. Radioligand specificity, and P-glycoprotein involvement in brain uptake, was also assessed. Results: 2-Cl-[{sup 11}C]-(-)-NPA and [{sup 11}C]-(-)-NPA were produced in high specific activity and purity. 2-Cl-[{sup 11}C]-(-)-NPA accumulated slower in the striatum than [{sup 11}C]-(-)-NPA, reaching maximum concentrations after 30 min. The maximal striatal uptake of 2-Cl-[{sup 11}C]-(-)-NPA (standard uptake value 0.72{+-}0.24) was approximately half that of [{sup 11}C]-(-)-NPA (standard uptake value 1.37{+-}0.18). Nonspecific uptake was similar for the two compounds. 2-Cl-[{sup 11}C]-(-)-NPA was metabolized quickly, leaving only 17% of the parent compound in the plasma after 30 min. The specific binding of 2-Cl-[{sup 11}C]-(-)-NPA was completely blocked and inhibition of P-glycoprotein did not alter the brain uptake. Conclusion: Ex vivo experiments showed, despite a favorable D{sub 2}/D{sub 3} selectivity, that 2-Cl-[{sup 11}C]-(-)-NPA is inferior to [{sup 11}C]-(-)-NPA as a PET tracer in rat, because of slower brain uptake and lower specific to

  3. Radiosynthesis and ex vivo evaluation of (R)-(-)-2-chloro-N-[1-11C-propyl]n-propylnorapomorphine

    International Nuclear Information System (INIS)

    Introduction: Several dopamine D2 agonist radioligands have been used with positron emission tomography (PET), including [11C-]-(-)-MNPA, [11C-]-(-)-NPA and [11C]-(+)-PHNO. These radioligands are considered particularly powerful for detection of endogenous dopamine release, but they either provide PET brain images with limited contrast or have affinity for both D2 and D3 receptors. We here present the carbon-11 radiolabeling and ex vivo evaluation of 2-Cl-(-)-NPA, a novel PET-tracer candidate with high in vitro D2/D3 selectivity. Methods: 2-Cl-[11C]-(-)-NPA and [11C]-(-)-NPA were synthesized by a two step N-acylation-reduction process using [11C]-propionyl chloride. Awake rats were injected with either tracer, via the tail vein. The rats were decapitated at various times, the brains were removed and quickly dissected, and plasma metabolites were measured. Radioligand specificity, and P-glycoprotein involvement in brain uptake, was also assessed. Results: 2-Cl-[11C]-(-)-NPA and [11C]-(-)-NPA were produced in high specific activity and purity. 2-Cl-[11C]-(-)-NPA accumulated slower in the striatum than [11C]-(-)-NPA, reaching maximum concentrations after 30 min. The maximal striatal uptake of 2-Cl-[11C]-(-)-NPA (standard uptake value 0.72±0.24) was approximately half that of [11C]-(-)-NPA (standard uptake value 1.37±0.18). Nonspecific uptake was similar for the two compounds. 2-Cl-[11C]-(-)-NPA was metabolized quickly, leaving only 17% of the parent compound in the plasma after 30 min. The specific binding of 2-Cl-[11C]-(-)-NPA was completely blocked and inhibition of P-glycoprotein did not alter the brain uptake. Conclusion: Ex vivo experiments showed, despite a favorable D2/D3 selectivity, that 2-Cl-[11C]-(-)-NPA is inferior to [11C]-(-)-NPA as a PET tracer in rat, because of slower brain uptake and lower specific to nonspecific binding ratio.

  4. Precision-cut kidney slices (PCKS to study development of renal fibrosis and efficacy of drug targeting ex vivo

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    Fariba Poosti

    2015-10-01

    Full Text Available Renal fibrosis is a serious clinical problem resulting in the greatest need for renal replacement therapy. No adequate preventive or curative therapy is available that could be clinically used to target renal fibrosis specifically. The search for new efficacious treatment strategies is therefore warranted. Although in vitro models using homogeneous cell populations have contributed to the understanding of the pathogenetic mechanisms involved in renal fibrosis, these models poorly mimic the complex in vivo milieu. Therefore, we here evaluated a precision-cut kidney slice (PCKS model as a new, multicellular ex vivo model to study the development of fibrosis and its prevention using anti-fibrotic compounds. Precision-cut slices (200-300 μm thickness were prepared from healthy C57BL/6 mouse kidneys using a Krumdieck tissue slicer. To induce changes mimicking the fibrotic process, slices were incubated with TGFβ1 (5 ng/ml for 48 h in the presence or absence of the anti-fibrotic cytokine IFNγ (1 µg/ml or an IFNγ conjugate targeted to PDGFRβ (PPB-PEG-IFNγ. Following culture, tissue viability (ATP-content and expression of α-SMA, fibronectin, collagen I and collagen III were determined using real-time PCR and immunohistochemistry. Slices remained viable up to 72 h of incubation, and no significant effects of TGFβ1 and IFNγ on viability were observed. TGFβ1 markedly increased α-SMA, fibronectin and collagen I mRNA and protein expression levels. IFNγ and PPB-PEG-IFNγ significantly reduced TGFβ1-induced fibronectin, collagen I and collagen III mRNA expression, which was confirmed by immunohistochemistry. The PKCS model is a novel tool to test the pathophysiology of fibrosis and to screen the efficacy of anti-fibrotic drugs ex vivo in a multicellular and pro-fibrotic milieu. A major advantage of the slice model is that it can be used not only for animal but also for (fibrotic human kidney tissue.

  5. Ex vivo localization and immunohistochemical detection of sentinel lymph node micrometastasis in patients with colorectal cancer can upgrade tumor staging

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    Wang Fu-Long

    2012-06-01

    Full Text Available Abstract Background It is not clear if sentinel lymph node (SLN mapping can improve outcomes in patients with colorectal cancers. The purpose of this study was to determine the prognostic values of ex vivo sentinel lymph node (SLN mapping and immunohistochemical (IHC detection of SLN micrometastasis in colorectal cancers. Methods Colorectal cancer specimens were obtained during radical resections and the SLN was identified by injecting a 1% isosulfan blue solution submucosally and circumferentially around the tumor within 30 min after surgery. The first node to stain blue was defined as the SLN. SLNs negative by hematoxylin and eosin (HE staining were further examined for micrometastasis using cytokeratin IHC. Results A total of 54 patients between 25 and 82 years of age were enrolled, including 32 males and 22 females. More than 70% of patients were T3 or above, about 86% of patients were stage II or III, and approximately 90% of patients had lesions grade II or above. Sentinel lymph nodes were detected in all 54 patients. There were 32 patients in whom no lymph node micrometastasis were detected by HE staining and 22 patients with positive lymph nodes micrometastasis detected by HE staining in non-SLNs. In contrast only 7 SLNs stained positive with HE. Using HE examination as the standard, the sensitivity, non-detection rate, and accuracy rate of SLN micrometastasis detection were 31.8% (7/22, 68.2% (15/22, and 72.2%, respectively. Micrometastasis were identified by ICH in 4 of the 32 patients with HE-negative stained lymph nodes, resulting in an upstaging rate 12.5% (4/32. The 4 patients who were upstaged consisted of 2 stage I patients and 2 stage II patients who were upstaged to stage III. Those without lymph node metastasis by HE staining who were upstaged by IHC detection of micrometastasis had a significantly poorer disease-free survival (p = 0.001 and overall survival (p = 0.004. Conclusion Ex vivo localization and

  6. The Use of MTT Assay, In Vitro and Ex Vivo, to Predict the Radiosensitivity of Colorectal Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Eun; Kim, Mi Sook; Kang, Chang Mo; Shin, Hye Kyung; Choi, Chul Won; Seo, Young Seok; Ji, Young Hoon [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Kim, Jong Il [Seoul Women' s University College of Medicine, Seoul (Korea, Republic of)

    2008-09-15

    The measurement of radiosensitivity of individuals is useful in radiation therapy. Unfortunately, the measurement of radiation survival using a clonogenic assay, which is the established standard, can be difficult and time consuming. The aim of this study is to compare radiosensitivity results obtained from the MTT and clonogenic assays, and to evaluate whether the MTT assay can be used on clinical specimens. Materials and Methods: HCT-8, LoVo, CT-26, and WiDr were the colon cancer cell lines used for this study. The clonogenic assay was performed to obtain the cell survival curves and surviving fractions at a dose of 2 Gy (SF2) as the standard technique for radiosensitivity. Also, the MTT assay was performed for each of the cell lines (in vitro). To simulate clinical specimens, the cell lines were inoculated into nude mice, removed when the tumors reached 1 cm in diameter, and chopped. Next, the tumors were subjected to the same process involved with the MTT assay in vitro. The inhibition rates (IR) of 10 Gy or 20 Gy of irradiation for in vitro and ex vivo were calculated based on the optical density of the MTT assay, respectively. Results: According to SF2 and the cell survival curve, the HCT-8 and WiDr cell lines were more resistant to radiation than LoVo and CT-26 (p<0.05). The IR was measured by in vitro. The MTT assay IR was 17.3%, 21%, 30% and 56.5% for the WiDr, HCT-8, LoVo and CT-26 cell lines, respectively. In addition, the IR measured ex vivo by the MTT assay was 23.5%, 26%, 38% and 53% in the HCT-8, WiDr, LoVo and CT-26 tumors, respectively. Conclusion: The radiosensitivity measured by the MTT assay was correlated with the measures obtained from the clonogenic assay. This result highlights the possibility that the MTT assay could be used in clinical specimens for individual radiosensitivity assays.

  7. Moving-shot versus fixed electrode techniques for radiofrequency ablation: Comparison in an ex-vivo bovine liver tissue model

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Eun Ju; Baek, Jung Hwan; Lee, Jeong Hyun [Dept. of Radiology and the Research Institute of Radiology, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of)

    2014-12-15

    To compare the ablation characteristics of the moving-shot technique (MST) and the fixed electrode technique (FET) for radiofrequency (RF) ablation in an ex-vivo bovine liver tissue model. We performed RF ablation using FET in 110 bovine liver blocks using 11 different ablation times ranging from 5 seconds to 5 minutes (10 blocks per each time duration). Ten bovine liver blocks at each ablation time of 1- or 2-minute, were ablated with MST, which treated conceptual ablation units by moving the electrode tip. We evaluated the ablation volume obtained with FET across ablation time lengths. The results of FET and MST performed with the same ablation time lengths, i.e., 1- and 2-minute ablation time were also compared. The ablation volume achieved with FET gradually increased with increasing ablation time; however, the pair-wise statistical comparison between 2 neighboring ablation time lengths was not significant after 30 seconds. MST with either 1- or 2-minute ablation time achieved larger ablation volumes (1.1 +/- 0.2 mL vs. 2.7 +/- 0.3 mL, p < 0.001; and 1.4 +/- 0.2 mL vs. 5.6 +/- 0.4 mL, p < 0.001, respectively), longer true RF times (46.7 +/- 4.6 seconds vs. 60 seconds, p < 0.001; and 64.8 +/- 4.6 seconds vs. 120 seconds, p < 0.001, respectively), fewer numbers of RF cut-offs (1.6 +/- 0.5 vs. 0, p < 0.001; and 5.5 +/- 0.5 vs. 0, p < 0.001, respectively), and greater energy deposition (2050.16 +/- 209.2 J vs. 2677.76 +/- 83.68 J, p < 0.001; and 2970.64 +/- 376.56 J vs. 5564.72 +/- 5439.2 J, p < 0.001, respectively), than FET. The MST can achieve a larger ablation volume by preventing RF cut-off, compared with the FET in an ex-vivo bovine liver model.

  8. Root canals decontamination by coherent photons initiated photoacustic streaming (PIPS) of irrigants: an ex-vivo study

    International Nuclear Information System (INIS)

    The aim of this ex vivo study was to assess the antibacterial effectiveness of coherent photon initiated photoacoustic streaming (PIPS) of irrigants using an Er:YAG laser equipped with a newly designed, stripped and tapered, tip in extracted teeth with infected root canals. One hundred-forty-eight single-rooted extracted teeth were prepared using a rotary abrasive instrument providing a root channel with a suitable size. The samples were sterilized and all teeth except ten (negative control group) were inoculated with Enterococcus faecalis and incubated in a CO2 chamber at 37°C for 15 days in Eppendorff tubes filled with trypticase soy broth medium changed every 2 days. Infected teeth were then randomly divided into 4 test groups (n=32 for each): pulsed erbium:YAG laser at non-ablative settings for 30 seconds with sterile bi-distilled water (Group A) or 5% sodium hypochlorite (NaOCl) (Group B); without laser activated sterile bi-distilled water irrigation for 30 seconds (Group C) or 5% NaOCl irrigation for 30 seconds (Group D); the positive control group received no treatment in infected teeth (n=10). Colony-forming units (CFUs) were counted from bacteriologic samples taken before (S1) and after treatment (S2). Data were analyzed by Kruskal-Wallis and post hoc Dunn's multiple comparison tests. CFU counts were significantly lower in groups B and D than in group C (P<0.001). Moreover, there was a significant difference between Group A and C (P<0.001). Group B showed the highest CFU reduction, which was significantly greater than that evident in groups A or C (P<0.001). There were no statistically significant differences between group B and D (P>0.05). None of the four groups predictably generated negative samples. Under the conditions of this ex vivo study, statistically significant difference wasn't found in planktonic bacteria reduction between the laser and NaOCl or NaOCl alone groups.

  9. Relevance of sunscreen application method, visible light and sunlight intensity to free-radical protection: A study of ex vivo human skin.

    Science.gov (United States)

    Haywood, Rachel

    2006-01-01

    With the continued rise in skin cancers worldwide there is a need for effective skin protection against sunlight damage. It was shown previously that sunscreens, which claimed UVA protection (SPF 20+), provided limited protection against UV-induced ascorbate radicals in human skin. Here the results of an electron spin resonance (ESR) investigation to irradiate ex vivo human skin with solar-simulated light are reported. The ascorbate radical signal in the majority of skin samples was directly proportional to the irradiance over relevant sunlight intensities (0.9-2.9 mW cm(-2)). Radical production (substratum-corneum) by UV (wavelengths 400 nm) was approximately 67% and 33% respectively. Ascorbate radicals were in steady state concentration at low irradiance (approximately 1 mW cm(-2) equivalent to UK sunlight), but at higher irradiance (approximately 3 mW cm(-2)) decreased with time, suggesting ascorbate depletion. Radical protection by a four star-rated sunscreen (with UVA protection) was optimal when applied as a thin film (40-60% at 2 mg cm(-2)) but less so when rubbed into the skin (37% at 4 mg cm(-2) and no significant protection at 2 mg cm(-2)), possibly due to cream filling crevices, which reduced film thickness. This study validates ESR determinations of the ascorbate radical for quantitative protection measurements. Visible light contribution to radical production, and loss of protection when sunscreen is rubbed into skin, has implications for sunscreen design and use for the prevention of free-radical damage. PMID:17205635

  10. Ex vivo immunomodulatory effect of all-trans-retinoic acid during Behçet's disease: a study in Algerian patients.

    Science.gov (United States)

    Djeraba, Zineb; Boumedine, Karim; Arroul-Lammali, Amina; Otmani, Fifi; Belguendouz, Houda; Touil-Boukoffa, Chafia

    2014-02-01

    Uveitis, recurrent oral and genital ulcerations associated with skin lesions are the major symptoms of a chronic multisystemic inflammatory disorder known as Behçet's disease (BD). High prevalence of this dreaded disease has been observed in the Mediterranean basin, including Algeria and along the Silk Road. Although the etiologic agent of this disease remains uncertain, many hypotheses have been advanced in its pathogenesis. Our team has previously reported high levels of nitric oxide (NO) in sera of BD patients, suggesting its deleterious effect during chronic inflammation. In our current study, the aim is to investigate the ex vivo immunomodulatory effect of all-trans-retinoic acid (ATRA) on NO pathway in Algerian BD patients. First, peripheral blood mononuclear cells isolated from active and inactive BD patients and healthy controls were cultured with different concentrations of ATRA. NO production was estimated with the Griess method. To elucidate the underlying mechanisms of ATRA effect on NO production, we analyze inducible nitric oxide synthase expression and nuclear factor-κB (NF-κB) activity by immunofluorescence test. Our results revealed a higher production of NO in active BD compared with the inactive stage and healthy controls. We observed that ATRA inhibits NO production in BD both in active and inactive stages and inhibits NF-κB translocation. In conclusion, we report a relationship between NO production and the disease activity. ATRA down-regulates NO production in BD patients. This immunomodulatory effect seems to be mediated through NF-κB pathway. All these findings suggest that ATRA could be considered as a promising therapy for BD. PMID:24369064

  11. An analytical method for assessing stage-specific drug activity in Plasmodium vivax malaria: implications for ex vivo drug susceptibility testing.

    Directory of Open Access Journals (Sweden)

    Douglas H Kerlin

    Full Text Available The emergence of highly chloroquine (CQ resistant P. vivax in Southeast Asia has created an urgent need for an improved understanding of the mechanisms of drug resistance in these parasites, the development of robust tools for defining the spread of resistance, and the discovery of new antimalarial agents. The ex vivo Schizont Maturation Test (SMT, originally developed for the study of P. falciparum, has been modified for P. vivax. We retrospectively analysed the results from 760 parasite isolates assessed by the modified SMT to investigate the relationship between parasite growth dynamics and parasite susceptibility to antimalarial drugs. Previous observations of the stage-specific activity of CQ against P. vivax were confirmed, and shown to have profound consequences for interpretation of the assay. Using a nonlinear model we show increased duration of the assay and a higher proportion of ring stages in the initial blood sample were associated with decreased effective concentration (EC(50 values of CQ, and identify a threshold where these associations no longer hold. Thus, starting composition of parasites in the SMT and duration of the assay can have a profound effect on the calculated EC(50 for CQ. Our findings indicate that EC(50 values from assays with a duration less than 34 hours do not truly reflect the sensitivity of the parasite to CQ, nor an assay where the proportion of ring stage parasites at the start of the assay does not exceed 66%. Application of this threshold modelling approach suggests that similar issues may occur for susceptibility testing of amodiaquine and mefloquine. The statistical methodology which has been developed also provides a novel means of detecting stage-specific drug activity for new antimalarials.

  12. Improving the ex vivo stability of drug ester compounds in rat and dog serum: inhibition of the specific esterases and implications on their identity.

    Science.gov (United States)

    Koitka, Matthias; Höchel, Joachim; Gieschen, Hille; Borchert, Hans-Hubert

    2010-02-01

    In drug development, it has been noticed that some drug compounds, especially esters, are unstable in serum samples ex vivo. This can lead to a substantial underestimation of the actual drug concentration. The rat and the dog, representing a rodent and non-rodent species, respectively, are widely used in preclinical studies. We studied the degradation of three structurally different drug esters in rat and dog serum. Moreover, the efficiency of selected enzyme inhibitors to prevent these degradations was investigated. Furthermore, we found indications of the identity of the drug-specific esterases by means of their inhibitor sensitivity as well as by protein purification and identification. The studied drugs were sagopilone, drospirenone, and methylprednisolone aceponate (MPA) all of which are used in (pre-)clinical drug development. The sagopilone-cleaving esterases in rat serum were inhibited by serine hydrolase inhibitors. We partly purified these esterases resulting in an activity yield of 5% and a purification factor of 472. Using matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS), the rat carboxylesterase isoenzyme ES-1 was identified in these fractions, thus pointing to its involvement in sagopilone cleavage. Drospirenone cleavage in rat serum was effected by butyrylcholinesterase (BChE) and paraoxonase 1 (PON1) as we deduced from the high efficacy of certain serine hydrolase and metallohydrolase inhibitors, respectively. Likewise, some inhibition characteristics implied that MPA was cleaved in rat serum by BChE and serine proteases. Partial purification of the MPA-specific esterases resulted in activity yields of 1-2%, exhibiting up to 10,000-fold purification. In dog serum, we found that sagopilone was not degraded which was in contrast to MPA and drospirenone. MPA degradation was mainly prevented by serine hydrolase inhibitors. We used a three-step purification to isolate the esterases cleaving MPA. This

  13. Ex-vivo HRMAS of adult brain tumours: metabolite quantification and assignment of tumour biomarkers.

    NARCIS (Netherlands)

    Wright, A.J.; Fellows, G.A.; Griffiths, J.R.; Wilson, M.; Bell, B.A.; Howe, F.A.

    2010-01-01

    BACKGROUND: High-resolution magic angle spinning (HRMAS) NMR spectroscopy allows detailed metabolic analysis of whole biopsy samples for investigating tumour biology and tumour classification. Accurate biochemical assignment of small molecule metabolites that are "NMR visible" will improve our inter

  14. Neuromuscular induced phonation in a human ex vivo perfused larynx preparation

    OpenAIRE

    Berke, Gerald; Mendelsohn, Abie H; Scott Howard, Nelson; Zhang, Zhaoyan

    2013-01-01

    Considering differences in laryngeal anatomy, degree of control, and range of voice qualities between animals and humans, investigations of the neuromuscular process of voice control are better conducted using a living human larynx in which parametric stimulation of individual laryngeal muscles is possible. Due to difficulties in access and monitoring of laryngeal muscle activities, such investigations are impossible in living human subject experiments. This study reports the recent success i...

  15. Evaluating acellular versus cellular perfusate composition during prolonged ex vivo lung perfusion after initial cold ischaemia for 24 hours.

    Science.gov (United States)

    Becker, Simon; Steinmeyer, Jasmin; Avsar, Murat; Höffler, Klaus; Salman, Jawad; Haverich, Axel; Warnecke, Gregor; Ochs, Matthias; Schnapper, Anke

    2016-01-01

    Normothermic ex vivo lung perfusion (EVLP) has developed as a powerful technique to evaluate particularly marginal donor lungs prior to transplantation. In this study, acellular and cellular perfusate compositions were compared in an identical experimental setting as no consensus has been reached on a preferred technique yet. Porcine lungs underwent EVLP for 12 h on the basis of an acellular or a cellular perfusate composition after 24 h of cold ischaemia as defined organ stress. During perfusion, haemodynamic and respiratory parameters were monitored. After EVLP, the lung condition was assessed by light and transmission electron microscopy. Aerodynamic parameters did not show significant differences between groups and remained within the in vivo range during EVLP. Mean oxygenation indices were 491 ± 39 in the acellular group and 513 ± 53 in the cellular group. Groups only differed significantly in terms of higher pulmonary artery pressure and vascular resistance in the cellular group. Lung histology and ultrastructure were largely well preserved after prolonged EVLP and showed only minor structural alterations which were similarly present in both groups. Prolonged acellular and cellular EVLP for 12 h are both feasible with lungs prechallenged by ischaemic organ stress. Physiological and ultrastructural analysis showed no superiority of either acellular or cellular perfusate composition.

  16. A novel dual ex vivo lung perfusion technique improves immediate outcomes in an experimental model of lung transplantation.

    Science.gov (United States)

    Tanaka, Y; Noda, K; Isse, K; Tobita, K; Maniwa, Y; Bhama, J K; D'Cunha, J; Bermudez, C A; Luketich, J D; Shigemura, N

    2015-05-01

    The lungs are dually perfused by the pulmonary artery and the bronchial arteries. This study aimed to test the feasibility of dual-perfusion techniques with the bronchial artery circulation and pulmonary artery circulation synchronously perfused using ex vivo lung perfusion (EVLP) and evaluate the effects of dual-perfusion on posttransplant lung graft function. Using rat heart-lung blocks, we developed a dual-perfusion EVLP circuit (dual-EVLP), and compared cellular metabolism, expression of inflammatory mediators, and posttransplant graft function in lung allografts maintained with dual-EVLP, standard-EVLP, or cold static preservation. The microvasculature in lung grafts after transplant was objectively evaluated using microcomputed tomography angiography. Lung grafts subjected to dual-EVLP exhibited significantly better lung graft function with reduced proinflammatory profiles and more mitochondrial biogenesis, leading to better posttransplant function and compliance, as compared with standard-EVLP or static cold preservation. Interestingly, lung grafts maintained on dual-EVLP exhibited remarkably increased microvasculature and perfusion as compared with lungs maintained on standard-EVLP. Our results suggest that lung grafts can be perfused and preserved using dual-perfusion EVLP techniques that contribute to better graft function by reducing proinflammatory profiles and activating mitochondrial respiration. Dual-EVLP also yields better posttransplant graft function through increased microvasculature and better perfusion of the lung grafts after transplantation.

  17. Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo.

    Science.gov (United States)

    Lee, Jun Ho; Lee, Seunghun; Yoon, Calvin J; Park, Jin Hyoung; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-02-01

    Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other. PMID:26977371

  18. Chitosan and Kappa-Carrageenan Vaginal Acyclovir Formulations for Prevention of Genital Herpes. In Vitro and Ex Vivo Evaluation

    Directory of Open Access Journals (Sweden)

    María-Pilar Sánchez-Sánchez

    2015-09-01

    Full Text Available Vaginal formulations for the prevention of sexually transmitted infections are currently gaining importance in drug development. Polysaccharides, such as chitosan and carrageenan, which have good binding capacity with mucosal tissues, are now included in vaginal delivery systems. Marine polymer-based vaginal mucoadhesive solid formulations have been developed for the controlled release of acyclovir, which may prevent the sexual transmission of the herpes simplex virus. Drug release studies were carried out in two media: simulated vaginal fluid and simulated vaginal fluid/simulated seminal fluid mixture. The bioadhesive capacity and permanence time of the bioadhesion, the prepared compacts, and compacted granules were determined ex vivo using bovine vaginal mucosa as substrate. Swelling processes were quantified to confirm the release data. Biocompatibility was evaluated through in vitro cellular toxicity assays, and the results showed that acyclovir and the rest of the materials had no cytotoxicity at the maximum concentration tested. The mixture of hydroxyl-propyl-methyl-cellulose with chitosan- or kappa-carrageenan-originated mucoadhesive systems that presented a complete and sustained release of acyclovir for a period of 8–9 days in both media. Swelling data revealed the formation of optimal mixed chitosan/hydroxyl-propyl-methyl-cellulose gels which could be appropriated for the prevention of sexual transmission of HSV.

  19. Lung transplantation with donation after circulatory determination of death donors and the impact of ex vivo lung perfusion.

    Science.gov (United States)

    Machuca, T N; Mercier, O; Collaud, S; Tikkanen, J; Krueger, T; Yeung, J C; Chen, M; Azad, S; Singer, L; Yasufuku, K; de Perrot, M; Pierre, A; Waddell, T K; Keshavjee, S; Cypel, M

    2015-04-01

    The growing demand for suitable lungs for transplantation drives the quest for alternative strategies to expand the donor pool. The aim of this study is to evaluate the outcomes of lung transplantation (LTx) with donation after circulatory determination of death (DCDD) and the impact of selective ex vivo lung perfusion (EVLP). From 2007 to 2013, 673 LTx were performed, with 62 (9.2%) of them using DCDDs (seven bridged cases). Cases bridged with mechanical ventilation/extracorporeal life support were excluded. From 55 DCDDs, 28 (51%) underwent EVLP. Outcomes for LTx using DCDDs and donation after neurological determination of death (DNDD) donors were similar, with 1 and 5-year survivals of 85% and 54% versus 86% and 62%, respectively (p = 0.43). Although comparison of survival curves between DCDD + EVLP versus DCDD-no EVLP showed no significant difference, DCDD + EVLP cases presented shorter hospital stay (median 18 vs. 23 days, p = 0.047) and a trend toward shorter length of mechanical ventilation (2 vs. 3 days, p = 0.059). DCDDs represent a valuable source of lungs for transplantation, providing similar results to DNDDs. EVLP seems an important technique in the armamentarium to safely increase lung utilization from DCDDs; however, further studies are necessary to better define the role of EVLP in this context. PMID:25772069

  20. Luciferase-expressing Leishmania infantum allows the monitoring of amastigote population size, in vivo, ex vivo and in vitro.

    Directory of Open Access Journals (Sweden)

    Grégory Michel

    2011-09-01

    Full Text Available Here we engineered transgenic Leishmania infantum that express luciferase, the objectives being to more easily monitor in real time their establishment either in BALB/c mice--the liver and spleen being mainly studied-or in vitro. Whatever stationary phase L. infantum promastigotes population--wild type or engineered to express luciferase-the parasite burden was similar in the liver and the spleen at day 30 post the intravenous inoculation of BALB/c mice. Imaging of L. infantum hosting BALB/C mice provided sensitivity in the range of 20,000 to 40,000 amastigotes/mg tissue, two tissues-liver and spleen-being monitored. Once sampled and processed ex vivo for their luciferin-dependent bioluminescence the threshold sensitivity was shown to range from 1,000 to 6,000 amastigotes/mg tissue. This model further proved to be valuable for in vivo measurement of the efficiency of drugs such as miltefosine and may, therefore, additionally be used to evaluate vaccine-induced protection.

  1. Protease-mediated release of chemotherapeutics from mesoporous silica nanoparticles to ex vivo human and mouse lung tumors.

    Science.gov (United States)

    van Rijt, Sabine H; Bölükbas, Deniz A; Argyo, Christian; Datz, Stefan; Lindner, Michael; Eickelberg, Oliver; Königshoff, Melanie; Bein, Thomas; Meiners, Silke

    2015-03-24

    Nanoparticles allow for controlled and targeted drug delivery to diseased tissues and therefore bypass systemic side effects. Spatiotemporal control of drug release can be achieved by nanocarriers that respond to elevated levels of disease-specific enzymes. For example, matrix metalloproteinase 9 (MMP9) is overexpressed in tumors, is known to enhance the metastatic potency of malignant cells, and has been associated with poor prognosis of lung cancer. Here, we report the synthesis of mesoporous silica nanoparticles (MSNs) tightly capped by avidin molecules via MMP9 sequence-specific linkers to allow for site-selective drug delivery in high-expressing MMP9 tumor areas. We provide proof-of-concept evidence for successful MMP9-triggered drug release from MSNs in human tumor cells and in mouse and human lung tumors using the novel technology of ex vivo 3D lung tissue cultures. This technique allows for translational testing of drug delivery strategies in diseased mouse and human tissue. Using this method we show MMP9-mediated release of cisplatin, which induced apoptotic cell death only in lung tumor regions of Kras mutant mice, without causing toxicity in tumor-free areas or in healthy mice. The MMP9-responsive nanoparticles also allowed for effective combinatorial drug delivery of cisplatin and proteasome inhibitor bortezomib, which had a synergistic effect on the (therapeutic) efficiency. Importantly, we demonstrate the feasibility of MMP9-controlled drug release in human lung tumors.

  2. Scopoletin from the flower buds of Magnolia fargesii inhibits protein glycation, aldose reductase, and cataractogenesis ex vivo.

    Science.gov (United States)

    Lee, Jun; Kim, Nan Hee; Nam, Joo Won; Lee, Yun Mi; Jang, Dae Sik; Kim, Young Sook; Nam, Sang Hae; Seo, Eun-Kyoung; Yang, Min Suk; Kim, Jin Sook

    2010-09-01

    Five compounds previously known structures, scopoletin (1), northalifoline (2), stigmast-4-en-3-one (3), tiliroside (4), and oplopanone (5) were obtained from the flower buds of Magnolia fargesii using chromatographic separation methods. The structures of 1-5 were identified by the interpretation of their spectroscopic data including 1D- and 2D-NMR as well as by comparison with reported values. Three compounds 1-3 were found from M. fargesii for the first time in this study. All the isolates (1-5) were subjected to in vitro bioassays to evaluate the inhibitory activity on advanced glycation end products formation and rat lens aldose reductase (RLAR). Compound 1 showed a remarkable inhibitory activity on advanced glycation end products formation with IC(50) value of 2.93 μM (aminoguanidine: 961 μM), and showed a significant RLAR inhibitory activity with IC(50) value of 22.5 μM (3.3-tetramethyleneglutaric acid: 28.7 μM). Compound 4 exhibited potent inhibitory activity against RLAR (IC(50) = 14.9 μM). In the further experiment ex vivo, cataractogenesis of rat lenses induced with xylose was significantly inhibited by compound 1 treatment.

  3. Ex vivo lung perfusion to improve donor lung function and increase the number of organs available for transplantation.

    Science.gov (United States)

    Valenza, Franco; Rosso, Lorenzo; Coppola, Silvia; Froio, Sara; Palleschi, Alessandro; Tosi, Davide; Mendogni, Paolo; Salice, Valentina; Ruggeri, Giulia M; Fumagalli, Jacopo; Villa, Alessandro; Nosotti, Mario; Santambrogio, Luigi; Gattinoni, Luciano

    2014-06-01

    This paper describes the initial clinical experience of ex vivo lung perfusion (EVLP) at the Fondazione Ca' Granda in Milan between January 2011 and May 2013. EVLP was considered if donor PaO2 /FiO2 was below 300 mmHg or if lung function was doubtful. Donors with massive lung contusion, aspiration, purulent secretions, pneumonia, or sepsis were excluded. EVLP was run with a low-flow, open atrium and low hematocrit technique. Thirty-five lung transplants from brain death donors were performed, seven of which after EVLP. EVLP donors were older (54 ± 9 years vs. 40 ± 15 years, EVLP versus Standard, P transplantation was higher (79 [40-84] vs. 39 [36-46], P transplantation, primary graft dysfunction (PGD72 grade 3, 32% vs. 28%, EVLP versus Standard, P = 1), mortality at 30 days (0% vs. 0%, P = 1), and overall survival (71% vs. 86%, EVLP versus Standard P = 0.27) were not different between groups. EVLP enabled a 20% increase in available donor organs and resulted in successful transplants with lungs that would have otherwise been rejected (ClinicalTrials.gov number: NCT01967953).

  4. Ex vivo analysis of human memory B lymphocytes specific for A and B influenza hemagglutinin by polychromatic flow-cytometry.

    Directory of Open Access Journals (Sweden)

    Monia Bardelli

    Full Text Available Understanding the impact that human memory B-cells (MBC, primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA⁺ B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses.

  5. Ex vivo catheter-based imaging of coronary atherosclerosis using multimodality OCT and NIRAF excited at 633 nm

    Science.gov (United States)

    Wang, Hao; Gardecki, Joseph A.; Ughi, Giovanni J.; Jacques, Paulino Vacas; Hamidi, Ehsan; Tearney, Guillermo J.

    2015-01-01

    While optical coherence tomography (OCT) has been shown to be capable of imaging coronary plaque microstructure, additional chemical/molecular information may be needed in order to determine which lesions are at risk of causing an acute coronary event. In this study, we used a recently developed imaging system and double-clad fiber (DCF) catheter capable of simultaneously acquiring both OCT and red excited near-infrared autofluorescence (NIRAF) images (excitation: 633 nm, emission: 680nm to 900nm). We found that NIRAF is elevated in lesions that contain necrotic core – a feature that is critical for vulnerable plaque diagnosis and that is not readily discriminated by OCT alone. We first utilized a DCF ball lens probe and a bench top setup to acquire en face NIRAF images of aortic plaques ex vivo (n = 20). In addition, we used the OCT-NIRAF system and fully assembled catheters to acquire multimodality images from human coronary arteries (n = 15) prosected from human cadaver hearts (n = 5). Comparison of these images with corresponding histology demonstrated that necrotic core plaques exhibited significantly higher NIRAF intensity than other plaque types. These results suggest that multimodality intracoronary OCT-NIRAF imaging technology may be used in the future to provide improved characterization of coronary artery disease in human patients. PMID:25909020

  6. Diffuse reflectance spectroscopy for optical nerve identification. Preliminary ex vivo results for feedback controlled oral and maxillofacial laser surgery

    Science.gov (United States)

    Stelzle, Florian; Zam, Azhar; Adler, Werner; Douplik, Alexandre; Tangermann-Gerk, Katja; Nkenke, Emeka; Neukam, Friedrich Wilhelm; Schmidt, Michael

    Objective: Laser surgery has many advantages. However, due to a lack of haptic feedback it is accompanied by the risk of iatrogenic nerve damage. The aim of this study was to evaluate the possibilities of optical nerve identification by diffuse reflectance spectroscopy to set the base for a feedback control system to enhance nerve preservation in oral and maxillofacial laser surgery. Materials and Methods: Diffuse reflectance spectra of nerve tissue, skin, mucosa, fat tissue, muscle, cartilage and bone (15120 spectra) of ex vivo pig heads were acquired in the wavelength range of 350-650 nm. Tissue differentiation was performed by principal components analysis (PCA) followed by linear discriminant analysis (LDA). Specificity and sensitivity were calculated by receiver operating characteristic (ROC) analysis and the area under curve (AUC). Results: Nerve tissue could correctly be identified and differed from skin, mucosa, fat tissue, muscle, cartilage and bone in more than 90% of the cases (AUC results) with a specificity of over 78% and a sensitivity of more than 86%. Conclusion: Nerve tissue can be identified by diffuse reflectance spectroscopy with high precision and reliability. The results may set the base for a feedback system to prevent iatrogenic nerve damage performing oral and maxillofacial laser surgery.

  7. Development and ex vivo evaluation of 5-aminolevulinic acid-loaded niosomal formulations for topical photodynamic therapy.

    Science.gov (United States)

    Bragagni, Marco; Scozzafava, Andrea; Mastrolorenzo, Antonio; Supuran, Claudiu T; Mura, Paola

    2015-10-15

    The objective of this study was the development of a niosomal formulation for improving skin permeation and penetration of 5-aminolevulinic acid (ALA) in the treatment of skin malignancies by photodynamic therapy (PDT). Different niosomal dispersions were prepared, using two different preparation methods. The effect of addition to a classic formulation, consisting in an equimolar Span 60-cholesterol mixture, of two different edge activators, dicethyl-phosphate (DCP) and sodium cholate (SC), and of the presence of ethanol on the vesicle properties and stability was evaluated. Selected formulations were loaded with the drug and evaluated for physicochemical and stability properties and encapsulation efficiency. Classic and elastic DCP-containing niosomes were the only formulations able to effectively incorporate the drug without instability problems. Ex vivo permeation and penetration studies through excised human skin showed that both the niosomal formulations were significantly more effective in improving ALA skin delivery than the simple aqueous drug solution commonly used in clinical practice, allowing, respectively, an increase of about 80 and 40% of the drug permeated amount and of about 100 and 50% of the drug retained into the skin. These results lead to consider the developed formulations potentially useful for improving ALA bioavailability and therapeutic effectiveness in skin malignancies treatment by topical PDT. PMID:26283280

  8. Vascular effects of sildenafil in patients with pulmonary fibrosis and pulmonary hypertension: an ex vivo/in vitro study.

    Science.gov (United States)

    Milara, Javier; Escrivá, Juan; Ortiz, José Luis; Juan, Gustavo; Artigues, Enrique; Morcillo, Esteban; Cortijo, Julio

    2016-06-01

    Sildenafil improves the 6-min walking distance in patients with idiopathic pulmonary fibrosis (IPF) and right-sided ventricular systolic dysfunction.We analysed the previously unexplored role of sildenafil on vasoconstriction and remodelling of pulmonary arteries from patients with IPF and pulmonary hypertension (PH) ex vivo Pulmonary arteries from 18 donors without lung disease, nine IPF, eight PH+IPF and four PH patients were isolated to measure vasodilator and anti-contractile effects of sildenafil in isometric organ bath. Ventilation/perfusion was explored in an animal model of bleomycin lung fibrosis.Sildenafil relaxed serotonin (5-HT) pre-contracted pulmonary arteries in healthy donors and IPF patients and, to a lesser extent, in PH+IPF and PH. Sildenafil inhibited 5-HT dose-response contraction curve mainly in PH+IPF and PH, but not in healthy donors. Sildenafil did not impair the ventilation/perfusion mismatching induced by bleomycin. Pulmonary arteries from PH+IPF patients showed a marked expression of phosphodiesterse-5 and extracellular matrix components. Sildenafil inhibited pulmonary artery endothelial and smooth muscle cell to mesenchymal transition by inhibition of extracellular regulated kinases 1 and 2 (ERK1/2) and SMAD3 phosphorylation.These results suggest an absence of direct relaxant effect and a prominent anti-contractile and anti-remodelling role of sildenafil in PH+IPF pulmonary arteries that could explain the beneficial effects of sildenafil in IPF with PH phenotype. PMID:27009174

  9. The development of a multiorgan ex vivo perfused model: results with the porcine liver-kidney circuit over 24 hours.

    Science.gov (United States)

    Chung, Wen Yuan; Gravante, Gianpiero; Al-Leswas, Dhya; Arshad, Ali; Sorge, Roberto; Watson, Chris C; Pollard, Cristina; Metcalfe, Matthew S; Dennison, Ashley R

    2013-05-01

    We already developed an ex vivo liver-kidney model perfused for 6 h in which the kidney acted as a homeostatic organ to improve the circuit milieu compared to liver alone. In the current study, we extended the multiorgan perfusions to 24 h to evaluate the results and eventual pitfalls manifesting with longer durations. Five livers and kidneys were harvested from female pigs and perfused over 24 h. The extracorporeal circuit included a centrifugal pump, heat exchanger, and oxygenator. The primary end point of the study was the evaluation of the organ functions as gathered from biochemical and acid-base parameters. In the combined liver-kidney circuit, the organs survived and maintained an acceptable homeostasis for different lengths of time, longer for the liver (up to 19-23 h of perfusions) than the kidney (9-13 h of perfusions). Furthermore, glucose and creatinine values decreased significantly over time (from the 5th and 9th hour of perfusion onward). The addition of a kidney to the perfusion circuit improved the biochemical environment by removing excess products from ongoing metabolic processes. The consequence is a more physiological milieu that could improve results from future experimental studies. However, it is likely that long perfusions require some nutritional support over the hours to maintain the organ's vitality and functionality throughout the experiments. PMID:23489088

  10. Recovery and Biodistribution of Ex Vivo Expanded Human Erythroblasts Injected into NOD/SCID/IL2Rγnull mice

    Directory of Open Access Journals (Sweden)

    Barbara Ghinassi

    2011-01-01

    Full Text Available Ex vivo expanded erythroblasts (EBs may serve as advanced transfusion products provided that lodgment occurs in the macrophage-niche of the marrow permitting maturation. EBs expanded from adult and cord blood expressed the receptors (CXCR4, VLA-4, and P-selectin ligand 1 necessary for interaction with macrophages. However, 4-days following transfusion to intact NOD/SCID/IL2Rγnull mice, CD235apos EBs were observed inside CD235aneg splenic cells suggesting that they underwent phagocytosis. When splenectomized and intact NOD/SCID/IL2Rγnull mice were transfused using retrovirally labeled human EBs, human cells were visualized by bioluminescence imaging only in splenectomized animals. Four days after injection, human CD235apos cells were detected in marrow and liver of splenectomized mice but only in spleen of controls. Human CD235apos erythrocytes in blood remained low in all cases. These studies establish splenectomized NOD/SCID/IL2Rγnull mice as a suitable model for tracking and quantification of human EBs in vivo.

  11. Prospective, Randomized Ex Vivo Trial to Assess the Ideal Stapling Site for Endoscopic Fundoplication with Medigus Ultrasonic Surgical Endostapler.

    Science.gov (United States)

    Gweon, Tae-Geun; Matthes, Kai

    2016-01-01

    Background and Aims. Endoscopic fundoplication is an emerging technique for the treatment of gastroesophageal reflux disease (GERD). The aim of this study is to determine the ideal position of the staples in relation to gastroesophageal junction (GEJ). Methods. Ten endoscopic fundoplication procedures were performed in each group using fresh ex vivo porcine stomachs: Group A: 2 staples each at 3 cm above the GEJ and 180° apart; Group B: 2 staples at 3 cm and 90° apart; Group C: 2 staples at 4 cm and 180° apart; Group D: 3 staples at 3 cm with 90° between each staple (180° total). After the procedure, the stomach was gradually filled with water. Gastric yield pressure (GYP) was determined by detection of reflux of the water in esophagus or by rupture of staples. Results. Mean increase of GYPs (±SD) after the procedure was as follows: Group A: 16.9 ± 8.7; Group B: 8.1 ± 7.9; Group C: 12.2 ± 9.4; Group D: 22.7 ± 13.3. GYP in Group A and Group D was higher than Group B (p = 0.03 and p = 0.01, resp.). Conclusions. We recommend the placement of 3 staples at 3 cm distance from the GEJ, which resulted in the highest increase of GYP. PMID:27547219

  12. Ex Vivo Expanded Allogeneic Mesenchymal Stem Cells With Bone Marrow Transplantation Improved Osteogenesis in Infants With Severe Hypophosphatasia.

    Science.gov (United States)

    Taketani, Takeshi; Oyama, Chigusa; Mihara, Aya; Tanabe, Yuka; Abe, Mariko; Hirade, Tomohiro; Yamamoto, Satoshi; Bo, Ryosuke; Kanai, Rie; Tadenuma, Taku; Michibata, Yuko; Yamamoto, Soichiro; Hattori, Miho; Katsube, Yoshihiro; Ohnishi, Hiroe; Sasao, Mari; Oda, Yasuaki; Hattori, Koji; Yuba, Shunsuke; Ohgushi, Hajime; Yamaguchi, Seiji

    2015-01-01

    Patients with severe hypophosphatasia (HPP) develop osteogenic impairment with extremely low alkaline phosphatase (ALP) activity, resulting in a fatal course during infancy. Mesenchymal stem cells (MSCs) differentiate into various mesenchymal lineages, including bone and cartilage. The efficacy of allogeneic hematopoietic stem cell transplantation for congenital skeletal and storage disorders is limited, and therefore we focused on MSCs for the treatment of HPP. To determine the effect of MSCs on osteogenesis, we performed multiple infusions of ex vivo expanded allogeneic MSCs for two patients with severe HPP who had undergone bone marrow transplantation (BMT) from asymptomatic relatives harboring the heterozygous mutation. There were improvements in not only bone mineralization but also muscle mass, respiratory function, and mental development, resulting in the patients being alive at the age of 3. After the infusion of MSCs, chimerism analysis of the mesenchymal cell fraction isolated from bone marrow in the patients demonstrated that donor-derived DNA sequences existed. Adverse events of BMT were tolerated, whereas those of MSC infusion did not occur. However, restoration of ALP activity was limited, and normal bony architecture could not be achieved. Our data suggest that multiple MSC infusions, following BMT, were effective and brought about clinical benefits for patients with lethal HPP. Allogeneic MSC-based therapy would be useful for patients with other congenital bone diseases and tissue disorders if the curative strategy to restore clinically normal features, including bony architecture, can be established.

  13. An ex vivo comparison of detection ability of three methods in discovering of MB2 canal in maxillary molars

    Directory of Open Access Journals (Sweden)

    Ghorbanzadeh A.

    2009-11-01

    Full Text Available "nBackground and Aim: A considerable percentage of failure in Endodontic treatments in maxillary molars is attributed to undiscovered second mesiobuccal canal (MB2.There are different methods for discovering and accessing to this canal. The purpose of this ex vivo study was to compare the detection ability of three methods (direct look, fiberoptic loup and surgical microscope to find MB2 after troughing with ultrasonic. "nMaterials and Methods: In this experimental study, we selected 90 extracted maxillary molars (45 first and 45 second molars in which after access cavity preparation MB2 canal was not discovered by direct vision and endodontic explorer. They were divided into 3 groups (n=30. The dentinal shelf between mesiobuccal and palatal canals was eliminated by an endodontic ultrasonic tip (troughing. After that, first group was searched by direct vision, second group by a loup and fiberoptic light and third group by dental operating microscope. Data were analyzed, specificity and sensitivity were calculated. "nResults: The results showed that 21%, 61%, and 92% of MB2 canals after troughing was found by direct vision, fiberoptic loup, and surgical microscope, respectively. "nConclusion: Based on the results of this study, surgical microscope and loup with fiberoptic are preferred methods for discovering MB2 canal. Troughing with ultrasonic can help find MB2 canal in all methods.

  14. Ex vivo treatment response of primary tumors and/or associated metastases for preclinical and clinical development of therapeutics.

    Science.gov (United States)

    Corben, Adriana D; Uddin, Mohammad M; Crawford, Brooke; Farooq, Mohammad; Modi, Shanu; Gerecitano, John; Chiosis, Gabriela; Alpaugh, Mary L

    2014-10-02

    The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

  15. Development of a double-monoclonal antibody sandwich ELISA: Tool for chicken interferon-γ detection ex vivo

    Science.gov (United States)

    Dai, Hua; Xu, Zheng-zhong; Wang, Meiling; Chen, Jun-hua; Chen, Xiang; Pan, Zhi-ming; Jiao, Xin-an

    2016-01-01

    The aim of the present work was to develop reagents to set up a chicken interferon-γ (ChIFN-γ) assay. Four monoclonal antibodies (mAbs) specific for ChIFN-γ were generated to establish sandwich ELISA based on 2 different mAbs. To improve the detection sensitivity of ChIFN-γ, a double-monoclonal antibody sandwich ELISA was developed using mAb 3E5 as capture antibody and biotinylated mAb 3E3 as a detection reagent. The results revealed that this ELISA has high sensitivity, allowing for the detection of 125 to 500 pg/mL of recombinant ChIFN-γ, and also has an excellent capacity for detecting native ChIFN-γ. This ELISA was then used to detect ChIFN-γ level in chickens immunized with a Newcastle disease virus (NDV) vaccine, the immunized chicken splenocytes were stimulated by NDV F protein as recall antigen. From our results, it appears that the sensitivity range of this sandwich ELISA test is adequate to measure the ex vivo release of ChIFN-γ. PMID:27127340

  16. Detection of Hepatic Fibrosis in Ex Vivo Liver Samples Using an Open-Photoacoustic-Cell Method: Feasibility Study

    Science.gov (United States)

    Stolik, S.; Fabila, D. A.; de la Rosa, J. M.; Escobedo, G.; Suárez-Álvarez, K.; Tomás, S. A.

    2015-09-01

    Design of non-invasive and accurate novel methods for liver fibrosis diagnosis has gained growing interest. Different stages of liver fibrosis were induced in Wistar rats by intraperitoneally administering different doses of carbon tetrachloride. The liver fibrosis degree was conventionally determined by means of histological examination. An open-photoacoustic-cell (OPC) technique for the assessment of liver fibrosis was developed and is reported here. The OPC technique is based on the fact that the thermal diffusivity can be accurately measured by photoacoustics taking into consideration the photoacoustic signal amplitude versus the modulation frequency. This technique measures directly the heat generated in a sample, due to non-radiative de-excitation processes, following the absorption of light. The thermal diffusivity was measured with a home-made open-photoacoustic-cell system that was specially designed to perform the measurement from ex vivo liver samples. The human liver tissue showed a significant increase in the thermal diffusivity depending on the fibrosis stage. Specifically, liver samples from rats exhibiting hepatic fibrosis showed a significantly higher value of the thermal diffusivity than for control animals.

  17. Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo

    Science.gov (United States)

    Lee, Jun Ho; Lee, Seunghun; Yoon, Calvin J.; Park, Jin Hyoung; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-01-01

    Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other. PMID:26977371

  18. Basic and clinical investigation of T3 immunoassay kit

    International Nuclear Information System (INIS)

    T3 immunoassay kit was investigated basically and clinically. A good result was obtained at the prescribed incubation temperature and for 16 hours of incubation time. Moreover, it was thought to be possible that incubation time could be shortened to 1 - 4 hours at 370C. Specificity of antibody was good. Recovery of added T3 was 100+-5 (S.D.) % on an average and parallel of dilution curve of high T3 serum was also good. Variation coefficient of accuracy of this kit was 1.5 - 2.1 % and that of reproducibility was 1.3 - 6.6 %. Mild hemolysis did not affect measurement value. Serum T3 level in normals, untreated patients with Basedow's disease and patients with primary hypothyroidism was 142+-21 ng/100 ml, 452+-156 ng/100 ml and 67+-17 ng/100 ml, respectively. Serum T3 level in patients with Hashimoto's disease was distributed to a wide extent, but that of patients with goiter and simple goiter ranged within normal range. On the other side, serum T3 level of normal pregnant woman was high and that of patients with anorexia nervosa showed low level. From the above mentioned results, it was concluded that this kit was simple in method and good in sensitivity, specificity and reproducibility and it was also useful for clinical applications. (M. Tsunoda)

  19. Encrustation of urologic double pigtail catheters-an ex vivo optical coherence tomography (OCT) study.

    Science.gov (United States)

    Bader, Markus J; Zilinberg, Katja; Weidlich, Patrick; Waidelich, Raphaela; Püls, Michaela; Gratzke, Christian; Stief, Christian G; Stepp, Herbert; Sroka, Ronald

    2013-05-01

    This study aims to evaluate whether optical coherence tomography (OCT) using both the surface and the endoluminal technique is feasible to investigate the locations and degree of encrustation process in clinically used ureteral stents. After removal from patients, 14 polyurethane JJ stents were investigated. A fresh JJ served as a control. The external surfaces were examined using an endoscopic surface OCT whereas the intraluminal surfaces were investigated by an endoluminal radial OCT device. The focus was on detection of encrustation or crystalline sedimentation. In 12 female and two male patients, the median indwelling time of the ureteral catheter was 100 days (range, 19-217). Using the endoluminal OCT, the size and grade of intraluminal encrustation could be expressed as a percentage relating to the open lumen of the reference stent. The maximum encrustation observed resulted in a remaining unrestricted lumen of 15-35 % compared to the reference. The luminal reduction caused by encrustation was significantly higher at the proximal end of the ureteral stent as compared to its distal part. The extraluminal OCT investigations facilitated the characterization of extraluminal encrustation. OCT techniques were feasible and facilitated the detection of encrustation of double pigtail catheters on both the extra and intra luminal surface. Quantitative expression of the degree of intraluminal encrustation could be achieved, with the most dense and thickened occurrence of intraluminal incrustation in the upper curl of the JJ stent. PMID:22869160

  20. Salivary MUC5B-mediated adherence (ex vivo) of Heliocobacter pylori during acute stress.

    NARCIS (Netherlands)

    J.A. Bosch; E.J.C. de Geus; T.J.M. Ligtenberg; K. Nazmi; E.C.I. Veerman; J. Hoogstraten; A.V. Amerongen Nieuwland

    2000-01-01

    Biochemical host defenses at mucosal sites, such as the oral cavity, play a key role in the regulation of microbial ecology and the prevention of infectious disease. This study investigated the effects of acute stress on the salivary levels of the carbohydrate structure sulfo-Lewis-super(a ) (SL), w

  1. Placental transport of large molecules –a study using human ex vivo placental perfusion

    DEFF Research Database (Denmark)

    Mathiesen, Line

    2011-01-01

    molecules, either by passive or facilitated diffusion or active transport systems. This makes placental transport studies interesting when investigating fetal exposure to foreign or innate substances. The aim of this thesis is to investigate the transport of selected substances across the human placenta...... and extrapolation to the in vivo situation critical. In my PhD study I have focused on validation and studies with placental perfusion of substances with a high molecular weight, which require transport or carrier molecules to be transported from the maternal to the fetal side, and longer perfusion time demanding...... within two hours of perfusion with a fetal flow rate of 3 mL/min. Negative controls are added to ensure that substance transfer is not due to leakage, e.g. high molecular weight substances that only pass the placental barrier with bulk flow through a leakage in the fetal system. Dextran (40kD) can...

  2. Ex vivo coronary atherosclerotic plaque characterization with multi-detector-row CT

    International Nuclear Information System (INIS)

    Multi-detector-row CT angiography (CTA) is a new technology that allows for non-invasive investigation of coronary atherosclerosis in patients. The relation between the morphology of atherosclerotic plaques assessed by CTA and histopathology is unknown. We investigated 11 human cadaver heart specimens. A mixture of methylcellulose and CT contrast media was injected into the coronary arteries to achieve in-vivo-like contrast enhancement within the coronary artery lumen. The morphologic pattern of atherosclerotic lesions found on CTA images and the tissue attenuation of non-calcified plaques were determined. After CTA imaging, atherosclerotic lesions in the coronary arteries were macroscopically identified and characterized histopathologically according to American Heart Association criteria. A total of 50 and 40 lesions were found macroscopically and by CTA, respectively. Thirty-three lesions could have been compared directly. The sensitivity of CTA compared with macroscopic detection of atheromas, fibroatheromas, fibrocalcified, and calcified lesions was 73, 70, 86, and 100%, respectively. The mean CT attenuation of predominantly lipid-rich and fibrous-rich plaques was significantly different (47±9 and 104±28 HU, respectively; p<0.01). Atherosclerotic coronary plaques detected by CTA may represent different stages of coronary atherosclerosis. The tissue attenuation of non-calcified plaques may allow for assessment of their predominant component. (orig.)

  3. Evaluation of sample holders designed for long-lasting X-ray micro-tomographic scans of ex-vivo soft tissue samples

    Science.gov (United States)

    Dudak, J.; Zemlicka, J.; Krejci, F.; Karch, J.; Patzelt, M.; Zach, P.; Sykora, V.; Mrzilkova, J.

    2016-03-01

    X-ray microradiography and microtomography are imaging techniques with increasing applicability in the field of biomedical and preclinical research. Application of hybrid pixel detector Timepix enables to obtain very high contrast of low attenuating materials such as soft biological tissue. However X-ray imaging of ex-vivo soft tissue samples is a difficult task due to its structural instability. Ex-vivo biological tissue is prone to fast drying-out which is connected with undesired changes of sample size and shape producing later on artefacts within the tomographic reconstruction. In this work we present the optimization of our Timepix equipped micro-CT system aiming to maintain soft tissue sample in stable condition. Thanks to the suggested approach higher contrast of tomographic reconstructions can be achieved while also large samples that require detector scanning can be easily measured.

  4. Evaluation of sample holders designed for long-lasting X-ray micro-tomographic scans of ex-vivo soft tissue samples

    International Nuclear Information System (INIS)

    X-ray microradiography and microtomography are imaging techniques with increasing applicability in the field of biomedical and preclinical research. Application of hybrid pixel detector Timepix enables to obtain very high contrast of low attenuating materials such as soft biological tissue. However X-ray imaging of ex-vivo soft tissue samples is a difficult task due to its structural instability. Ex-vivo biological tissue is prone to fast drying-out which is connected with undesired changes of sample size and shape producing later on artefacts within the tomographic reconstruction. In this work we present the optimization of our Timepix equipped micro-CT system aiming to maintain soft tissue sample in stable condition. Thanks to the suggested approach higher contrast of tomographic reconstructions can be achieved while also large samples that require detector scanning can be easily measured

  5. A computational atlas of the hippocampal formation using ex vivo, ultra-high resolution MRI: Application to adaptive segmentation of in vivo MRI

    DEFF Research Database (Denmark)

    Iglesias, Juan Eugenio; Augustinack, Jean C.; Nguyen, Khoa;

    2015-01-01

    level using ultra-high resolution, ex vivo MRI. Fifteen autopsy samples were scanned at 0.13 mm isotropic resolution (on average) using customized hardware. The images were manually segmented into 13 different hippocampal substructures using a protocol specifically designed for this study; precise...... from the in vivo and ex vivo data were combined into a single computational atlas of the hippocampal formation with a novel atlas building algorithm based on Bayesian inference. The resulting atlas can be used to automatically segment the hippocampal subregions in structural MRI images, using...... datasets with different types of MRI contrast. The results show that the atlas and companion segmentation method: 1) can segment T1 and T2 images, as well as their combination, 2) replicate findings on mild cognitive impairment based on high-resolution T2 data, and 3) can discriminate between Alzheimer...

  6. Temperature elevation by HIFU in ex vivo porcine muscle: MRI measurement and simulation study

    Energy Technology Data Exchange (ETDEWEB)

    Solovchuk, Maxim A., E-mail: solovchuk@gmail.com [Center for Advanced Study in Theoretical Sciences (CASTS), National Taiwan University, Taipei 10617, Taiwan (China); Hwang, San Chao; Chang, Hsu [Medical Engineering Research Division, National Health Research Institute, Miaoli 35053, Taiwan (China); Thiriet, Marc [Sorbonne Universités, UPMC Univ Paris 06, UMR 7598, Laboratoire Jacques-Louis Lions, F-75005, Paris (France); Sheu, Tony W. H., E-mail: twhsheu@ntu.edu.tw [Department of Engineering Science and Ocean Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan, Republic of China and Center for Advanced Study in Theoretical Sciences (CASTS), National Taiwan University, Taipei 10617, Taiwan (China)

    2014-05-15

    Purpose: High-intensity focused ultrasound is a rapidly developing medical technology with a large number of potential clinical applications. Computational model can play a pivotal role in the planning and optimization of the treatment based on the patient's image. Nonlinear propagation effects can significantly affect the temperature elevation and should be taken into account. In order to investigate the importance of nonlinear propagation effects, nonlinear Westervelt equation was solved. Weak nonlinear propagation effects were studied. The purpose of this study was to investigate the correlation between the predicted and measured temperature elevations and lesion in a porcine muscle. Methods: The investigated single-element transducer has a focal length of 12 cm, an aperture of 8 cm, and frequency of 1.08 MHz. Porcine muscle was heated for 30 s by focused ultrasound transducer with an acoustic power in the range of 24–56 W. The theoretical model consists of nonlinear Westervelt equation with relaxation effects being taken into account and Pennes bioheat equation. Results: Excellent agreement between the measured and simulated temperature rises was found. For peak temperatures above 85–90 °C “preboiling” or cavitation activity appears and lesion distortion starts, causing small discrepancy between the measured and simulated temperature rises. From the measurements and simulations, it was shown that distortion of the lesion was caused by the “preboiling” activity. Conclusions: The present study demonstrated that for peak temperatures below 85–90 °C numerical simulation results are in excellent agreement with the experimental data in three dimensions. Both temperature rise and lesion size can be well predicted. Due to nonlinear effect the temperature in the focal region can be increased compared with the linear case. The current magnetic resonance imaging (MRI) resolution is not sufficient. Due to the inevitable averaging the measured

  7. Distinguishing human normal or cancerous esophagus tissue ex vivo using multiphoton microscopy

    International Nuclear Information System (INIS)

    Application of multiphoton microscopy (MPM) to clinical cancer research has greatly developed over the last few years. In this paper, we mainly focus on two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) for investigating esophageal cancer. We chiefly discuss the SHG/TPEF image and spectral characteristics of normal and cancerous esophagus submucosa with the combined multi-channel imaging mode and Lambda mode of a multiphoton microscope (LSM 510 META). Great differences can be detected, such as collagen content and morphology, glandular-shaped cancer cells, TPEF/SHG intensity ratio, and so on, which demonstrate that the multiphoton imaging technique has the potential ability for minimally-invasive early cancer diagnosis. (paper)

  8. Extracellular DNA contributes to dental biofilm formation: an ex vivo study

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke Louise; Dige, Irene;

    The extracellular matrix of dental biofilms plays an important role during caries development. It increases the mechanical stability of the biofilm, it prevents desiccation, it serves as a reservoir for nutrients and it contributes to the long-term preservation of acidic microenvironments. Research......, and that the enzymatic removal of extracellular DNA might be used as a therapeutic approach to biofilm diseases. Here, we investigate the effect of treatment with DNase I (100 Kunitz) on in vivo grown young dental biofilms. A total of 300 biofilm samples were grown on glass slabs placed on acrylic splints for 2.5, 5, 7...... results make a strong case for an increased research focus on extracellular DNA in dental biofilms and might pave the way for new, non-bactericidal therapeutic approaches to caries control based on DNase treatment....

  9. Proton Magnetic Resonance and Human Thyroid Neoplasia III. Ex VivoChemical-Shift Microimaging

    Science.gov (United States)

    Rutter, Allison; Künnecke, Basil; Dowd, Susan; Russell, Peter; Delbridge, Leigh; Mountford, Carolyn E.

    1996-03-01

    Magnetic-resonance chemical-shift microimaging, with a spatial resolution of 40 × 40 μm, is a modality which can detect alterations to cellular chemistry and hence markers of pathological processes in human tissueex vivo.This technique was used as a chemical microscope to assess follicular thyroid neoplasms, lesions which are unsatisfactorily investigated using standard histopathological techiques or water-based magnetic-resonance imaging. The chemical-shift images at the methyl frequency (0.9 ppm) identify chemical heterogeneity in follicular tumors which are histologically homogeneous. The observed changes to cellular chemistry, detectable in foci of approximately 100 cells or less, support the existence of a preinvasive state hitherto unidentified by current pathological techniques.

  10. Distinguishing human normal or cancerous esophagus tissue ex vivo using multiphoton microscopy

    Science.gov (United States)

    Liu, N. R.; Chen, G. N.; Wu, S. S.; Chen, R.

    2014-02-01

    Application of multiphoton microscopy (MPM) to clinical cancer research has greatly developed over the last few years. In this paper, we mainly focus on two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) for investigating esophageal cancer. We chiefly discuss the SHG/TPEF image and spectral characteristics of normal and cancerous esophagus submucosa with the combined multi-channel imaging mode and Lambda mode of a multiphoton microscope (LSM 510 META). Great differences can be detected, such as collagen content and morphology, glandular-shaped cancer cells, TPEF/SHG intensity ratio, and so on, which demonstrate that the multiphoton imaging technique has the potential ability for minimally-invasive early cancer diagnosis.

  11. High Glucose Impairs Insulin Signaling in the Glomerulus: An In Vitro and Ex Vivo Approach

    Science.gov (United States)

    Katsoulieris, Elias N.; Drossopoulou, Garyfalia I.; Kotsopoulou, Eleni S.; Vlahakos, Dimitrios V.; Lianos, Elias A.; Tsilibary, Effie C.

    2016-01-01

    Objective Chronic hyperglycaemia, as seen in type II diabetes, results in both morphological and functional impairments of podocytes in the kidney. We investigated the effects of high glucose (HG) on the insulin signaling pathway, focusing on cell survival and apoptotic markers, in immortalized human glomerular cells (HGEC; podocytes) and isolated glomeruli from healthy rats. Methods and Findings HGEC and isolated glomeruli were cultured for various time intervals under HG concentrations in the presence or absence of insulin. Our findings indicated that exposure of HGEC to HG led to downregulation of all insulin signaling markers tested (IR, p-IR, IRS-1, p-Akt, p-Fox01,03), as well as to increased sensitivity to apoptosis (as seen by increased PARP cleavage, Casp3 activation and DNA fragmentation). Short insulin pulse caused upregulation of insulin signaling markers (IR, p-IR, p-Akt, p-Fox01,03) in a greater extent in normoglycaemic cells compared to hyperglycaemic cells and for the case of p-Akt, in a PI3K-dependent manner. IRS-1 phosphorylation of HG-treated podocytes was negatively regulated, favoring serine versus tyrosine residues. Prolonged insulin treatment caused a significant decrease of IR levels, while alterations in glucose concentrations for various time intervals demonstrated changes of IR, p-IR and p-Akt levels, suggesting that the IR signaling pathway is regulated by glucose levels. Finally, HG exerted similar effects in isolated glomeruli. Conclusions These results suggest that HG compromises the insulin signaling pathway in the glomerulus, promoting a proapoptotic environment, with a possible critical step for this malfunction lying at the level of IRS-1 phosphorylation; thus we herein demonstrate glomerular insulin signaling as another target for investigation for the prevention and/ or treatment of diabetic nephropathy. PMID:27434075

  12. Plasmodium falciparum susceptibility to anti-malarial drugs in Dakar, Senegal, in 2010: an ex vivo and drug resistance molecular markers study

    OpenAIRE

    Fall, Bécaye; Pascual, Aurélie; Sarr, Fatoumata; Wurtz, Nathalie; Richard, Vincent; Baret, Eric; Diémé, Yaya; Briolant, Sébastien; Bercion, Raymond; Wade, Boubacar; Tall, Adama; Pradines, Bruno

    2013-01-01

    BACKGROUND: In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria. Since the introduction of ACT, there have been very few reports on the level of resistance of P. falciparum to anti-malarial drugs. To determine whether parasite susceptibility has been affected by the new anti-malarial policies, an ex vivo susceptibility and drug resistance molecular marker study was conducted on...

  13. Changes in Global Gene Expression Associated with 3D Structure of Tumors: An Ex Vivo Matrix-Free Mesothelioma Spheroid Model

    OpenAIRE

    Heungnam Kim; Yen Phung; Mitchell Ho

    2012-01-01

    Tumor microenvironments present significant barriers to anti-tumor agents. Molecules involved in multicellular tumor microenvironments, however, are difficult to study ex vivo. Here, we generated a matrix-free tumor spheroid model using the NCI-H226 mesothelioma cell line and compared the gene expression profiles of spheroids and monolayers using microarray analysis. Microarray analysis revealed that 142 probe sets were differentially expressed between tumor spheroids and monolayers. Gene ont...

  14. Expansion of human and murine hematopoietic stem and progenitor cells ex vivo without genetic modification using MYC and Bcl-2 fusion proteins.

    Directory of Open Access Journals (Sweden)

    Gregory A Bird

    Full Text Available The long-term repopulating hematopoietic stem cell (HSC population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs. We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use.

  15. Results from in vitro and ex vivo skin aging models assessing the antiglycation and anti-elastase MMP-12 potential of glycylglycine oleamide

    Directory of Open Access Journals (Sweden)

    Bogdanowicz P

    2016-06-01

    Full Text Available Patrick Bogdanowicz, Marie-José Haure, Isabelle Ceruti, Sandrine Bessou-Touya, Nathalie Castex-Rizzi Department of Pharmacology, Pierre Fabre Dermo-Cosmétique, Toulouse, France Background: Glycation is an aging reaction of naturally occurring sugars with dermal proteins. Type I collagen and elastin are most affected by glycation during intrinsic chronological aging. Aim: To study the in vitro and ex vivo assays in human skin cells and explants and the antiaging effects of glycylglycine oleamide (GGO. Materials and methods: The antiglycation effect of GGO was assessed in a noncellular in vitro study on collagen and, ex vivo, by immunohistochemical staining on human skin explants (elastin network glycation. The ability of GGO to contract fibroblasts was assessed in a functional assay, and its anti-elastase (MMP-12 activity was compared to that of oleic acid alone, glycylglycine (GG alone, and oleic acid associated with GG. Results: In vitro, GGO reduced the glycation of type I collagen. Ex vivo, GGO restored the expression of fibrillin-1 inhibited by glycation. Furthermore, GGO induced a tissue retraction of almost 30%. Moreover, the MMP-12 activity was inhibited by up to 60%. Conclusion: Under the present in vitro and ex vivo conditions, GGO prevents glycation of the major structural proteins of the dermis, helping to reduce the risk of rigidification. By maintaining the elastic function of the skin, GGO may be a promising sparring partner for other topical antiaging agents. Keywords: extracellular matrix, glycylglycine oleamide, glycation, fibrillin-1, matrix metalloproteinase-12, skin aging

  16. Nigrosome-1 on Susceptibility Weighted Imaging to Differentiate Parkinson’s Disease From Atypical Parkinsonism: An In Vivo and Ex Vivo Pilot Study

    Science.gov (United States)

    Meijer, Frederick J.A.; Steens, Stefan C.; van Rumund, Anouke; van Cappellen van Walsum, Anne-Marie; Küsters, Benno; Esselink, Rianne A.J.; Verbeek, Marcel M.; Bloem, Bastiaan R.; Goraj, Bożena

    2016-01-01

    Summary Background Previous case-control studies have suggested that the absence of a swallow-tail appearance in the substantia nigra on high-resolution SWI, representing nigrosome-1, has high accuracy to identify Parkinson’s disease (PD). The first goal of our study was to evaluate nigrosome-1 ex vivo using optimized high-resolution susceptibility sensitive MRI. Our second goal was to evaluate its diagnostic value in vivo using a clinical 3T SWI sequence to differentiate between PD and atypical parkinsonism (AP) in a cohort of patients with early-stage parkinsonism. Material/Methods Case-control pilot study to evaluate nigrosome-1 ex vivo (2 PD, 2 controls), using high-resolution susceptibility sensitive sequences at 11.7 T MRI. Next, evaluation of nigrosome-1 in vivo using a clinical 3 T SWI sequence in a prospective cohort of 60 patients with early-stage parkinsonism (39 PD, 21 AP). Moreover, 12 control subjects were scanned. The bilateral substantia nigra was evaluated by two neuroradiologists for the presence, absence or indecisive presence of nigrosome-1. The discriminative power was evaluated by Receiver-Operating Characteristic. Results We identified nigrosome-1 in ex vivo control subjects. Nigrosome-1 was not identified in the ex vivo PD cases. In our prospective clinical cohort study, the AUC for the swallow-tail sign to discriminate between PD and AP was 0.56 (0.41–0.71) for reader 1 and 0.68 (0.55–0.82) for reader 2. Conclusions The diagnostic accuracy of the swallow-tail sign was marginal to discriminate between PD and AP using our clinical 3 T SWI sequence.

  17. Live-cell imaging of the early stages of colony development in Fusarium oxysporum in vitro and ex vivo during infection of a human corneal model

    OpenAIRE

    Kurian, Smija Mariam

    2016-01-01

    ABSTRACTThe University of ManchesterName: Smija Mariam KurianDegree title: Doctor of PhilosophyResearch title: Live-cell imaging of the early stages of colony development in Fusarium oxysporum in vitro and ex vivo during infection of a human corneal modelDate: May 2016Abstract: Fusarium oxysporum is a major fungal plant pathogen and emerging human pathogen. It has been hypothesised that conidial anastomosis tube (CAT) fusion may facilitate horizontal gene/chromosome transfer that could result...

  18. Analysis of Dermal Papilla Cell Interactome Using STRING Database to Profile the ex Vivo Hair Growth Inhibition Effect of a Vinca Alkaloid Drug, Colchicine

    OpenAIRE

    Ching-Wu Hsia; Ming-Yi Ho; Hao-Ai Shui; Chong-Bin Tsai; Min-Jen Tseng

    2015-01-01

    Dermal papillae (DPs) control the formation of hair shafts. In clinical settings, colchicine (CLC) induces patients’ hair shedding. Compared to the control, the ex vivo hair fiber elongation of organ cultured vibrissa hair follicles (HFs) declined significantly after seven days of CLC treatment. The cultured DP cells (DPCs) were used as the experimental model to study the influence of CLC on the protein dynamics of DPs. CLC could alter the morphology and down-regulate the expression of alkal...

  19. Evaluation of primary stability of innovated orthodontic miniscrew system (STS): An ex-vivo study

    Science.gov (United States)

    Seifi, Massoud

    2016-01-01

    Background Stability is determined as one of the requirements in use of Temporary Anchorage Devices (TAD) in orthodontics. Miniscrew has been a widely used Bone Anchor. Compared with mini-implant that necessitates osseointegration; mechanical retention is a determining factor for primary stability of miniscrew. Studies investigated various ways to increase primary stability. The aim of this study is to introduce a new configuration of miniscrew system which is believed to obtain more primary stability. Material and Methods Freshly ovine mandibles were cut in blocks. Twenty-seven miniscrews (diameter 1.6 × 8 mm; G2, Dual Top Anchor System, Jeil Medical, Seoul, Korea) were inserted in the blocks and divided in 2 experimental groups: single miniscrew and the innovated design “Seifi Twin Screw (STS)”. Primary stability was evaluated by Periotest “M”® device. Results Independent t-test showed a significant difference between 2 experimental groups in periotest evaluation (p< 0.05). STS demonstrated higher primary stability due to its mechanical configuration and design. Conclusions The STS provides higher primary stability and was found to be effective in increased success rate of miniscrew systems from the standpoint of primary stability. Key words:Anchorage procedures, anchorage techniques, orthodontic anchorage procedures, miniscrews, temporary anchorage device. PMID:27398174

  20. Ex Vivo Costimulatory Blockade to Generate Regulatory T Cells From Patients Awaiting Kidney Transplantation.

    Science.gov (United States)

    Guinan, E C; Cole, G A; Wylie, W H; Kelner, R H; Janec, K J; Yuan, H; Oppatt, J; Brennan, L L; Turka, L A; Markmann, J

    2016-07-01

    Short-term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen-related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end-stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3(+) CD4(+) CD25(+) CD127(lo) surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg-specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation. PMID:26790369

  1. Ex vivo preparations of the intact vomeronasal organ and accessory olfactory bulb.

    Science.gov (United States)

    Doyle, Wayne I; Hammen, Gary F; Meeks, Julian P

    2014-01-01

    The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior. PMID:25145699

  2. Development of buccal adhesive tablet with prolonged antifungal activity: Optimization and ex vivo deposition studies

    Directory of Open Access Journals (Sweden)

    Madgulkar A

    2009-01-01

    Full Text Available The purpose of the present work was to prepare buccal adhesive tablets of miconazole nitrate. The simplex centroid experimental design was used to arrive at optimum ratio of carbopol 934P, hydroxypropylmethylcellulose K4M and polyvinylpyrollidone, which will provide desired drug release and mucoadhesion. Swelling index, mucoadhesive strength and in vitro drug release of the prepared tablet was determined. The drug release and bioadhesion was dependent on type and relative amounts of the polymers. The optimized combination was subjected to in vitro antifungal activity, transmucosal permeation, drug deposition in mucosa, residence time and bioadhesion studies. IR spectroscopy was used to investigate any interaction between drug and excipients. Dissolution of miconazole from tablets was sustained for 6 h. based on the results obtained, it can be concluded that the prepared slow release buccoadhesive tablets of miconazole would markedly prolong the duration of antifungal activity. Comparison of in vitro antifungal activity of tablet with marketed gel showed that drug concentrations above the minimum inhibitory concentration were achieved immediately from both formulations but release from tablet was sustained up to 6 h, while the gel showed initially fast drug release, which did not sustain later. Drug permeation across buccal mucosa was minimum from the tablet as well as marketed gel; the deposition of drug in mucosa was higher in case of tablet. In vitro residence time and bioadhesive strength of tablet was higher than gel. Thus the buccoadhesive tablet of miconazole nitrate may offer better control of antifungal activity as compared to the gel formulation.

  3. Ex-vivo HRMAS of adult brain tumours: metabolite quantification and assignment of tumour biomarkers

    Directory of Open Access Journals (Sweden)

    Wilson M

    2010-03-01

    Full Text Available Abstract Background High-resolution magic angle spinning (HRMAS NMR spectroscopy allows detailed metabolic analysis of whole biopsy samples for investigating tumour biology and tumour classification. Accurate biochemical assignment of small molecule metabolites that are "NMR visible" will improve our interpretation of HRMAS data and the translation of NMR tumour biomarkers to in-vivo studies. Results 1D and 2D 1H HRMAS NMR was used to determine that 29 small molecule metabolites, along with 8 macromolecule signals, account for the majority of the HRMAS spectrum of the main types of brain tumour (astrocytoma grade II, grade III gliomas, glioblastomas, metastases, meningiomas and also lymphomas. Differences in concentration of 20 of these metabolites were statistically significant between these brain tumour types. During the course of an extended 2D data acquisition the HRMAS technique itself affects sample analysis: glycine, glutathione and glycerophosphocholine all showed small concentration changes; analysis of the sample after HRMAS indicated structural damage that may affect subsequent histopathological analysis. Conclusions A number of small molecule metabolites have been identified as potential biomarkers of tumour type that may enable development of more selective in-vivo 1H NMR acquisition methods for diagnosis and prognosis of brain tumours.

  4. Curcumin-loaded colloidal carrier system: formulation optimization, mechanistic insight, ex vivo and in vivo evaluation

    Directory of Open Access Journals (Sweden)

    Naz Z

    2015-07-01

    Full Text Available Zrien Naz, Farhan Jalees AhmadNanomedicine Research Lab, Faculty of Pharmacy, Jamia Hamdard, New Delhi, IndiaAbstract: The present work investigated the topical delivery potential of nanoemulsion gel loaded with curcumin (CR. CR nanoemulsion (CR-NE was prepared by spontaneous emulsification method using oil (Labrafac PG/glyceryl triacetate, surfactant:cosurfactant (Smix (tween 80/polyethylene glycol [PEG] 400 and water. The pseudo-ternary phase diagrams were constructed and thermodynamic stability testing was performed. Droplet size and zeta potential were evaluated using photon correlation spectroscopy and transmission electron spectroscopy. Six formulations selected with an average droplet size ≤70±2.72 nm showed a fourfold increase in skin permeation as compared to crude CR solution in oil. The formulation CR-NE4 having a flux of 117.04±2.32 µg/cm2/h and with maximum retention (42.87% was selected, characterized (droplet size =41.13±3.34 nm and zeta potential =-33.1±1.45 mV, and incorporated into gel using carbopol-980 (1% w/v. Skin dynamics analyzed by confocal laser scanning microscopy showed maximum deposition of CR up to a depth of 86.98 µm and was in concordance with differential scanning calorimetry and Fourier transform infrared spectroscopy studies that confirmed lipid bilayer disruption, enhancing permeation. A 28-day anti-arthritic evaluation (body weight, paw edema, tibiotarsal joint thickness, TNF-α and IL-1β levels, and histopathology on Freund’s complete adjuvant induced arthritic rat model after topical application of CR-NE gel in Wistar rats demonstrated substantial reversal of arthritic symptoms. Thus, CR-NE gel possesses potential for therapeutic effects locally in inflammatory arthritic disorders with improved topical bioavailability.Keywords: anti-arthritic, anti-inflammatory, curcumin, nanoemulsion, skin dynamics, topical

  5. Photoactivated disinfection (PAD) of dental root canal system – An ex-vivo study

    Science.gov (United States)

    Mohan, Dennis; Maruthingal, Sunith; Indira, Rajamani; Divakar, Darshan Devang; Al Kheraif, Abdulaziz Abdullah; Ramakrishnaiah, Ravikumar; Durgesh, B.H.; Basavarajappa, Santhosh; John, Jacob

    2015-01-01

    Aim To investigate the efficacy of photo activated disinfection (PAD) in reducing colony-forming unit (CFU) counts of Enterococcus faecalis (E. faecalis) in infected dental root canals. The study compared the efficacy of PAD with conventional endodontic treatment (CET) and also a combination of CET along with PAD. Material and Methods 53 maxillary incisors were taken for the study. Teeth were divided into 3 groups, CET (Group I) (n = 11), PAD (Group II) (n = 21), and a combination of CET and PAD (Group III) which consisted of (n = 21) samples, Group II and Group III were further divided into 2 subgroups, Group IIa, IIb and Group IIIa, IIIb. Strains of E. faecalis were inoculated in all the root canals. CET group samples were treated by chemo-mechanical preparation (CMP) alone, PAD samples were treated with laser alone at 2 different exposure time (4 min and 2 min). In the combination treatment, samples were treated initially by CET and then by PAD for a time period of 4 min and 2 min. Contents of the root canal were aspirated, diluted and plated in Tryptone Soya Broth (TSB) and plates were incubated for 24 h to observe the bacterial regrowth. Results Showed PAD used along with CMP reduced the bacterial load of E. faecalis by 99.5% at 4 min and 98.89% at 2 min. Conclusion PAD may be an adjunctive procedure to kill residual bacteria in the dental root canal systems after standard endodontic root canal preparation. PMID:26858548