WorldWideScience

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. 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.

  3. ex vivo DNA assembly

    Directory of Open Access Journals (Sweden)

    Adam B Fisher

    2013-10-01

    Full Text Available Even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. Advances in cloning techniques have resulted in powerful in vitro and in vivo assembly of DNA. However, monetary and time costs have limited these approaches. Here, we report an ex vivo DNA assembly method that uses cellular lysates derived from a commonly used laboratory strain of Escherichia coli for joining double-stranded DNA with short end homologies embedded within inexpensive primers. This method concurrently shortens the time and decreases costs associated with current DNA assembly methods.

  4. 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.

  5. Concise review: ex vivo expansion of cord blood-derived hematopoietic stem and progenitor cells: basic principles, experimental approaches, and impact in regenerative medicine.

    Science.gov (United States)

    Flores-Guzmán, Patricia; Fernández-Sánchez, Verónica; Mayani, Hector

    2013-11-01

    Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play key roles in the production of mature blood cells and in the biology and clinical outcomes of hematopoietic transplants. The numbers of these cells, however, are extremely low, particularly in umbilical cord blood (UCB); thus, ex vivo expansion of human UCB-derived HSCs and HPCs has become a priority in the biomedical field. Expansion of progenitor cells can be achieved by culturing such cells in the presence of different combinations of recombinant stimulatory cytokines; in contrast, expansion of actual HSCs has proved to be more difficult because, in addition to needing recombinant cytokines, HSCs seem to deeply depend on the presence of stromal cells and/or elements that promote the activation of particular self-renewal signaling pathways. Hence, there is still controversy regarding the optimal culture conditions that should be used to achieve this. To date, UCB transplants using ex vivo-expanded cells have already been performed for the treatment of different hematological disorders, and although results are still far from being optimal, the advances are encouraging. Recent studies suggest that HSCs may also give rise to nonhematopoietic cells, such as neural, cardiac, mesenchymal, and muscle cells. Such plasticity and the possibility of producing nonhematopoietic cells at the clinical scale could bring new alternatives for the treatment of neural, metabolic, orthopedic, cardiac, and neoplastic disorders. Once standardized, ex vivo expansion of human HSCs/HPCs will surely have a positive impact in regenerative medicine.

  6. Comparison of In-Vitro and Ex-Vivo Wound Healing Assays for the Investigation of Diabetic Wound Healing and Demonstration of a Beneficial Effect of a Triterpene Extract

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    Ueck, Christopher; Volksdorf, Thomas; Houdek, Pia; Vidal-y-Sy, Sabine; Sehner, Susanne; Ellinger, Bernhard; Lobmann, Ralf; Larena-Avellaneda, Axel; Reinshagen, Konrad; Ridderbusch, Ina; Kohrmeyer, Klaas; Moll, Ingrid; Daniels, Rolf; Werner, Philipp; Merfort, Irmgard; Brandner, Johanna M.

    2017-01-01

    Diabetes mellitus is a frequent cause for chronic, difficult-to-treat wounds. New therapies for diabetic wounds are urgently needed and in-vitro or ex-vivo test systems are essential for the initial identification of new active molecules. The aim of this study is to compare in-vitro and ex-vivo test systems for their usability for early drug screening and to investigate the efficacy of a birch bark triterpene extract (TE) that has been proven ex-vivo and clinically to accelerate non-diabetic wound healing (WH), in a diabetic context. We investigated in-vitro models for diabetic WH, i.e. scratch assays with human keratinocytes from diabetic donors or cultured under hyperglycaemic conditions and a newly developed porcine ex-vivo hyperglycaemic WH model for their potential to mimic delayed diabetic WH and for the influence of TE in these test systems. We show that keratinocytes from diabetic donors often fail to exhibit significantly delayed WH. For cells under hyperglycaemic conditions significant decrease is observed but is influenced by choice of medium and presence of supplements. Also, donor age plays a role. Interestingly, hyperglycaemic effects are mainly hyperosmolaric effects in scratch assays. Ex-vivo models under hyperglycaemic conditions show a clear and substantial decrease of WH, and here both glucose and hyperosmolarity effects are involved. Finally, we provide evidence that TE is also beneficial for ex-vivo hyperglycaemic WH, resulting in significantly increased length of regenerated epidermis to 188±16% and 183±11% (SEM; p<0.05) compared to controls when using two different TE formulations. In conclusion, our results suggest that microenvironmental influences are important in WH test systems and that therefore the more complex hyperglycaemic ex-vivo model is more suitable for early drug screening. Limitations of the in-vitro and ex-vivo models are discussed. Furthermore our data recommend TE as a promising candidate for in-vivo testings in diabetic

  7. Investigation of the differentiation of ex vivo nerve and fat tissues using laser-induced breakdown spectroscopy (LIBS): Prospects for tissue-specific laser surgery.

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    Mehari, Fanuel; Rohde, Maximillian; Kanawade, Rajesh; Knipfer, Christian; Adler, Werner; Klämpfl, Florian; Stelzle, Florian; Schmidt, Michael

    2016-10-01

    In the present study, the elemental compositions of fat and nerve tissue during their plasma mediated laser ablation are studied in the context of tissue differentiation for laser surgery applications by using Laser-Induced Breakdown Spectroscopy (LIBS). Tissue samples of porcine fat and nerve were prepared as ex vivo experimental objects. Plasma mediated laser ablation is performed using an Nd : YAG laser in open air and under normal stray light conditions. The performed measurements suggest that the two tissue types show a high similarity in terms of qualitative elemental composition while at the same time revealing a distinct difference in the concentration of the constituent elements. Different analysis approaches are evaluated and discussed to optimize the tissue-differentiation performance of the LIBS approach. Plasma mediated laser tissue ablation.

  8. X-ray cell tracking: from ex-vivo to in-vivo experiments

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    Astolfo, A.; Schültke, E.; Menk, R.-H.; Hall, C.; Juurlink, B.; Arfelli, F.

    2013-06-01

    The capacity to track cells (cell tracking) using x-rays on ex-vivo specimens of both malignant and non-malignant cell lines on small animals has been demonstrated recently. Gold nanoparticles have been used as a cellular contrast agent to render cells visible in x-ray microCT acquisitions. The limits of the technique proposed are basically driven by the imaging system used. Single cell resolution can be achieved using synchrotron radiation in-vitro or ex-vivo samples. Micro-focus x-ray tubes can be used to obtain high resolution cell tracking but with some limitations. However, the translation from ex-vivo to in-vivo experiments is not straightforward. The dose restrictions required for in-vivo longitudinal experiments set severe limitations on the technique. Here we present a detailed investigation showing a significant reduction of x-ray dose for the tracking of brain tumour cells. Monte Carlo simulations have been performed considering different spatial resolutions, photon fluence, number of projections, lesion dimension and cell contrast dilution. The findings are compared with real samples imaged using the same parameters. A pioneering in-vivo experiment conducted at the SYRMEP beamline (Elettra, Basovizza, Italy) is presented here as proof of principle of in-vivo longitudinal x-ray cell tracking experiments on small animals at low x-ray doses.

  9. Ebola Virus Persistence in Semen Ex Vivo.

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    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.

  10. 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

  11. Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.

    Science.gov (United States)

    Bosch, Daniel; Asede, Douglas; Ehrlich, Ingrid

    2016-04-05

    Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

  12. 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%.

  13. 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.

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

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    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

  15. 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

  16. 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...

  17. 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.

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

    Science.gov (United States)

    Mundargi, Raghavendra; Venkataraman, Divya; Kumar, Saranya; Mogal, Vishal; Ortiz, Raphael; Loo, Joachim; Venkatraman, Subbu; Steele, Terry

    2015-01-01

    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.

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

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    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.

  20. Optimization of SCF feeding regimen for ex vivo expansion of cord blood hematopoietic stem cells.

    Science.gov (United States)

    Du, Zheng; Cai, Haibo; Ye, Zhaoyang; Tan, Wen-Song

    2012-12-15

    Stem cell factor (SCF) plays important roles in ex vivo expansion of hematopoietic stem cells (HSCs). In this study, the effects of dose and feeding time of SCF on ex vivo expansion of CD34(+) cells were investigated in serum-free medium supplemented with a cytokine cocktail composed of SCF, thrombopoietin (TPO) and flt3-ligand (FL). Among the four tested doses (0, 5, 50 and 500ng/mL), a SCF dose of 50ng/mL was demonstrated to be most favorable for ex vivo expansion of CD34(+) cells, which resulted in 34.22±10.80 and 8.89±1.25 folds of expansion regarding total cells and CD34(+) cells, respectively. Meanwhile, the specific growth rate of cells, the consumption rate of SCF and the percentage of CD34(+)c-kit(+) cells during the 21-day culture process were analyzed. The results indicated that initial 4-day period was a critical stage for SCF functioning on CD34(+) cells during ex vivo expansion. Based on this, a modified SCF feeding regimen was proposed, in which SCF (50ng/mL) was only supplemented on day 0 in the cytokine cocktail and cells were then fed with TPO and FL till the end of culture. It was found that this SCF feeding regimen could expand CD34(+) cells efficiently, thus providing a cost-effect expansion protocol for HSCs.

  1. 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.

  2. 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.

  3. Photodynamic diagnosis of bladder cancer in ex vivo urine cytology

    Science.gov (United States)

    Fu, C. Y.; Ng, B. K.; Razul, S. Gulam; Olivo, Malini C.; Lau, Weber K. O.; Tan, P. H.; Chin, William

    2006-02-01

    Bladder cancer is the fourth common malignant disease worldwide, accounting for 4% of all cancer cases. In Singapore, it is the ninth most common form of cancer. The high mortality rate can be reduced by early treatment following precancerous screening. Currently, the gold standard for screening bladder tumors is histological examination of biopsy specimen, which is both invasive and time-consuming. In this study ex vivo urine fluorescence cytology is investigated to offer a timely and biopsy-free means for detecting bladder cancers. Sediments in patients' urine samples were extracted and incubated with a novel photosensitizer, hypericin. Laser confocal microscopy was used to capture the fluorescence images at an excitation wavelength of 488 nm. Images were subsequently processed to single out the exfoliated bladder cells from the other cells based on the cellular size. Intensity histogram of each targeted cell was plotted and feature vectors, derived from the histogram moments, were used to represent each sample. A difference in the distribution of the feature vectors of normal and low-grade cancerous bladder cells was observed. Diagnostic algorithm for discriminating between normal and low-grade cancerous cells is elucidated in this paper. This study suggests that the fluorescence intensity profiles of hypericin in bladder cells can potentially provide an automated quantitative means of early bladder cancer diagnosis.

  4. 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.

  5. Ex vivo-expanded cynomolgus macaque regulatory T cells are resistant to alemtuzumab-mediated cytotoxicity

    OpenAIRE

    2013-01-01

    Alemtuzumab (Campath-1H) is a humanized monoclonal antibody (Ab) directed against CD52 that depletes lymphocytes and other leukocytes, mainly by complement-dependent mechanisms. We investigated the influence of alemtuzumab (i) on ex vivo-expanded cynomolgus monkeys regulatory T cells (Treg) generated for prospective use in adoptive cell therapy and (ii) on naturally-occurring Treg following alemtuzumab infusion. Treg were isolated from PBMC and lymph nodes and expanded for two rounds. CD52 ex...

  6. Ex vivo culture of human fetal gonads

    DEFF Research Database (Denmark)

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

    2015-01-01

    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......-treatment mediates initiation of meiosis in human fetal ovary ex vivo. STUDY DESIGN, SIZE, DURATION: This was a controlled ex vivo study of human fetal gonads treated with RA in 'hanging-drop' tissue cultures. The applied experimental set-up preserves germ cell-somatic niche interactions and the investigated...

  7. Light Emission Requires Exposure to the Atmosphere in Ex Vivo Bioluminescence Imaging

    Directory of Open Access Journals (Sweden)

    Yusuke Inoue

    2006-04-01

    Full Text Available The identification of organs bearing luciferase activity by in vivo bioluminescence imaging (BLI is often difficult, and ex vivo imaging of excised organs plays a complementary role. This study investigated the importance of exposure to the atmosphere in ex vivo BLI. Mice were inoculated with murine pro-B cell line Ba/F3 transduced with firefly luciferase and p190 BCR-ABL. They were killed following in vivo BLI, and whole-body imaging was done after death and then after intraperitoneal air injection. In addition, the right knee was exposed and imaged before and after the adjacent bones were cut. Extensive light signals were seen on in vivo imaging. The luminescence disappeared after the animal was killed, and air injection restored the light emission from the abdomen only, suggesting a critical role of atmospheric oxygen in luminescence after death. Although no substantial light signal at the right knee was seen before bone cutting, light emission was evident after cutting. In conclusion, in ex vivo BLI, light emission requires exposure to the atmosphere. Bone destruction is required to demonstrate luciferase activity in the bone marrow after death.

  8. In vitro and ex vivo angiogenic effects of roxarsone on rat endothelial cells.

    Science.gov (United States)

    Zhu, Jiaqiao; Cui, Weibo; Liu, Xue; Ying, Jun; Hu, Chengyun; Zhang, Yumei

    2013-11-25

    Roxarsone, a feed additive, is being used worldwide to promote animal growth. However, the potential effect of roxarsone on angiogenesis has not been extensively characterized. We examined the ability of roxarsone to promote angiogenesis of rat endothelial cells in vitro and from rat aorta rings ex vivo. Endothelial cells from rats were exposed to 0.01-10.00μM roxarsone, 5ng/mL vascular endothelial growth factor (VEGF) as a positive control or phosphate buffer saline (PBS) as a negative control. Cell proliferation was measured by MTT assay, and the content of VEGF in supernatants was measured by enzyme-linked immunosorbent assay and Western blotting. A Matrigel-induced tube formation assay was used to evaluate the effects of roxarsone on endothelial cells. Additionally, the total number and length of microvessels sprouted from rat aortic rings were measured for ex vivo investigation of angiogenesis. Results showed that the cell viability and total number and length of capillary-like tube formations after roxarsone treatment was significantly higher than that of negative (Proxarsone was significantly higher than that of PBS treatment, with a peak value of 1.0μM. These results further demonstrate the potential of roxarsone to promote angiogenesis in vitro and ex vivo.

  9. 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

  10. In and ex vivo breast disease study by Raman spectroscopy

    DEFF Research Database (Denmark)

    Raniero, L.; Canevari, R. A.; Ramalho, L. N. Z.

    2011-01-01

    %, while the in vivo skin-removed normal and abnormal tissues were correctly classified again based on their measured Raman spectra with a discriminant proportion of 86%. This result reveals a strong influence due to the skin of the breast, which decreased the specificity by 11%. Finally, the results from......In this work, Raman spectra in the 900-1,800 cm(-1) wavenumber region of in vivo and ex vivo breast tissues of both healthy mice (normal) and mice with induced mammary gland tumors (abnormal) were measured. In the case of the in vivo tissues, the Raman spectra were collected for both transcutaneous...... ex vivo measurements gave the highest specificity and sensitivity: 96 and 97%, respectively, as well as a largest percentage for correct discrimination: 94%. Now that the important bands have been experimentally determined in this and other works, what remains is for first principles molecular...

  11. 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.

  12. Ex Vivo Metrics, a preclinical tool in new drug development.

    Science.gov (United States)

    Curtis, C Gerald; Bilyard, Kevin; Stephenson, Hugo

    2008-01-23

    Among the challenges facing translational medicine today is the need for greater productivity and safety during the drug development process. To meet this need, practitioners of translational medicine are developing new technologies that can facilitate decision making during the early stages of drug discovery and clinical development. Ex Vivo Metrics is an emerging technology that addresses this need by using intact human organs ethically donated for research. After hypothermic storage, the organs are reanimated by blood perfusion, providing physiologically and biochemically stable preparations. In terms of emulating human exposure to drugs, Ex Vivo Metrics is the closest biological system available for clinical trials. Early application of this tool for evaluating drug targeting, efficacy, and toxicity could result in better selection among promising drug candidates, greater drug productivity, and increased safety.

  13. Ex Vivo Metrics™, a preclinical tool in new drug development

    Directory of Open Access Journals (Sweden)

    Bilyard Kevin

    2008-01-01

    Full Text Available Abstract Among the challenges facing translational medicine today is the need for greater productivity and safety during the drug development process. To meet this need, practitioners of translational medicine are developing new technologies that can facilitate decision making during the early stages of drug discovery and clinical development. Ex Vivo Metrics™ is an emerging technology that addresses this need by using intact human organs ethically donated for research. After hypothermic storage, the organs are reanimated by blood perfusion, providing physiologically and biochemically stable preparations. In terms of emulating human exposure to drugs, Ex Vivo Metrics is the closest biological system available for clinical trials. Early application of this tool for evaluating drug targeting, efficacy, and toxicity could result in better selection among promising drug candidates, greater drug productivity, and increased safety.

  14. 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

  15. 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

  16. 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...

  17. Bioluminescence-mediated longitudinal monitoring of adipose-derived stem cells in a large mammal ex vivo organ culture.

    Science.gov (United States)

    Peeters, Mirte; van Rijn, Sjoerd; Vergroesen, Pieter-Paul A; Paul, Cornelis P L; Noske, David P; Vandertop, W Peter; Wurdinger, Thomas; Helder, Marco N

    2015-09-09

    Recently, ex vivo three-dimensional organ culture systems have emerged to study the physiology and pathophysiology of human organs. These systems also have potential as a translational tool in tissue engineering; however, this potential is limited by our ability to longitudinally monitor the fate and action of cells used in regenerative therapies. Therefore, we investigated luciferase-mediated bioluminescence imaging (BLI) as a non-invasive technique to continuously monitor cellular behavior in ex vivo whole organ culture. Goat adipose-derived stem cells (gADSCs) were transduced with either Firefly luciferase (Fluc) or Gaussia luciferase (Gluc) reporter genes and injected in isolated goat intervertebral discs (IVD). Luciferase activity was monitored by BLI for at least seven days of culture. Additionally, possible confounders specific to avascular organ culture were investigated. Gluc imaging proved to be more suitable compared to Fluc in monitoring gADSCs in goat IVDs. We conclude that BLI is a promising tool to monitor spatial and temporal cellular behavior in ex vivo organ culture. Hence, ex vivo organ culture systems allow pre-screening and pre-validation of novel therapeutic concepts prior to in vivo large animal experimentation. Thereby, organ culture systems can reduce animal use, and improve the speed of innovation by overcoming technological, ethical and financial challenges.

  18. 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.

  19. Functional genetic targeting of embryonic kidney progenitor cells ex vivo.

    Science.gov (United States)

    Junttila, Sanna; Saarela, Ulla; Halt, Kimmo; Manninen, Aki; Pärssinen, Heikki; Lecca, M Rita; Brändli, André W; Sims-Lucas, Sunder; Skovorodkin, Ilya; Vainio, Seppo J

    2015-05-01

    The embryonic mammalian metanephric mesenchyme (MM) is a unique tissue because it is competent to generate the nephrons in response to Wnt signaling. An ex vivo culture in which the MM is separated from the ureteric bud (UB), the natural inducer, can be used as a classic tubule induction model for studying nephrogenesis. However, technological restrictions currently prevent using this model to study the molecular genetic details before or during tubule induction. Using nephron segment-specific markers, we now show that tubule induction in the MM ex vivo also leads to the assembly of highly segmented nephrons. This induction capacity was reconstituted when MM tissue was dissociated into a cell suspension and then reaggregated (drMM) in the presence of human recombinant bone morphogenetic protein 7/human recombinant fibroblast growth factor 2 for 24 hours before induction. Growth factor-treated drMM also recovered the capacity for organogenesis when recombined with the UB. Cell tracking and time-lapse imaging of chimeric drMM cultures indicated that the nephron is not derived from a single progenitor cell. Furthermore, viral vector-mediated transduction of green fluorescent protein was much more efficient in dissociated MM cells than in intact mesenchyme, and the nephrogenic competence of transduced drMM progenitor cells was preserved. Moreover, drMM cells transduced with viral vectors mediating Lhx1 knockdown were excluded from the nephric tubules, whereas cells transduced with control vectors were incorporated. In summary, these techniques allow reproducible cellular and molecular examinations of the mechanisms behind nephrogenesis and kidney organogenesis in an ex vivo organ culture/organoid setting.

  20. Lactobacillus rhamnosus GG increases Toll-like receptor 3 gene expression in murine small intestine ex vivo and in vivo.

    Science.gov (United States)

    Aoki-Yoshida, A; Saito, S; Fukiya, S; Aoki, R; Takayama, Y; Suzuki, C; Sonoyama, K

    2016-06-01

    Administration of Lactobacillus rhamnosus GG (LGG) has been reported to be therapeutically effective against acute secretory diarrhoea resulting from the structural and functional intestinal mucosal lesions induced by rotavirus infection; however, the underlying mechanisms remain to be completely elucidated. Because Toll-like receptor 3 (TLR3) plays a key role in the innate immune responses following the recognition of rotavirus, the present study examined whether LGG influences TLR3 gene expression in murine small intestine ex vivo and in vivo. We employed cultured intestinal organoids derived from small intestinal crypts as an ex vivo tissue model. LGG supplementation increased TLR3 mRNA levels in the intestinal organoids, as estimated by quantitative real-time polymerase chain reaction. Likewise, single and 7-day consecutive daily administrations of LGG increased TLR3 mRNA levels in the small intestine of C57BL/6N mice. The mRNA levels of other TLRs were not substantially altered both ex vivo and in vivo. In addition, LGG supplementation increased the mRNA levels of an antiviral type 1 interferon, interferon-α (IFN-α), and a neutrophil chemokine, CXCL1, upon stimulation with a synthetic TLR3 ligand, poly(I:C) in the intestinal organoids. LGG administration did not alter IFN-α and CXCL1 mRNA levels in the small intestine in vivo. Supplementation of other bacterial strains, Bifidobacterium bifidum and Lactobacillus paracasei, failed to increase TLR3 and poly(I:C)-stimulated CXCL1 mRNA levels ex vivo. We propose that upregulation of TLR3 gene expression may play a pivotal role in the therapeutic efficacy of LGG against rotavirus-associated diarrhoea. In addition, we demonstrated that intestinal organoids may be a promising ex vivo tissue model for investigating host-pathogen interactions and the antiviral action of probiotics in the intestinal epithelium.

  1. Ex vivo gene therapy for HIV-1 treatment.

    Science.gov (United States)

    Scherer, Lisa J; Rossi, John J

    2011-04-15

    Until recently, progress in ex vivo gene therapy (GT) for human immunodeficiency virus-1 (HIV-1) treatment has been incremental. Long-term HIV-1 remission in a patient who received a heterologous stem cell transplant for acquired immunodeficiency syndrome-related lymphoma from a CCR5(-/-) donor, even after discontinuation of conventional therapy, has energized the field. We review the status of current approaches as well as future directions in the areas of therapeutic targets, combinatorial strategies, vector design, introduction of therapeutics into stem cells and enrichment/expansion of gene-modified cells. Finally, we discuss recent advances towards clinical application of HIV-1 GT.

  2. In vitro and ex vivo toxicological testing of sildenafil-loaded solid lipid nanoparticles.

    Science.gov (United States)

    Paranjpe, M; Neuhaus, V; Finke, J H; Richter, C; Gothsch, T; Kwade, A; Büttgenbach, S; Braun, A; Müller-Goymann, C C

    2013-08-01

    The aim of this study was to investigate the potential cytotoxicity of solid lipid nanoparticles (SLN) loaded with sildenafil. The SLNs were tested as a new drug delivery system (DDS) for the inhalable treatment of pulmonary hypertension in human lungs. Solubility of sildenafil in SLN lipid matrix (30:70 phospholipid:triglyceride) was determined to 1% sildenafil base and 0.1% sildenafil citrate, respectively. Sildenafil-loaded SLN with particle size of approximately 180 nm and monomodal particle size distribution were successfully manufactured using a novel microchannel homogenization method and were stable up to three months. Sildenafil-loaded SLN were then used in in vitro and ex vivo models representing lung and heart tissue. For in vitro models, human alveolar epithelial cell line (A459) and mouse heart endothelium cell line (MHEC5-T) were used. For ex vivo models, rat precision cut lung slices (PCLS) and rat heart slices (PCHS) were used. All the models were treated with plain SLN and sildenafil-loaded SLN in a concentration range of 0-5000 µg/ml of lipid matrix. The toxicity was evaluated in vitro and ex vivo by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Median lethal dose 50% (LD50) values for A549 cells and PCLS were found to be in the range of 1200-1900 µg/ml while for MHEC5-T cells and precision cut heart slices values were found between 1500 and 2800 µg/ml. PCHS showed slightly higher LD50 values in comparison to PCLS. Considering the toxicological aspects, sildenafil-loaded SLN could have potential in the treatment of pulmonary hypertension via inhalation route.

  3. Ex vivo MRI of axillary lymph nodes in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Luciani, Alain [Department of Radiology, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France)], E-mail: luciani@hmn.ap-hop-paris.fr; Pigneur, Frederic [Department of Radiology, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France); Ghozali, Faridah [Department of Pathology, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France); Dao, Thu-Ha [Department of Radiology, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France); Cunin, Patrick [Unite de Recherche Clinique, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France); Meyblum, Evelyne [Department of Radiology, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France); De Baecque-Fontaine, Cecile [Department of Pathology, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France); Alamdari, Ali [Department of Plastic Surgery, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France); Maison, Patrick [Unite de Recherche Clinique, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France); Deux, Jean Francois [Department of Radiology, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France); Lagrange, Jean Leon [Department of Radiotherapy, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France); Lantieri, Laurent [Department of Plastic Surgery, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France); Rahmouni, Alain [Department of Radiology, AP-HP, Groupe Henri Mondor-Albert Chenevier, 94010 Creteil (France)

    2009-01-15

    Purpose: To provide a strategy for precise co-localization of lymph nodes on axillary lymph-node dissection (ALND) specimens both on pathology and MR. To identify nodal features suggestive of metastatic involvement on a node-to-node basis. Materials and methods: National Institutional review-board approved this prospective study of 18 patients with breast cancer referred for ALND. Ex vivo T1 and inversion recovery (IR) T2 WI of ALND specimens tightly positioned within scaled plastic cranes was performed immediately after surgery. The correspondence of MR-based or pathologically based nodes location was assessed. The MR size and morphological presentation of metastatic and normal nodes were compared (Student's t-test or Mann-Whitney test). Quantitative variables were compared using Pearson coefficient. Results: 207 nodes were retrieved on pathology and 165 on MR. MR-pathological correlation of nodes location was high regarding MR-identified nodes (r = 0.755). An MR short axis threshold of 4 mm yielded the best predictive value for metastatic nodal involvement (Se = 78.6%; Sp = 62.3%). Irregular contours (Se = 35.7%; Sp = 96.7%), central nodal hyper-intensity on IR T2 WI (Se = 57.1%; Sp = 91.4%), and a cortical thickness above 3 mm (Se = 63.6%; Sp = 83.2%) were significantly associated with metastatic involvement. Conclusion: Ex vivo MR allows node-to-node correlation with pathology. Morphological MR criteria can suggest metastatic involvement.

  4. 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.

  5. Ex-Vivo percutaneous absorption of enrofloxacin: Comparison of LMOG organogel vs. pentravan cream.

    Science.gov (United States)

    Kirilov, Plamen; Tran, Van Hung; Ducrotté-Tassel, Alban; Salvi, Jean-Paul; Perrot, Sébastien; Haftek, Marek; Boulieu, Roselyne; Pirot, Fabrice

    2016-02-10

    The objective of this study was to investigate the percutaneous absorption of enrofloxacin from two base formulations, Pentravan cream and LMOG organogel. Ex-vivo experiments were carried out on pig ear skin. The percutaneous permeation through pig skin of two formulations containing 5 wt% of enrofloxacin was measured and compared using Franz diffusion cells. At appropriate intervals up to 120 h, diffusion samples were taken and analyzed using HPLC assays. Permeation profiles were established and the parameters Tlag and flux values were calculated. In this ex-vivo study, the flux values were 0.35 μgcm(-2)h(-1) for Pentravan and 1.22 μgcm(-2)h(-1) for LMOG organogel, corresponding respectively to 7.9 % and 29.3 % of enrofloxacin absorbed after 120 h by these formulations. The lag time (T lag) of Pentravan and organogel were 6.32 and 0.015 h respectively. The absorption time to reach the antibiotic concentration of enrofloxacin (2 μgmL(-1)) in the receptor was 60 h with Pentravan and 30 h with the organogel, suggesting more effective treatment by the latter. Enrofloxacin contained in organogel could be absorbed through pig ear skin 3.7 times greater than that in Pentravan (commercial formulation). This study demonstrates the perspective of organogel formulations as potential drug delivery systems.

  6. 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.

  7. 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.

  8. Ex vivo-expanded cynomolgus macaque regulatory T cells are resistant to alemtuzumab-mediated cytotoxicity.

    Science.gov (United States)

    Dons, E M; Raimondi, G; Zhang, H; Zahorchak, A F; Bhama, J K; Lu, L; Ezzelarab, M; Ijzermans, J N M; Cooper, D K C; Thomson, A W

    2013-08-01

    Alemtuzumab (Campath-1H) is a humanized monoclonal antibody (Ab) directed against CD52 that depletes lymphocytes and other leukocytes, mainly by complement-dependent mechanisms. We investigated the influence of alemtuzumab (i) on ex vivo-expanded cynomolgus monkey regulatory T cells (Treg) generated for prospective use in adoptive cell therapy and (ii) on naturally occurring Treg following alemtuzumab infusion. Treg were isolated from PBMC and lymph nodes and expanded for two rounds. CD52 expression, binding of alemtuzumab and both complement-mediated killing and Ab-dependent cell-mediated cytotoxicity (ADCC) were compared between freshly isolated and expanded Treg and effector T cells. Monkeys undergoing allogeneic heart transplantation given alemtuzumab were monitored for Treg and serum alemtuzumab activity. Ex vivo-expanded Treg showed progressive downregulation of CD52 expression, absence of alemtuzumab binding, minimal change in complement inhibitory protein (CD46) expression and no complement-dependent killing or ADCC. Infusion of alemtuzumab caused potent depletion of all lymphocytes, but a transient increase in the incidence of circulating Treg. After infusion of alemtuzumab, monkey serum killed fresh PBMC, but not expanded Treg. Thus, expanded cynomolgus monkey Treg are resistant to alemtuzumab-mediated, complement-dependent cytotoxicity. Furthermore, our data suggest that these expanded monkey Treg can be infused into graft recipients given alemtuzumab without risk of complement-mediated killing.

  9. 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.

  10. 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 two experimental models. The results from the in vitro studies were in excellent agreement with the ex vivo results (caffeine approximately antipyrine>benzoic acid>glyphosate). However the transfer rate was much slower in the BeWo cells compared to the perfusion system. The advantages and limitations....... 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...... across the BeWo cells was observed in the rank order of caffeine>antipyrine>benzoic acid>glyphosate in terms of both the apparent permeability coefficient and the initial slope, defined as the linear rate of substance transferred to the fetal compartment as percent per time, a parameter used to compare...

  11. Transplant of ex vivo incubated bone marrow with rIL -7 for the enhancement of immuno-hematopoietic reconstitution.

    Science.gov (United States)

    Abdul-Hai, Ali; Ben Yehuda, Aryeh; Slavin, Shimon; Or, Reuven

    2015-01-01

    Interleukin-7 (IL-7) is a critical cytokine in early B and T cell development. Peripheral T cell expansion and thymopoiesis and is a result of the ongoing reconstitution from uncommitted stem cells after transplant. We investigated the efficacy of ex vivo incubated bone marrow cells treated with recombinant human IL-7 (rIL-7) on subsequent in vivo murine models of syngeneic bone marrow (BM) transplant. After ex vivo culture with rIL-7, we observed a 1½-fold increase in BM cellularity; this increase was associated with an enhanced reconstitution of bone marrow cells and thymocytes at 45 days post-transplant. In addition to increased cellularity, lymphocytes from mice transplanted with cultured rIL-7 showed enhanced proliferative responses to mitogenic stimulation. These findings suggest rIL-7 to be a promising agent for the clinical application of treating immune deficiency and enhancing immuno-hematopoietic reconstitution of the stem cell auto/allograft.

  12. 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

  13. 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.

  14. In vitro-ex vivo correlations between a cell-laden hydrogel and mucosal tissue for screening composite delivery systems.

    Science.gov (United States)

    Blakney, Anna K; Little, Adam B; Jiang, Yonghou; Woodrow, Kim A

    2016-11-01

    Composite delivery systems where drugs are electrospun in different layers and vary the drug stacking-order are posited to affect bioavailability. We evaluated how the formulation characteristics of both burst- and sustained-release electrospun fibers containing three physicochemically diverse drugs: dapivirine (DPV), maraviroc (MVC) and tenofovir (TFV) affect in vitro and ex vivo release. We developed a poly(hydroxyethyl methacrylate) (pHEMA) hydrogel release platform for the rapid, inexpensive in vitro evaluation of burst- and sustained-release topical or dermal drug delivery systems with varying microarchitecture. We investigated properties of the hydrogel that could recapitulate ex vivo release into nonhuman primate vaginal tissue. Using a dimethyl sulfoxide extraction protocol and high-performance liquid chromatography analysis, we achieved >93% recovery from the hydrogels and >88% recovery from tissue explants for all three drugs. We found that DPV loading, but not stacking order (layers of fiber containing a single drug) or microarchitecture (layers with isolated drug compared to all drugs in the same layer) impacted the burst release in vitro and ex vivo. Our burst-release formulations showed a correlation for DPV accumulation between the hydrogel and tissue (R(2)=( )0.80), but the correlation was not significant for MVC or TFV. For the sustained-release formulations, the PLGA/PCL content did not affect TFV release in vitro or ex vivo. Incorporation of cells into the hydrogel matrix improved the correlation between hydrogel and tissue explant release for TFV. We expect that this hydrogel-tissue mimic may be a promising preclinical model to evaluate topical or transdermal drug delivery systems with complex microarchitectures.

  15. Uptake of phenothiazines by the harvested chylomicrons ex vivo model: influence of self-nanoemulsifying formulation design.

    Science.gov (United States)

    Shahnaz, Gul; Hartl, Markus; Barthelmes, Jan; Leithner, Katharina; Sarti, Federica; Hintzen, Fabian; Rahmat, Deni; Salvenmoser, Willi; Bernkop-Schnürch, Andreas

    2011-09-01

    The aim of this study was to examine the potential of self-nanoemulsifying drug delivery systems (SNEDDS) on the uptake of the lipophilic and poorly water soluble phenothiazines thioridazine and chlorpromazine with the isolated plasma derived chylomicron (CM) ex vivo model. The multi-component delivery systems were optimized by evaluating their ability to self-emulsify when introduced to an aqueous medium under gentle agitation. The uptake of phenothiazines by isolated plasma derived chylomicrons was investigated with short chain triglyceride (SCT) SNEDDS, medium chain triglyceride (MCT) SNEDDS, and long chain triglyceride (LCT) SNEDDS. SNEDDS were also evaluated for their stabilities, dispersibilities, percentage transmittances and by particle size analyses. For thioridazine a 5.6-fold and for chlorpromazine a 3.7-fold higher CM uptake could be observed using a LCT-SNEDDS formulation compared to the drugs without formulation. In contrast, ex vivo uptake by isolated CM was not significantly increased by SNEDDS formulations based on MCT and SCT. Compared with isolated CM, the CM sizes were increased 2.5-fold in LCT-SNEDDS, whereas in MCT-SNEDDS or SCT-SNEDDS only a small, non-significant (P<0.05) increase in CM size was observed. These results show that distinct SNEDDS formulations containing phenothiazines are efficiently uptaken by plasma derived chylomicrons ex vivo.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. Ex vivo expansion of human peripheral blood progenitors.

    Science.gov (United States)

    Chabannon, C; Herrera-Rodriguez, D; Bardin, F; Mouren, M; Novakovitch, G; Blaise, D; Maraninchi, D; Mannoni, P

    1995-01-01

    Culture of human hematopoietic progenitors on a large scale could lead to several clinical applications within the near future, including the production of differentiated and functional cells, the increase in the number of early progenitors, especially stem cells, with such use as gene transfer, or the improvement of grafts used to limit the hematological toxicity associated with high-dose chemotherapy. In this case, one can still distinguish different objectives: improvement of grafts that contain low numbers of progenitors because of prior chemotherapies or because of marrow involvement for example, and qualitative changes in the graft content that would allow to envision the disappearance, or the further reduction, in the duration of absolute neutropenia that follows delivery of high dose chemotherapy ("nadir rescue"), despite substitution of mobilized blood cells to marrow cells and the in vivo use of hematopoietic growth factors. Additional advantages may be related to tumor purging in autologous expanded cells, and to the change in the ratio between hematopoietic progenitors and immunocompetent cells in allogeneic expanded populations. Therefore it appears that in vitro expansion currently raises two types of questions: the first ones are related to the definition of clinical or biological endpoints to be achieved, the second ones are related to "bioengineering", and deal with the efficiency and safety of progenitor cell cultures to be used for clinical applications. We here present preliminary results preparing future pilot clinical studies with ex vivo cultured human hematopoietic cells.

  1. Corneal morphology after ex-vivo UV and mid-infrared laser ablation

    Science.gov (United States)

    Spyratou, E.; Voloudakis, G. E.; Moutsouris, K.; Asproudis, I.; Baltatzis, S.; Makropoulou, M.; Bacharis, C.; Serafetinides, A. A.

    2008-12-01

    In this work, ablation experiments of ex vivo porcine cornea tissue were conducted with two solid state lasers (an Er:YAG laser and the 4th harmonic of an Nd:YAG laser, both in the ns pulse width range) emitting in mid infrared and ultraviolet part of the spectrum respectively, at moderate laser fluences. The cornea epithelium of each porcine eye was manually removed before the ablation. Histology analysis of the specimens was performed, in order to examine the microscopic appearance of the ablated craters and the existence of any thermal or mechanical damage caused by the midinfrared and the UV laser irradiation. For a detailed and complete examination of the morphology of the laser ablated corneal tissue, the surface roughness was investigated by scanning electron microscopy.

  2. 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...

  3. Fusing in vivo and ex vivo NMR sources of information for brain tumor classification

    Science.gov (United States)

    Croitor-Sava, A. R.; Martinez-Bisbal, M. C.; Laudadio, T.; Piquer, J.; Celda, B.; Heerschap, A.; Sima, D. M.; Van Huffel, S.

    2011-11-01

    In this study we classify short echo-time brain magnetic resonance spectroscopic imaging (MRSI) data by applying a model-based canonical correlation analyses algorithm and by using, as prior knowledge, multimodal sources of information coming from high-resolution magic angle spinning (HR-MAS), MRSI and magnetic resonance imaging. The potential and limitations of fusing in vivo and ex vivo nuclear magnetic resonance sources to detect brain tumors is investigated. We present various modalities for multimodal data fusion, study the effect and the impact of using multimodal information for classifying MRSI brain glial tumors data and analyze which parameters influence the classification results by means of extensive simulation and in vivo studies. Special attention is drawn to the possibility of considering HR-MAS data as a complementary dataset when dealing with a lack of MRSI data needed to build a classifier. Results show that HR-MAS information can have added value in the process of classifying MRSI data.

  4. 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.

  5. 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.

  6. 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....

  7. Equivalent metabolic acidosis with four colloids and saline on ex vivo haemodilution.

    Science.gov (United States)

    Morgan, T J; Vellaichamy, M; Cowley, D M; Weier, S L; Venkatesh, B; Jones, M A

    2009-05-01

    Colloid infusions can cause metabolic acidosis. Mechanisms and relative severity with different colloids are incompletely understood. We compared haemodilution acid-base effects of 4% albumin, 3.5% polygeline, 4% succinylated gelatin (all weak acid colloids, strong ion difference 12 mEq/l, 17.6 mEq/l and 34 mEq/l respectively), 6% hetastarch (non-weak acid colloid, strong ion difference zero) and 0.9% saline (crystalloid, strong ion difference zero). Gelatin weak acid properties were tracked via the strong ion gap. Four-step ex vivo dilutions of pre-oxygenated human venous blood were performed to a final [Hb] near 50% baseline. With each fluid, base excess fell to approximately -13 mEq/l. Base excess/[Hb] relationships across dilution were linear and direct (R2 > or = 0.96), slopes and intercepts closely resembling saline. Baseline strong ion gap was -0.3 (2.1) mEq/l. Post-dilution increases occurred in three groups: small with saline, hetastarch and albumin (to 3.5 (02) mEq/l, 4.3 (0.3) mEq/l, 3.3 (1.4) mEq/l respectively), intermediate with polygeline (to 12.2 (0.9) mEq/l) and greatest with succinylated gelatin (to 20.8 (1.4) mEq/l). We conclude that, despite colloid weak acid activity ranging from zero (hydroxyethyl starch) to greater than that of albumin with both gelatin preparations, ex vivo dilution causes a metabolic acidosis of identical severity to saline in each case. This uniformity reflects modifications to the albumin and gelatin saline vehicles, in part aimed at pH correction. By proportionally increasing the strong ion difference, these modifications counter deviations from pure saline effects caused by colloid weak acid activity. Extrapolation in vivo requires further investigation.

  8. Microultrasound characterisation of ex vivo porcine tissue for ultrasound capsule endoscopy

    Science.gov (United States)

    Lay, H. S.; Cox, B. F.; Sunoqrot, M.; Démoré, C. E. M.; Näthke, I.; Gomez, T.; Cochran, S.

    2017-01-01

    Gastrointestinal (GI) disease development and progression is often characterised by cellular and tissue architectural changes within the mucosa and sub-mucosa layers. Current clinical capsule endoscopy and other approaches are heavily reliant on optical techniques which cannot detect disease progression below the surface layer of the tissue. To enhance the ability of clinicians to detect cellular changes earlier and more confidently, both quantitative and qualitative microultrasound (μUS) techniques are investigated in healthy ex vivo porcine GI tissue. This work is based on the use of single-element, focussed μUS transducers made with micromoulded piezocomposite operating at around 48 MHz. To explore the possibility that μUS can detect Crohn’s disease and other inflammatory bowel diseases, ex vivo porcine small bowel tissue samples were cannulised and perfused with phosphate-buffered saline followed by various dilutions of polystyrene microspheres. Comparison with fluorescent imaging showed that the microspheres had infiltrated the microvasculature of the samples and that μUS was able to successfully detect this as a mimic of inflammation. Samples without microspheres were analysed using quantitative ultrasound to assess mechanical properties. Attenuation coefficients of 1.78 ± 0.66 dB/mm and 1.92 ± 0.77 dB/mm were obtained from reference samples which were surgically separated from the muscle layer. Six intact samples were segmented using a software algorithm and the acoustic impedance, Z, for varying tissue thicknesses, and backscattering coefficient, BSC, were calculated using the reference attenuation values and tabulated.

  9. 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.

  10. 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.

  11. Ex vivo quantitative multiparametric MRI mapping of human meniscus degeneration

    Energy Technology Data Exchange (ETDEWEB)

    Nebelung, Sven; Kuhl, Christiane; Truhn, Daniel [Aachen University Hospital, Department of Diagnostic and Interventional Radiology, Aachen (Germany); Tingart, Markus; Jahr, Holger [Aachen University Hospital, Department of Orthopaedics, Aachen (Germany); Pufe, Thomas [RWTH Aachen University, Institute of Anatomy and Cell Biology, Aachen (Germany)

    2016-12-15

    To evaluate the diagnostic performance of T1, T1ρ, T2, T2*, and UTE-T2* (ultrashort-echo time-enhanced T2*) mapping in the refined graduation of human meniscus degeneration with histology serving as standard-of-reference. This IRB-approved intra-individual comparative ex vivo study was performed on 24 lateral meniscus body samples obtained from 24 patients undergoing total knee replacement. Samples were assessed on a 3.0-T MRI scanner using inversion-recovery (T1), spin-lock multi-gradient-echo (T1ρ), multi-spin-echo (T2) and multi-gradient-echo (T2* and UTE-T2*) sequences to determine relaxation times of quantitative MRI (qMRI) parameters. Relaxation times were calculated on the respective maps, averaged to the entire meniscus and to its zones. Histologically, samples were analyzed on a four-point score according to Williams (0-III). QMRI results and Williams (sub)scores were correlated using Spearman's ρ, while Williams grade-dependent differences were assessed using Kruskal-Wallis and Dunn's tests. Sensitivities and specificities in the detection of intact (Williams grade [WG]-0) and severely degenerate meniscus (WG-II-III) were calculated. Except for T2*, significant increases in qMRI parameters with increasing Williams grades were observed. T1, T1ρ, T2, and UTE-T2* exhibited high sensitivity and variable specificity rates. Significant marked-to-strong correlations were observed for these parameters with each other, with histological WGs and the subscores tissue integrity and cellularity. QMRI mapping holds promise in the objective evaluation of human meniscus. Although sufficient discriminatory power of T1, T1ρ, T2, and UTE-T2* was only demonstrated for the histological extremes, these data may aid in the future MRI-based parameterization and quantification of human meniscus degeneration. (orig.)

  12. 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

  13. 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.

  14. 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.

  15. Bone marrow-derived dendritic cells pulsed with tumor lysates induce anti-tumor immunity against gastric cancer ex vivo

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    AIM: To investigate whether bone marrow-derived denritic cells pulsed with tumor lysates induce immunity against gastric cancer ex vivo. METHODS: c-kit+ hematopoietic progenitor cells were magnetically isolated with a MiniMACS separator from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFα to induce their maturation. They were analysed by morphological observation, phenotype analysis, and mixed lymphocyte reaction (MLR). Bone marrow-derived DCs (BM-DCs) were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1:3 DC:tumor cell ratio. Finally, cytotoxic T lymphocyte (CTL) activity and interferon gamma (IFNy) secretion was evaluated ex vivo.RESULTS: c-kit+ hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 d showed the character of typical mature DCs. Norphologically, observed by light microscope, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed.high levels of Ia, DEC-205, CD11b, CD80 and CD86 antigen, moderate levels of CD40, and negative for F4/80. Functionally, these ceils gained the capacity to stimulate allogeneic T cells in MLR assay. However, immature DCs cultured with cytokines for 5 d did not have typical DCs phenotypic markers and could not stimulate allogeneic T cells. Ex vivo primed T cells with SGC-7901 tumor cell lysate-pulsed (TP) DCs were able to induce effective CTL activity against SGC-7901 tumor cells (E:T = 100:1, 69.55% ± 6.05% specific lysis), but not B16 tumor cells, and produced higher levels of IFNγ, when stimulated with SGC-7901 tumor cells but not when stimulated with B16 tumor cells (1575.31 ± 60.25 pg/mL in SGC-7901 group vs 164.11 ± 18.52 pg/mL in B16 group, P < 0.01).CONCLUSION: BM-derived DCs pulsed with tumor lysates can induce anti-tumor immunity specific to gastric cancer ex vivo.

  16. 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.

  17. Behavior of tip-steerable needles in ex vivo and in vivo tissue.

    Science.gov (United States)

    Majewicz, Ann; Marra, Steven P; van Vledder, Mark G; Lin, MingDe; Choti, Michael A; Song, Danny Y; Okamura, Allison M

    2012-10-01

    Robotic needle steering is a promising technique to improve the effectiveness of needle-based clinical procedures, such as biopsies and ablation, by computer-controlled, curved insertions of needles within solid organs. In this paper, we explore the capabilities, challenges, and clinical relevance of asymmetric-tip needle steering through experiments in ex vivo and in vivo tissue. We evaluate the repeatability of needle insertion in inhomogeneous biological tissue and compare ex vivo and in vivo needle curvature and insertion forces. Steerable needles curved more in kidney than in liver and prostate, likely due to differences in tissue properties. Pre-bent needles produced higher insertion forces in liver and more curvature in vivo than ex vivo. When compared to straight stainless steel needles, steerable needles did not cause a measurable increase in tissue damage and did not exert more force during insertion. The minimum radius of curvature achieved by prebent needles was 5.23 cm in ex vivo tissue, and 10.4 cm in in vivo tissue. The curvatures achieved by bevel tip needles were negligible for in vivo tissue. The minimum radius of curvature for bevel tip needles in ex vivo tissue was 16.4 cm; however, about half of the bevel tip needles had negligible curvatures. We also demonstrate a potential clinical application of needle steering by targeting and ablating overlapping regions of cadaveric canine liver.

  18. Visualizing Oxazine 4 nerve-specific fluorescence ex vivo in frozen tissue sections

    Science.gov (United States)

    Barth, Connor W.; Gibbs, Summer L.

    2016-03-01

    Nerve damage plagues surgical outcomes and remains a major burden for patients, surgeons, and the healthcare system. Fluorescence image-guided surgery using nerve specific small molecule fluorophores offers a solution to diminish surgical nerve damage through improved intraoperative nerve identification and visualization. Oxazine 4 has shown superior nerve specificity in initial testing in vivo, while exhibiting a red shifted excitation and emission spectra compared to other nerve-specific fluorophores. However, Oxazine 4 does not exhibit near-infrared (NIR) excitation and emission, which would be ideal to improve penetration depth and nerve signal to background ratios for in vivo imaging. Successful development of a NIR nerve-specific fluorophore will require understanding of the molecular target of fluorophore nerve specificity. While previous small molecule nerve-specific fluorophores have demonstrated excellent ex vivo nerve specificity, Oxazine 4 ex vivo nerve specific fluorescence has been difficult to visualize. In the present study, we examined each step of the ex vivo fluorescence microscopy sample preparation procedure to discover how in vivo nerve-specific fluorescence is changed during ex vivo tissue sample preparation. Through step-by-step examination we found that Oxazine 4 fluorescence was significantly diminished by washing and mounting tissue sections for microscopy. A method to preserve Oxazine 4 nerve specific fluorescence ex vivo was determined, which can be utilized for visualization by fluorescence microscopy.

  19. 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.

  20. Ex vivo encapsulation of dexamethasone sodium phosphate into human autologous erythrocytes using fully automated biomedical equipment.

    Science.gov (United States)

    Mambrini, Giovanni; Mandolini, Marco; Rossi, Luigia; Pierigè, Francesca; Capogrossi, Giovanni; Salvati, Patricia; Serafini, Sonja; Benatti, Luca; Magnani, Mauro

    2017-01-30

    Erythrocyte-based drug delivery systems are emerging as potential new solutions for the release of drugs into the bloodstream. The aim of the present work was to assess the performance of a fully automated process (EDS) for the ex-vivo encapsulation of the pro-drug dexamethasone sodium phosphate (DSP) into autologous erythrocytes in compliance with regulatory requirements. The loading method was based on reversible hypotonic hemolysis, which allows the opening of transient pores in the cell membrane to be crossed by DSP. The efficiency of encapsulation and the biochemical and physiological characteristics of the processed erythrocytes were investigated in blood samples from 34 healthy donors. It was found that the processed erythrocytes maintained their fundamental properties and the encapsulation process was reproducible. The EDS under study showed greater loading efficiency and reduced variability compared to previous EDS versions. Notably, these results were confirmed using blood samples from Ataxia Telangiectasia (AT) patients, 9.33±1.40 and 19.41±2.10mg of DSP (mean±SD, n=134) by using 62.5 and 125mg DSP loading quantities, respectively. These results support the use of the new EDS version 3.2.0 to investigate the effect of erythrocyte-delivered dexamethasone in regulatory trials in patients with AT.

  1. 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 skin, negating the requirement for animal models in this context, prior to study in a clinical trial environment.

  2. 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.

  3. 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.

  4. Quantification of fibronectin as a method to assess ex vivo extracellular matrix remodeling

    DEFF Research Database (Denmark)

    Bager, Cecilie Liv; Gudmann, N.; Willumsen, N.

    2016-01-01

    of the ECM is fibronectin. Fibronectin serves as an adhesion molecule anchoring cells to the underlying basement membrane through direct interaction with integrin receptors. Fibronectin hereby modulates the properties of the ECM and affects cellular processes. Quantification of fibronectin remodeling could...... therefore be used to assess the changes in the ECM that occur during progression of fibro-proliferative pathologies. Ex vivo models are becoming state-of-the-art tools to study ECM remodeling as the cellular composition and the organization of the ECM are preserved. Ex vivo models may therefore...... be a valuable tool to study the ECM remodeling that occurs during progression of fibro-proliferative pathologies. The aim of this study was to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. A competitive The enzyme-linked immunosorbent assay (ELISA) against the C...

  5. A new veno-venous bypass type for ex-vivo liver resection in dogs

    Institute of Scientific and Technical Information of China (English)

    Peng Lei; Shi-Qi Liu; Xiao-Hai Cui; Yi Lv; Ge Zhao; Jian-Hui Li

    2013-01-01

    Ex-vivo liver resection is a procedure in which the liver is completely removed, perfused and after bench surgery, the liver is autotransplanted to the original site. Ex-vivo liver resection is an important treatment for unresectable liver tumors. This surgical procedure requires long operation time, during which blood flow must be carefully maintained to avoid venous congestion. An effective veno-venous bypass (VVB) may meet this requirement. The present study was to test our new designed VVB device which comprised one heparinized polyvinylchloride tube and three magnetic rings. The efficacy of this device was tested in five dogs. A VVB was established in 6-10 minutes. There was no leakage during the procedure. Hemodynamics was stable at anhepatic phase, which indicated that the bypass was successful. This newly-developed VVB device maintained circulation stability during ex-vivo liver resection in our dog model and thus, this VVB device significantly shortened the operation time.

  6. Ex-vivo-Expansion perikardialer Patchstrukturen : zur Frage des Einflusses von zyklischem Stress und laminaren Strömungsbedingungen auf das Ex-vivo-Remodelling perikardialer Patchstrukturen

    OpenAIRE

    Hesse, Björn

    2009-01-01

    BACKGROUND: Pericardium is widely used as a patch structure in paediatric cardiovascular heart surgery. However, the major problem still is the restricted availability and level of quality of autologous pericardium. Based on those limiting factors, a new concept has already been tested in a pilot study combining the mesh graft technique of burn surgery with the principles of tissue engineering to expand pericardial tissue ex vivo. The aims of this dissertation were to verify the preliminary r...

  7. Biomonitoring and hormone-disrupting effect biomarkers of persistent organic pollutants in vitro and ex vivo.

    Science.gov (United States)

    Bonefeld-Jørgensen, Eva C; Ghisari, Mandana; Wielsøe, Maria; Bjerregaard-Olesen, Christian; Kjeldsen, Lisbeth S; Long, Manhai

    2014-07-01

    Persistent organic pollutants (POPs) include lipophilic legacy POPs and the amphiphilic perfluorinated alkyl acids (PFAAs). They have long half-lives and bioaccumulate in the environment, animals and human beings. POPs possess toxic, carcinogenic and endocrine-disrupting potentials. Endocrine-disrupting chemicals (EDCs) are compounds that either mimic or block endogenous hormones and thus disrupt the normal hormone homeostasis. Biomonitoring assesses the internal doses of a person to provide information about chemical exposures. Effect biomarkers assess chemicals potential to affect cellular functions in vivo/ex vivo. Human beings are exposed to complex mixtures of chemicals, having individually very different biological potentials and effects. Therefore, the assessment of the combined, integrated biological effect of the actual chemical mixture in human blood is important. In vitro and ex vivo cell systems have been introduced for the assessment of the integrated level of xenobiotic cellular effects in human beings. Ex vivo studies have shown geographical differences in bioaccumulated POP serum levels, being reflected by the combined biomarker effects of the complex mixture extracted from human serum. Xenohormone receptor transactivities can be used as an ex vivo integrated biomarker of POP exposure and effects. Epidemiological and in vitro/ex vivo studies have supported the potential impact of the combined effect of serum POPs on the activity of hormone and/or dioxin receptors as a risk factor for human health. With focus on hormone disruption, this MiniReview will give an update on recent POP-related endocrine-disrupting effects in vitro/ex vivo/in vivo and some related genetic data.

  8. 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.

  9. 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.

  10. Biosafety in Ex Vivo Gene Therapy and Conditional Ablation of Lentivirally Transduced Hepatocytes in Nonhuman Primates

    Science.gov (United States)

    Menzel, Olivier; Birraux, Jacques; Wildhaber, Barbara E; Jond, Caty; Lasne, Françoise; Habre, Walid; Trono, Didier; Nguyen, Tuan H; Chardot, Christophe

    2009-01-01

    Ex vivo gene therapy is an interesting alternative to orthotopic liver transplantation (OLT) for treating metabolic liver diseases. In this study, we investigated its efficacy and biosafety in nonhuman primates. Hepatocytes isolated from liver lobectomy were transduced in suspension with a bicistronic liver-specific lentiviral vector and immediately autotransplanted (SLIT) into three cynomolgus monkeys. The vector encoded cynomolgus erythropoietin (EPO) and the conditional suicide gene herpes simplex virus-thymidine kinase (HSV-TK). Survival of transduced hepatocytes and vector dissemination were evaluated by detecting transgene expression and vector DNA. SLIT was safely performed within a day in all three subjects. Serum EPO and hematocrit rapidly increased post-SLIT and their values returned to baseline within about 1 month. Isoforms of EPO detected in monkeys' sera differed from the physiological renal EPO. In liver biopsies at months 8 and 15, we detected EPO protein, vector mRNA and DNA, demonstrating long-term survival and functionality of transplanted lentivirally transduced hepatocytes. Valganciclovir administration resulted in complete ablation of the transduced hepatocytes. We demonstrated the feasibility and biosafety of SLIT, and the long term (>1 year) functionality of lentivirally transduced hepatocytes in nonhuman primates. The HSV-TK/valganciclovir suicide strategy can increase the biosafety of liver gene therapy protocols by safely and completely ablating transduced hepatocytes on demand. PMID:19568222

  11. 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.

  12. Ex-vivo holographic microscopy and spectroscopic analysis of head and neck cancer

    Science.gov (United States)

    Holler, Stephen; Wurtz, Robert; Auyeung, Kelsey; Auyeung, Kris; Paspaley-Grbavac, Milan; Mulroe, Brigid; Sobrero, Maximiliano; Miles, Brett

    2015-03-01

    Optical probes to identify tumor margins in vivo would greatly reduce the time, effort and complexity in the surgical removal of malignant tissue in head and neck cancers. Current approaches involve visual microscopy of stained tissue samples to determine cancer margins, which results in the excision of excess of tissue to assure complete removal of the cancer. Such surgical procedures and follow-on chemotherapy can adversely affect the patient's recovery and subsequent quality of life. In order to reduce the complexity of the process and minimize adverse effects on the patient, we investigate ex vivo tissue samples (stained and unstained) using digital holographic microscopy in conjunction with spectroscopic analyses (reflectance and transmission spectroscopy) in order to determine label-free, optically identifiable characteristic features that may ultimately be used for in vivo processing of cancerous tissues. The tissue samples studied were squamous cell carcinomas and associated controls from patients of varying age, gender and race. Holographic microscopic imaging scans across both cancerous and non-cancerous tissue samples yielded amplitude and phase reconstructions that were correlated with spectral signatures. Though the holographic reconstructions and measured spectra indicate variations even among the same class of tissue, preliminary results indicate the existence of some discriminating features. Further analyses are presently underway to further this work and extract additional information from the imaging and spectral data that may prove useful for in vivo surgical identification.

  13. 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.

  14. Nasal administration of metoclopramide from different dosage forms: in vitro, ex vivo, and in vivo evaluation.

    Science.gov (United States)

    Tas, Cetin; Ozkan, Cansel Kose; Savaser, Ayhan; Ozkan, Yalcin; Tasdemir, Umut; Altunay, Hikmet

    2009-04-01

    Nasal drug delivery is an interesting route of administration for metoclopramide hydrochloride (MTC) in preventing different kind of emesis. Currently, the routes of administration of antiemetics are oral or intravenous, although patient compliance is often impaired by the difficulties associated with acute emesis or invasiveness of parenteral administration. In this perspective, nasal dosage forms (solution, gel, and lyophilized powder) of MTC were prepared by using a mucoadhesive polymer sodium carboxymethylcellulose (NaCMC). In vitro and ex vivo drug release studies were performed in a modified horizontal diffusion chamber with cellulose membrane and excised cattle nasal mucosa as diffusion barriers. The tolerance of nasal mucosa to the formulation and its components were investigated using light microscopy. In vivo studies were carried out for the optimized formulations in sheep and the pharmacokinetics parameters were compared with oral solution and IV dosage form. The release of MTC from solution and powder formulations was found to be higher than gel formulation (p nasal bioavailability of gel is higher than those of solution and powder (p nasal residence time and thereby nasal absorption.

  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. 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

  17. Broadly Neutralizing Anti-HIV Antibodies Prevent HIV Infection of Mucosal Tissue Ex Vivo.

    Science.gov (United States)

    Scott, Yanille M; Park, Seo Young; Dezzutti, Charlene S

    2016-02-01

    Broadly neutralizing monoclonal antibodies (nAbs) specific for HIV are being investigated for use in HIV prevention. Due to their ability to inhibit HIV attachment to and entry into target cells, nAbs may be suitable for use as topical HIV microbicides. As such, they would present an alternative intervention for individuals who may not benefit from using antiretroviral-based products for HIV prevention. We theorize that nAbs can inhibit viral transmission through mucosal tissue, thus reducing the incidence of HIV infection. The efficacy of the PG9, PG16, VRC01, and 4E10 antibodies was evaluated in an ex vivo human model of mucosal HIV transmission. nAbs reduced HIV transmission, causing 1.5- to 2-log10 reductions in HIV replication in ectocervical tissues and ≈3-log10 reductions in HIV replication in colonic tissues over 21 days. These antibodies demonstrated greater potency in colonic tissues, with a 50-fold higher dose being required to reduce transmission in ectocervical tissues. Importantly, nAbs retained their potency and reduced viral transmission in the presence of whole semen. No changes in tissue viability or immune activation were observed in colonic or ectocervical tissue after nAb exposure. Our data suggest that topically applied nAbs are safe and effective against HIV infection of mucosal tissue and support further development of nAbs as a topical microbicide that could be used for anal as well as vaginal protection.

  18. 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.

  19. Ex vivo generation of dendritic cells from cryopreserved, post-induction chemotherapy, mobilized leukapheresis from pediatric patients with medulloblastoma.

    Science.gov (United States)

    Nair, Smita K; Driscoll, Timothy; Boczkowski, David; Schmittling, Robert; Reynolds, Renee; Johnson, Laura A; Grant, Gerald; Fuchs, Herbert; Bigner, Darell D; Sampson, John H; Gururangan, Sridharan; Mitchell, Duane A

    2015-10-01

    Generation of patient-derived, autologous dendritic cells (DCs) is a critical component of cancer immunotherapy with ex vivo-generated, tumor antigen-loaded DCs. An important factor in the ability to generate DCs is the potential impact of prior therapies on DC phenotype and function. We investigated the ability to generate DCs using cells harvested from pediatric patients with medulloblastoma for potential evaluation of DC-RNA based vaccination approach in this patient population. Cells harvested from medulloblastoma patient leukapheresis following induction chemotherapy and granulocyte colony stimulating factor mobilization were cryopreserved prior to use in DC generation. DCs were generated from the adherent CD14+ monocytes using standard procedures and analyzed for cell recovery, phenotype and function. To summarize, 4 out of 5 patients (80%) had sufficient monocyte recovery to permit DC generation, and we were able to generate DCs from 3 out of these 4 patient samples (75%). Overall, we successfully generated DCs that met phenotypic requisites for DC-based cancer therapy from 3 out of 5 (60%) patient samples and met both phenotypic and functional requisites from 2 out of 5 (40%) patient samples. This study highlights the potential to generate functional DCs for further clinical treatments from refractory patients that have been heavily pretreated with myelosuppressive chemotherapy. Here we demonstrate the utility of evaluating the effect of the currently employed standard-of-care therapies on the ex vivo generation of DCs for DC-based clinical studies in cancer patients.

  20. Ex-vivo assessment and non-invasive in vivo imaging of internal hemorrhages in Aga2/+ mutant mice

    Energy Technology Data Exchange (ETDEWEB)

    Ermolayev, Vladimir [Institute for Biological and Medical Imaging, Helmholtz Zentrum München, Building 56, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany); Cohrs, Christian M. [Institute for Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany); Mohajerani, Pouyan; Ale, Angelique [Institute for Biological and Medical Imaging, Helmholtz Zentrum München, Building 56, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany); Hrabé de Angelis, Martin [Institute for Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany); Ntziachristos, Vasilis, E-mail: v.ntziachristos@tum.de [Institute for Biological and Medical Imaging, Helmholtz Zentrum München, Building 56, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany)

    2013-03-08

    Highlights: ► Aga2/+ mice, model for Osteogenesis imperfecta, have type I collagen mutation. ► Aga2/+ mice display both moderate and severe phenotypes lethal 6–11th postnatal. ► Internal hemorrhages studied in Aga2/+ vs. control mice at 6 and 9 days postnatal. ► Anatomical and functional findings in-vivo contrasted to the ex-vivo appearance. -- Abstract: Mutations in type I collagen genes (COL1A1/2) typically lead to Osteogenesis imperfecta, the most common heritable cause of skeletal fractures and bone deformation in humans. Heterozygous Col1a1{sup Aga2/+}, animals with a dominant mutation in the terminal C-propeptide domain of type I collagen develop typical skeletal hallmarks and internal hemorrhages starting from 6 day after birth. The disease progression for Aga2/+ mice, however, is not uniform differing between severe phenotype lethal at the 6–11th day of life, and moderate-to-severe one with survival to adulthood. Herein we investigated whether a new modality that combines X-ray computer tomography with fluorescence tomography in one hybrid system can be employed to study internal bleedings in relation to bone fractures and obtain insights into disease progression. The disease phenotype was characterized on Aga2/+ vs. wild type mice between 6 and 9 days postnatal. Anatomical and functional findings obtained in-vivo were contrasted to the ex-vivo appearance of the same tissues under cryo-slicing.

  1. Solubility and Bioavailability Enhancement of Poorly Aqueous Soluble Atorvastatin: In Vitro, Ex Vivo, and In Vivo Studies

    Directory of Open Access Journals (Sweden)

    Madhuri S. Rodde

    2014-01-01

    Full Text Available The objective of this investigation was to improve the solubility of the poorly water soluble drug atorvastatin (ATR, using solid dispersion (SD techniques, with Neem Gum (NG as a hydrophilic carrier. The effects of the polymer concentration and method of preparation on the solubility and dissolution rate were studied. The results showed that the solubility of ATR increases with increasing NG concentration. However, dissolution rate of ATR from its SD was dependent on the method used to prepare SD. An in vitro drug release study revealed that the solvent evaporation technique is a more convenient and effective method of preparing SD than kneading method. The SD was characterized using DSC, SEM, and XRD study. An in vivo study was performed in which the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG CoA reductase inhibition activity was measured. A significant reduction in HMG CoA reductase activity was observed with SD of ATR compared with the plain drug. An ex vivo absorption study was carried out using modified apparatus developed in our laboratory. The in vitro drug release and in vivo and ex vivo studies clearly demonstrated the potential of hydrophilic NG in enhancing the solubility, dissolution rate, and bioavailability of ATR.

  2. Assessment of Drug Sensitivity in Hematopoietic Stem and Progenitor Cells From Acute Myelogenous Leukemia and Myelodysplastic Syndrome Ex Vivo.

    Science.gov (United States)

    Knorr, Katherine L B; Finn, Laura E; Smith, B Douglas; Hess, Allan D; Foran, James M; Karp, Judith E; Kaufmann, Scott H

    2016-11-07

    : Current understanding suggests that malignant stem and progenitor cells must be reduced or eliminated for prolonged remissions in myeloid neoplasms such as acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS). Multicolor flow cytometry has been widely used to distinguish stem and myeloid progenitor cells from other populations in normal and malignant bone marrow. In this study, we present a method for assessing drug sensitivity in MDS and AML patient hematopoietic stem and myeloid progenitor cell populations ex vivo using the investigational Nedd8-activating enzyme inhibitor MLN4924 and standard-of-care agent cytarabine as examples. Utilizing a multicolor flow cytometry antibody panel for identification of hematopoietic stem cells, multipotent progenitors, common myeloid progenitors, granulocyte-monocyte progenitors, and megakaryocyte-erythroid progenitors present in mononuclear cell fractions isolated from bone marrow aspirates, we compare stem and progenitor cell counts after treatment for 24 hours with drug versus diluent. We demonstrate that MLN4924 exerts a cytotoxic effect on MDS and AML stem and progenitor cell populations, whereas cytarabine has more limited effects. Further application of this method for evaluating drug effects on these populations ex vivo and in vivo may inform rational design and selection of therapies in the clinical setting.

  3. In vitro and ex vivo microbial leakage assessment in endodontics: A literature review

    OpenAIRE

    Savadkouhi, Sohrab Tour; Bakhtiar, Hengameh; Ardestani, Safoura Emami

    2016-01-01

    The aim of this study was to perform a literature review of published in-vitro and ex-vivo studies, which evaluated microbial leakage in endodontics in the past 10 years. A comprehensive electronic literature search was carried out in PubMed database for English articles published from 2005 to 2016 using the keywords “endodontics,” “in vitro,” “ex vivo,” “microbial leakage,” “microbial penetration,” “saliva,” “Enterococcus faecalis,” “E. faecalis,” “endodontic sealers,” “temporary filling mat...

  4. [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.

  5. 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

  6. 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

  7. Assessment of thermal sensitivity of CT during heating of liver : an ex vivo study

    NARCIS (Netherlands)

    Pandeya, G. D.; Greuter, M. J. W.; Schmidt, B.; Flohr, T.; Oudkerk, M.

    2012-01-01

    Objectives: The purpose of this study was to assess the thermal sensitivity of CT during heating of ex-vivo animal liver. Methods: Pig liver was indirectly heated from 20 to 90 degrees C by passage of hot air through a plastic tube. The temperature in the heated liver was measured using calibrated t

  8. 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

  9. 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.

  10. Particle Bombardment of Ex Vivo Skin to Deliver DNA and Express Proteins

    NARCIS (Netherlands)

    Sokol, Ena; Nijenhuis, Miranda; Sjollema, Klaas A; Jonkman, Marcel F; Pas, Hendri H; Giepmans, Ben N G

    2017-01-01

    Particle bombardment of gold microparticles coated with plasmids, which are accelerated to high velocity, is used for transfection of cells within tissue. Using this method, cDNA encoding proteins of interest introduced into ex vivo living human skin enables studying of proteins of interest in real

  11. 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

    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...

  12. 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)

  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. Ex vivo and in vitro production of pro-inflammatory cytokines in Blau syndrome

    Directory of Open Access Journals (Sweden)

    P. Galozzi

    2015-03-01

    Full Text Available The objective was to study both ex vivo and in vitro secretion of pro-inflammatory cytokines in patients affected by Blau syndrome (BS and carrying p.E383K mutation in the CARD15/NOD2 gene associated with the disease. For ex vivo studies, peripheral blood mononuclear cells (PBMCs, serum from three patients and healthy controls have been collected. PBMCs have been cultured in the presence or absence of inflammatory enhancers, such as lipopolysaccharide (LPS and muramyl dipeptide (MDP. The levels of interleukin (IL-1β, IL-6, IL-8, tumor necrosis factor (TNF-α and interferon (IFN-γ were assayed by either immunoassay or array-based system. For in vitro studies, different constructs were created cloning human wild-type and p.E383K-mutated NOD2 cDNA into the expression vector pCMV-Tag2c. HEK293 cell lines were stably transfected, cultured with or without MDP and IL-8 level was assayed in their surnatants. Statistical analysis in both studies was performed using non-parametric tests. Both ex vivo and in vitro studies have not identified a significant increase in secretion of the analyzed proinflammatory cytokines. p.E383K-mutated NOD2 transfected cells express low level of IL-8. The ex vivo basal level results from both serum and PBMCs surnatants present similar levels of IL-1β, IL-6, TNF-α and IFN-γ in patients and controls. The presence of the stimulant agents (LPS and MDP, either individual or paired, does not lead to significant increases in all cytokines concentrations in patients compared to controls. Taken together, the ex vivo and in vitro data suggest that there is not a primary mediation of IL-1β and other pro-inflammatory cytokines in BS patients carrying p.E383K.

  15. 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.

  16. Pressure dependency of aortic pulse wave velocity in vivo is not affected by vasoactive substances that alter aortic wall tension ex vivo.

    Science.gov (United States)

    Butlin, Mark; Lindesay, George; Viegas, Kayla D; Avolio, Alberto P

    2015-05-15

    Aortic stiffness, a predictive parameter in cardiovascular medicine, is blood pressure dependent and experimentally requires isobaric measurement for meaningful comparison. Vasoactive drug administration to change peripheral resistance and blood pressure allows such isobaric comparison but may alter large conduit artery wall tension, directly changing aortic stiffness. This study quantifies effects of sodium nitroprusside (SNP, vasodilator) and phenylephrine (PE, vasoconstrictor) on aortic stiffness measured by aortic pulse wave velocity (aPWV) assessed by invasive pressure catheterization in anaesthetized Sprague-Dawley rats (n = 7). This was compared with nondrug-dependent alteration of blood pressure through reduced venous return induced by partial vena cava occlusion. In vivo drug concentration was estimated by modeling clearance rates. Ex vivo responses of excised thoracic and abdominal aortic rings to drugs was measured using myography. SNP administration did not alter aPWV compared with venous occlusion (P = 0.21-0.87). There was a 5% difference in aPWV with PE administration compared with venous occlusion (P arteries in vivo. However, similar drug concentrations affect aortic ring wall tension ex vivo. Future studies investigating in vivo and ex vivo kinetics will need to elucidate mechanisms for this marked difference.

  17. Characteristics of smooth muscle cells' shape and proliferation rate in novel short-term thermal angioplasty ex vivo and in vitro.

    Science.gov (United States)

    Kunio, Mie; Shimazaki, Natsumi; Ito, Arisa; Hayashi, Tomoaki; Arai, Tsunenori

    2010-01-01

    We investigated the influences on the smooth muscle cells of temporally heated arterial walls in both ex vivo and in vitro study to determine the optimum heat parameter of novel short-term thermal angioplasty, Photo-thermo Dynamic Balloon Angioplasty (PTDBA). Arterial heating dilatation was performed by the prototype PTDBA balloon ex vivo. We found that the smooth muscle cells in the vessel wall were stretch-fixed after the heating dilatation ex vivo. The stretch-fixing rate of these cells was increased with the temperature rise in the balloon of PTDBA from 60 °C to 70 °C. We measured the proliferation rate of the stretch-fixed smooth muscle cells, which were extracted from porcine arteries, on specially designed culture equipment in vitro. It was observed that the proliferation rate was inhibited at 20 % stretching compared to 10 % stretching. We think the stretch-fixing of the smooth muscle cells might not be harmful for PTDBA performances.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. EX VIVO STUDY OF QUANTITATIVE ULTRASOUND PARAMETERS IN FATTY RABBIT LIVERS

    Science.gov (United States)

    Ghoshal, Goutam; Lavarello, Roberto J.; Kemmerer, Jeremy P.; Miller, Rita J.; Oelze, Michael L.

    2012-01-01

    Nonalcoholic fatty liver disease (NAFLD) affects more than 30% of Americans, and with increasing problems of obesity in the United States, NAFLD is poised to become an even more serious medical concern. At present, accurate classification of steatosis (fatty liver) represents a significant challenge. In this study, the use of high-frequency (8 to 25 MHz) quantitative ultrasound (QUS) imaging to quantify fatty liver was explored. QUS is an imaging technique that can be used to quantify properties of tissue giving rise to scattered ultrasound. The changes in the ultrasound properties of livers in rabbits undergoing atherogenic diets of varying durations were investigated using QUS. Rabbits were placed on a special fatty diet for 0, 3, or 6 weeks. The fattiness of the livers was quantified by estimating the total lipid content of the livers. Ultrasonic properties, such as speed of sound, attenuation, and backscatter coefficients, were estimated in ex vivo rabbit liver samples from animals that had been on the diet for varying periods. Two QUS parameters were estimated based on the backscatter coefficient: effective scatterer diameter (ESD) and effective acoustic concentration (EAC), using a spherical Gaussian scattering model. Two parameters were estimated based on the backscattered envelope statistics (the k parameter and the μ parameter) according to the homodyned K distribution. The speed of sound decreased from 1574 to 1565 m/s and the attenuation coefficient increased from 0.71 to 1.27 dB/cm/MHz, respectively, with increasing fat content in the liver. The ESD decreased from 31 to 17 μm and the EAC increased from 38 to 63 dB/cm3 with increasing fat content in the liver. A significant increase in the μ parameter from 0.18 to 0.93 scatterers/mm3 was observed with increasing fat content in the liver samples. The results of this study indicate that QUS parameters are sensitive to fat content in the liver. PMID:23062376

  4. 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.

  5. Ex vivo and in vivo coherent Raman imaging of the peripheral and central nervous system

    Science.gov (United States)

    Huff, Terry Brandon

    A hallmark of nervous system disorders is damage or degradation of the myelin sheath. Unraveling the mechanisms underlying myelin degeneration and repair represent one of the great challenges in medicine. This thesis work details the development and utilization of advanced optical imaging methods to gain insight into the structure and function of myelin in both healthy and diseased states in the in vivo environment. This first part of this thesis discusses ex vivo studies of the effects of high-frequency stimulation of spinal tissues on the structure of the node of Ranvier as investigated by coherent anti-Stokes Raman scattering (CARS) imaging (manuscript submitted to Journal of Neurosciece). Reversible paranodal myelin retraction at the nodes of Ranvier was observed during 200 Hz electrical stimulation, beginning minutes after the onset and continuing for up to 10 min after stimulation was ceased. A mechanistic study revealed a Ca2+ dependent pathway: high-frequency stimulation induced paranodal myelin retraction via pathologic calcium influx into axons, calpain activation, and cytoskeleton degradation through spectrin break-down. Also, the construction of dual-scanning CARS microscope for large area mapping of CNS tissues is detailed (Optics Express, 2008, 16:19396-193409). A confocal scanning head equipped with a rotating polygon mirror provides high speed, high resolution imaging and is coupled with a motorized sample stage to generate high-resolution large-area images of mouse brain coronal section and guinea pig spinal cord cross section. The polygon mirror decreases the mosaic acquisition time significantly without reducing the resolution of individual images. The ex vivo studies are then extended to in vivo imaging of mouse sciatic nerve tissue by CARS and second harmonic generation (SHG) imaging (Journal of Microscopy, 2007, 225: 175-182). Following a minimally invasive surgery to open the skin, CARS imaging of myelinated axons and SHG imaging of the

  6. 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.

  7. 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.

  8. 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

    2015-01-01

    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. PMID:27877820

  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. Photobinding of ketoprofen in vitro and ex vivo

    NARCIS (Netherlands)

    Moser, J; Sarabia, Z; Minter, H; Lovell, WW; van Henegouwen, GMJB

    2000-01-01

    Ketoprofen (KP), a non-steroidal anti-inflammatory drug of the 2-aryl propionic class, has been shown to produce photoallergic side effects as well as cutaneous photosensitizing properties that induce other phototoxic effects. In the present study we investigated photobinding of ketoprofen to both h

  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. Ex-vivo expansion of red blood cells: how real for transfusion in humans?

    Science.gov (United States)

    Migliaccio, Anna Rita; Masselli, Elena; Varricchio, Lilian; Whitsett, Carolyn

    2012-03-01

    Blood transfusion is indispensable for modern medicine. In developed countries, the blood supply is adequate and safe but blood for alloimmunized patients is often unavailable. Concerns are increasing that donations may become inadequate in the future as the population ages prompting a search for alternative transfusion products. Improvements in culture conditions and proof-of-principle studies in animal models have suggested that ex-vivo expanded red cells may represent such a product. Compared to other cell therapies transfusion poses the unique challenge of requiring great cell doses (2.5×10(12) cells vs 10(7) cells). Although production of such cell numbers is theoretically possible, current technologies generate red cells in numbers sufficient only for safety studies. It is conceived that by the time these studies will be completed, technical barriers to mass cell production will have been eliminated making transfusion with ex-vivo generated red cells a reality.

  14. RADIOFREQUENCY ABLATION USING HYPERTONIC SALINE SOLUTION INSTILLATION: AN EX VIVO STUDY

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To determine whether radiofrequency ablation using hypertonic saline solution instillation can increase the extent of thermally mediated coagulation in ex vivo pig liver tissue. Methods Fifty-six radiofrequency ablation lesions were produced in fresh ex vivo pig's liver. According to different saline solutions, the lesions were divided into six groups: 25% acetic acid, 18% NaCl, 10% NaCl, 5% NaCl, 0.9% NaCl, and distilled water. After radiofrequency ablation, the lesions size and morphology were measured and compared.Results Using different instillation, the volume of coagulation necrosis was different: 25% acetic acid >18% NaCl >10% NaCl >5% NaCl >0.9% NaCl>distilled water. Conclusion Radiofrequency ablation using hypertonic saline solution instillation can increase the volume of radiofrequency ablation induced necrosis.

  15. Ex Vivo Aneurysm models mimicking real cases for the preoperative training of the clipping technique

    Directory of Open Access Journals (Sweden)

    Martin D.

    2016-06-01

    Full Text Available Training in a specialty like cerebrovascular neurosurgery becomes more and more difficult as the access to training is limited by the increasing number of neurosurgical departments and the lack of expert centers for specific pathology. This is why an increased investment in experimental training is encountered in many centers worldwide. The best models for training the clipping technique are ex Vivo on cadaveric heads aneurysm models, animal models or augmented reality models. We present a few ex Vivo models of aneurysms mimicking ACOA, ACM bifurcation and basil are tip aneurysms using a pulsed continuous perfusion system. Clipping training on aneurysm models is an invaluable tool both for the residents and for the specialists with a special interest in cerebrovascular surgery.

  16. 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).

  17. 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.

  18. 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....

  19. 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

    BACKGROUND: Placental malaria occurs when Plasmodium falciparum infected erythrocytes sequester in the placenta. Placental parasite isolates bind to chondroitin sulphate A (CSA) by expression of VAR2CSA on the surface of infected erythrocytes, but may sequester by other VAR2CSA mediated mechanisms...... placental tissue. RESULTS: The ex vivo placental perfusion model was modified to study adhesion of infected erythrocytes binding to CSA, endothelial protein C receptor (EPCR) or a transgenic parasite where P. falciparum erythrocyte membrane protein 1 expression had been shut down. Infected erythrocytes...... 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...

  20. 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.

  1. The effect of pentamidine on melanoma ex vivo.

    Science.gov (United States)

    Smith, Jason; Stewart, Benjamin J; Glaysher, Sharon; Peregrin, Katharine; Knight, Louise A; Weber, David J; Cree, Ian A

    2010-02-01

    Pentamidine is a small molecule inhibitor of the Ca(+)-binding protein S100B and disrupts the S100B-p53 protein-protein interaction; this is thought to restore wild-type p53 tumour suppressor function in melanoma. Additional anticancer effects may be the result of inhibition of regenerating liver family phosphatases. In this study, we have used a standardized ATP-tumour chemosensitivity assay to investigate the effect of pentamidine on cells derived from 18 skin melanoma samples and one uveal melanoma sample. The cells were tested at six concentrations from which the IC(50) and IC(90) were calculated. To allow comparison between samples, an index(sum) was calculated based on the percentage of tumour growth inhibition at each concentration. Of the skin melanoma samples tested, 78% exhibited an index(sum) less than 300 indicating strong inhibition. The median index(sum) of 237 also indicates considerable activity against these samples. The median IC(90) (30.2 micromol/l) may be clinically achievable in a proportion of patients. The uveal melanoma sample exhibited an index(sum) of 333 indicating moderate inhibition, and 86% inhibition at test drug concentration (37.96 micromol/l). These results show that pentamidine has activity against melanoma, and support the prospect of its development for therapeutic use.

  2. Photoacoustic imaging of prostate brachytherapy seeds in ex vivo prostate

    Science.gov (United States)

    Kuo, Nathanael; Kang, Hyun Jae; DeJournett, Travis; Spicer, James; Boctor, Emad

    2011-03-01

    The localization of brachytherapy seeds in relation to the prostate is a key step in intraoperative treatment planning (ITP) for improving outcomes in prostate cancer patients treated with low dose rate prostate brachytherapy. Transrectal ultrasound (TRUS) has traditionally been the modality of choice to guide the prostate brachytherapy procedure due to its relatively low cost and apparent ease of use. However, TRUS is unable to visualize seeds well, precluding ITP and producing suboptimal results. While other modalities such as X-ray and magnetic resonance imaging have been investigated to localize seeds in relation to the prostate, photoacoustic imaging has become an emerging and promising modality to solve this challenge. Moreover, photoacoustic imaging may be more practical in the clinical setting compared to other methods since it adds little additional equipment to the ultrasound system already adopted in procedure today, reducing cost and simplifying engineering steps. In this paper, we demonstrate the latest efforts of localizing prostate brachytherapy seeds using photoacoustic imaging, including visualization of multiple seeds in actual prostate tissue. Although there are still several challenges to be met before photoacoustic imaging can be used in the operating room, we are pleased to present the current progress in this effort.

  3. Ex vivo effectiveness of French over-the-counter products against head lice (Pediculus humanus capitis De Geer, 1778).

    Science.gov (United States)

    Combescot-Lang, Catherine; Vander Stichele, Robert H; Toubate, Berthine; Veirron, Emilie; Mumcuoglu, Kosta Y

    2015-05-01

    Head lice infestation is still a public health problem worldwide, with an intracountry and intercountry prevalence variation of 0.7 to 59%. There is a large variety of over-the-counter anti-louse products, but their efficacy is not always well assessed. Our objective was to test the pediculicidal and ovicidal efficacy of 21 over-the-counter head louse products, available in France during the period of 2008 to 2012. We tested children living in Tours City in central France and visiting preschools, primary schools, kindergarten, camps, and child care facilities, as well as children in their family houses, and were examined for the presence of lice. The products were collected from randomly selected pharmacies by covert investigators and then tested in the laboratory on an ex vivo sample of head lice and their eggs, collected from the hair of infested children. Living lice and unharmed eggs were collected from the scalps of 3-12 years old. The laboratory conditions for ex vivo testing mimicked the manufacturers' instructions for exposure time and application method. In 21 runs, 3919 living lice and 4321 undamaged living eggs were collected from the scalp of over 400 children. The 21 products were classified in three groups: 6 products in a group of potentially 100% pediculicidal activity and potentially 100% ovicidal activity, 8 products in a group of potentially 100% pediculicidal activity but insufficient ovicidal activity (including 2 products with claims of single application treatment), and 7 products in a group of insufficient pediculicidal activity and ovicidal activity. The pharmaceutical market for head lice products in France is swamped with poorly tested and ineffective products. Rigorous efficacy testing preregistration and periodic screening and testing of effectiveness in the post-registration period should be endorsed by the health authorities.

  4. 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

  5. Ex-vivo assessment of meningococcal resistance to natural immunity: serum bactericidal assays versus whole- blood assay

    Directory of Open Access Journals (Sweden)

    Nosratollah Saeed Adeli

    2009-01-01

    Full Text Available 1, 2,1 2 (Received 4 Dec, 2008; Accepted 14 Feb, 2009AbstractBackground and purpose: Literature review indicates that antibiotic resistance of bacteria may be associated with the resistance to host natural immunity. In the previous study we found that: all meningococcal isolates of meningitis cases are less sensitive to typical anti-meningococcal antibiotics. In present study, the association of the above characters was analyzed in an ex-vivo “case-control” study.Materials and methods: A randomized controlled trial compared effect two dietary treatments (low glycemic index & low fat in 46 adults, ages 18-55 years old, BMI >27,who proceed to motahary clinic in shiraz, Iran. Body weight, BMI, WHR (waist/hip ratio, fast and post-prandial Plasma lipid profile (Triacylglycerol, total Cholesterol, LDL-C, HDL-C concentration level of obese women measured at the beginning and at the end 6 weeks.Results: The resistance of both groups against selected sera were similar in the ratio 1:2- by SIC and SBC methods. The growth of “test” and “control” groups in the pooled sera were stopped at the ratios 1:512 and 1:1024 respectively. In SBA the population of “test” and “control” groups decreased in parallel to each other. However, the invasive (test meningococcal strains established slower reduction in WBA.Conclusion: Studies on the correlation between antibiotic resistance of bacteria and the resistance to the natural immunity have ended with different results. According to our experience, it seems that for ex- vivo investigation of natural immunity against meningococci the WBA is more reliable. J Mazand Univ Med Sci 2009; 19(68:48-57 (Persian

  6. 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

  7. 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.

  8. Ex-vivo expansion of red blood cells: How real for transfusion in humans?

    OpenAIRE

    Migliaccio, Anna Rita; Masselli, Elena; Varricchio, Lilian; Whitsett, Carolyn

    2011-01-01

    Blood transfusion is indispensable for modern medicine. In developed countries, the blood supply is adequate and safe but blood for alloimmunized patients is often unavailable. Concerns are increasing that donations may become inadequate in the future as the population ages prompting a search for alternative transfusion products. Improvements in culture conditions and proof-of-principle studies in animal models have suggested that ex-vivo expanded red cells may represent such a product. Compa...

  9. 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.

  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. 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.

  12. 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

  13. Modelo de conductancia hidráulica de la dentina humana ex vivo

    OpenAIRE

    Hevia, Javier; Fresno, Consuelo; Martin, Javier; Moncada,G; Letelier, Claudia; Oliveira Júnior, Osmir Batista de [UNESP; Fernández, Eduardo

    2013-01-01

    The main objective of this work was to mount and test an experimental model to measure the hydraulic conductance of ex vivo dentin. Seventeen healthy third molars, with indication of extraction of healthy donors aged between 15 and 30 years were obtained by informed consent. After cleaning them, disinfecting them, including them in resin epoxy and cutting them, there were 17 samples of dentin, corresponding to a disk of resin with a coronal section of tooth showing the dentin exposed on both ...

  14. 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.

  15. Parsley extract inhibits in vitro and ex vivo platelet aggregation and prolongs bleeding time in rats.

    Science.gov (United States)

    Gadi, Dounia; Bnouham, Mohamed; Aziz, Mohammed; Ziyyat, Abderrahim; Legssyer, Abdelkhaleq; Legrand, Chantal; Lafeve, Françoise Fauvel; Mekhfi, Hassane

    2009-08-17

    Many cardiovascular diseases are associated with an increase in blood platelet activity. In Morocco, parsley (Petroselinum crispum, Apiaceae) is one of the medicinal herbs used to treat cardiovascular diseases such as arterial hypertension. In this study, crude aqueous extract (CAE) of parsley was evaluated for its anti-platelet activity in experimental animals on platelet aggregation in vitro and ex vivo; and on bleeding time in vivo. The in vitro aggregation was monitored after pre-incubation of platelets with CAE. The bleeding time and ex vivo aggregation were performed after oral treatment. CAE inhibited dose dependently platelet aggregation in vitro induced by thrombin, ADP, collagen and epinephrine. The oral administration of CAE (3g/kg) inhibited significantly (p<0.001) platelet aggregation ex vivo and prolonged bleeding time (p<0.001) without changes in the platelet amount. The prolongation of bleeding time by CAE may be attributed to the observed inhibition of platelet aggregation. These effects could be related in part to the polyphenolic compounds present in the extract. These results support the hypothesis that the dietary intake of parsley may be benefit in the normalization of platelet hyperactivation, in the nutritional prevention of cardiovascular diseases and are potentially interesting in the development of new prevention strategies.

  16. Development of an ex vivo human-porcine respiratory model for preclinical studies.

    Science.gov (United States)

    Perinel, Sophie; Pourchez, Jérémie; Leclerc, Lara; Avet, John; Durand, Marc; Prévôt, Nathalie; Cottier, Michèle; Vergnon, Jean M

    2017-02-24

    Anatomical models to study aerosol delivery impose huge limitations and extrapolation to humans remains controversial. This study aimed to develop and validate an ex vivo human-like respiratory tract model easy to use and relevant to compare to in vivo human data. A human plastinated head is connected to an ex vivo porcine pulmonary tract ventilated artificially by passive expansion. A physiological study measures "pleural" depressions, tidal volumes, and minute ventilation for the respiratory rates chosen (10, 15, and 20 per minute) with three inspiratory/expiratory ratios (1/1, 1/2, and 1/3). Scintigraphy with (81m)Krypton assesses the homogeneity of the ventilation. Forty different experiments were set for validation, with 36 (90%) ventilating successfully. At a respiratory rate of 15/minute with inspiratory/expiratory ratio of 1/2, the tidal volume average was 824 mL (standard deviation, 207 mL). The scintigraphy performed on 16 ex vivo models (44.4%), showed homogenous ventilation with great similarity to human physiological studies. Ratio of the peripheral to central count rates were equally correlated with human data published in the literature. This new model, combining research feasibility and human physiology likeness, provides a realistic approach to human inhalation and therefore can be an interesting tool in aerosol regional deposition studies.

  17. Development of an ex vivo human-porcine respiratory model for preclinical studies

    Science.gov (United States)

    Perinel, Sophie; Pourchez, Jérémie; Leclerc, Lara; Avet, John; Durand, Marc; Prévôt, Nathalie; Cottier, Michèle; Vergnon, Jean M.

    2017-01-01

    Anatomical models to study aerosol delivery impose huge limitations and extrapolation to humans remains controversial. This study aimed to develop and validate an ex vivo human-like respiratory tract model easy to use and relevant to compare to in vivo human data. A human plastinated head is connected to an ex vivo porcine pulmonary tract ventilated artificially by passive expansion. A physiological study measures “pleural” depressions, tidal volumes, and minute ventilation for the respiratory rates chosen (10, 15, and 20 per minute) with three inspiratory/expiratory ratios (1/1, 1/2, and 1/3). Scintigraphy with 81mKrypton assesses the homogeneity of the ventilation. Forty different experiments were set for validation, with 36 (90%) ventilating successfully. At a respiratory rate of 15/minute with inspiratory/expiratory ratio of 1/2, the tidal volume average was 824 mL (standard deviation, 207 mL). The scintigraphy performed on 16 ex vivo models (44.4%), showed homogenous ventilation with great similarity to human physiological studies. Ratio of the peripheral to central count rates were equally correlated with human data published in the literature. This new model, combining research feasibility and human physiology likeness, provides a realistic approach to human inhalation and therefore can be an interesting tool in aerosol regional deposition studies. PMID:28233793

  18. 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.

  19. Pre vivo, ex vivo and in vivo evaluations of [{sup 68}Ga]-EDTMP

    Energy Technology Data Exchange (ETDEWEB)

    Mitterhauser, Markus [Department of Nuclear Medicine, Medical University of Vienna, Vienna 1090 (Austria) and Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Vienna 1090 (Austria) and Hospital Pharmacy, General Hospital of Vienna, Vienna 1090 (Austria)]. E-mail: markus.mitterhauser@meduniwien.ac.at; Toegel, Stefan [Department of Nuclear Medicine, Medical University of Vienna, Vienna 1090 (Austria); Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Vienna 1090 (Austria); Wadsak, Wolfgang [Department of Nuclear Medicine, Medical University of Vienna, Vienna 1090 (Austria); Department of Inorganic Chemistry, University of Vienna, Vienna 1090 (Austria); Lanzenberger, Rupert R. [Department of Psychiatry, Medical University of Vienna, Vienna 1090 (Austria); Mien, Leonhard-Key [Department of Nuclear Medicine, Medical University of Vienna, Vienna 1090 (Austria); Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Vienna 1090 (Austria); Kuntner, Claudia [Department of Radiopharmaceuticals, Austrian Research Centers, Seibersdorf 2443 (Austria); Wanek, Thomas [Department of Radiopharmaceuticals, Austrian Research Centers, Seibersdorf 2443 (Austria); Eidherr, Harald [Department of Nuclear Medicine, Medical University of Vienna, Vienna 1090 (Austria); Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Vienna 1090 (Austria); Ettlinger, Dagmar E. [Department of Nuclear Medicine, Medical University of Vienna, Vienna 1090 (Austria); Viernstein, Helmut [Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Vienna 1090 (Austria); Kluger, Rainer [Department of Orthopedics, Donauspital, Vienna 1220 (Austria); Dudczak, Robert [Department of Nuclear Medicine, Medical University of Vienna, Vienna 1090 (Austria); Kletter, Kurt [Department of Nuclear Medicine, Medical University of Vienna, Vienna 1090 (Austria)

    2007-05-15

    Introduction: The objectives of this study were to develop a simple preparation method for [{sup 68}Ga]-EDTMP and to evaluate the applicability of [{sup 68}Ga]-EDTMP as a potential positron emission tomography (PET) bone imaging agent using pre vivo, ex vivo and in vivo models. Methods: [{sup 68}Ga]-EDTMP was prepared using [{sup 68}Ga]-gallium chloride eluted from the {sup 68}Ge/{sup 68}Ga generator and commercially available Multibone kits. Binding affinity to bone compartments was evaluated using a recently established pre vivo model. In vivo (microPET) and ex vivo experiments were performed in mice, and the results of which were compared with those obtained with [{sup 18}F]-fluoride. Results: [{sup 68}Ga]-EDTMP was accessible via simple kit preparation and predominantly accumulated in bone tissue in vivo, ex vivo and pre vivo. Binding to mineral bone was irreversible, and low binding was observed in organic bone. In vivo microPET evaluation revealed predominant uptake in bone with renal excretion. Compared with [{sup 18}F]-fluoride, the uptake was lower and the PET image quality was reduced. Conclusions: From the present evaluation, apart from the autonomy for PET centers without an onsite cyclotron, the advantage of [{sup 68}Ga]-EDTMP over [{sup 18}F]-fluoride is not apparent and the future clinical prospect of [{sup 68}Ga]-EDTMP remains speculative.

  20. 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.

  1. 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.

  2. Prediction of intraocular antibody drug stability using ex-vivo ocular model.

    Science.gov (United States)

    Patel, Sulabh; Stracke, Jan Olaf; Altenburger, Ulrike; Mahler, Hanns-Christian; Metzger, Philipp; Shende, Pankaj; Jere, Dhananjay

    2017-03-01

    Following intravitreal (IVT) injection, therapeutic proteins get exposed to physiological pH, temperature and components in the vitreous humor (VH) for a significantly long time. Therefore, it is of interest to study the stability of the proteins in the VH. However, the challenge posed by the isolated VH (such as pH shift upon isolation and incubation due to the formation of smaller molecular weight (MW) degradation products) can result in artefacts when investigating protein stability in relevance for the actual in vivo situation. In this current study, an ex-vivo intravitreal horizontal stability model (ExVit-HS) has been successfully developed and an assessment of long-term stability of a bi-specific monoclonal antibody (mAb) drug in the isolated VH for 3months at physiological conditions has been conducted. The stability assessment was performed using various analytical techniques such as microscopy, UV visible for protein content, target binding ELISA, Differential Scanning Calorimetry (DSC), Capillary-electrophoresis-SDS, Size Exclusion (SEC) and Ion-exchange chromatography (IEC) and SPR-Biacore. The results show that the ExVit-HS model was successful in maintaining the VH at physiological conditions and retained a majority of protein in the VH-compartment throughout the study period. The mAb exhibited significantly less fragmentation in the VH relative to the PBS control; however, chemical stability of the mAb was equally compromised in VH and PBS. Interestingly, in the PBS control, mAb showed a rapid linear loss in the binding affinity. The loss in binding was almost 20% higher compared to that in VH after 3months. The results clearly suggest that the mAb has different degradation kinetics in the VH compared to PBS. These results suggest that it is beneficial to investigate the stability in the VH for drugs intended for IVT injection and that are expected longer residence times in the VH. The studies show that the ExVit-HS model may become a valuable tool

  3. 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

    2014-02-21

    .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.

  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

    .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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. An ex vivo approach to botanical-drug interactions: A proof of concept study

    Science.gov (United States)

    Wang, Xinwen; Zhu, Hao-Jie; Munoz, Juliana; Gurley, Bill J.; Markowitz, John S.

    2015-01-01

    Ethnopharmacological relevance Botanical medicines are frequently used in combination with therapeutic drugs, imposing a risk for harmful botanical-drug interactions (BDIs). Among the existing BDI evaluation methods, clinical studies are the most desirable, but due to their expense and protracted time-line for completion, conventional in vitro methodologies remain the most frequently used BDI assessment tools. However, many predictions generated from in vitro studies are inconsistent with clinical findings. Accordingly, the present study aimed to develop a novel ex vivo approach for BDI assessment and expand the safety evaluation methodoloy in applied ethnopharmacological research. Materials and Methods This approach differs from conventional in vitro methods in that rather than botanical extracts or individual phytochemicals being prepared in artificial buffers, human plasma/serum collected from a limited number of subjects administered botanical supplements was utilized to assess BDIs. To validate the methodology, human plasma/serum samples collected from healthy subjects administered either milk thistle or goldenseal extracts were utilized in incubation studies to determine their potential inhibitory effects on CYP2C9 and CYP3A4/5, respectively. Silybin A and B, two principal milk thistle phytochemicals, and hydrastine and berberine, the purported active constituents in goldenseal, were evaluated in both phosphate buffer and human plasma based in vitro incubation systems. Results Ex vivo study results were consistent with formal clinical study findings for the effect of milk thistle on the disposition of tolbutamide, a CYP2C9 substrate, and for goldenseal’s influence on the pharmacokinetics of midazolam, a widely accepted CYP3A4/5 substrate. Compared to conventional in vitro BDI methodologies of assessment, the introduction of human plasma into the in vitro study model changed the observed inhibitory effect of silybinA, silybin B and hydrastine and berberine

  10. Ex vivo Time Evolution of Thrombus Growth through Optical and Electrical Impedance data fusion

    Science.gov (United States)

    Affanni, A.; Specogna, R.; Trevisan, F.

    2013-09-01

    We designed a novel sensor specifically aimed at ex vivo measurements of white thrombus volume growth; a white thrombus is induced within an artificial micro-channel where hemostasis takes place starting from whole blood under flow conditions. The advantage of the proposed methodology is to identify the time evolution of the thrombus volume by means of an original data fusion methodology based on 2D optical and electrical impedance data simultaneously processed. On the contrary, the present state of the art optical imaging methodologies allow the thrombus volume estimation only at the end of the hemostatic process.

  11. Comparative study of temperature measurements in ex vivo swine muscle and a tissue-mimicking material during high intensity focused ultrasound exposures

    Science.gov (United States)

    Maruvada, S.; Liu, Y.; Pritchard, W. F.; Herman, B. A.; Harris, G. R.

    2012-01-01

    Tissue-mimicking materials (TMMs) can provide a convenient, stable, and reproducible means for testing high intensity focused ultrasound (HIFU) devices. When TMMs containing thermal sensors are used to measure ultrasound-induced temperature rise, it is important that measurement results reasonably represent those that occur in biological tissue. Therefore the aim of this paper is to compare the thermal behavior of the TMM under HIFU exposure to that of ex vivo tissue. This was accomplished using both a previously developed TMM and fresh ex vivo swine muscle that were instrumented with bare 50 µm thin wire thermocouples. HIFU at 825 kHz was focused at the thermocouple junction. 30 s exposures of increasing peak negative pressure (1 to 5 MPa) were applied and the temperature profile during and after sonication was recorded. B-mode imaging was used to monitor bubble activity during sonication. If bubble formation was noted during the sonication, the sonication was repeated at the same pressure levels two more times at 20 min intervals. Temperature traces obtained at various pressure levels demonstrated similar types of heating profiles in both the tissue and TMM, the exact nature of which depended on whether bubbles formed during the HIFU exposure. The onset of bubble activity occurred at lower ultrasonic pressures in the TMM, but the basic temperature rise features due to HIFU exposure were essentially the same for both materials.

  12. 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.

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

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    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.

  14. Sirolimus inhibits key events of restenosis in vitro/ex vivo: evaluation of the clinical relevance of the data by SI/MPL- and SI/DES-ratio's

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    Kountides Margaratis

    2007-05-01

    Full Text Available Abstract Background Sirolimus (SRL, Rapamycin has been used successfully to inhibit restenosis both in drug eluting stents (DES and after systemic application. The current study reports on the effects of SRL in various human in vitro/ex vivo models and evaluates the theoretical clinical relevance of the data by SI/MPL- and SI/DES-ratio's. Methods Definition of the SI/MPL-ratio: relation between significant inhibitory effects in vitro/ex vivo and the maximal plasma level after systemic administration in vivo (6.4 ng/ml for SRL. Definition of the SI/DES-ratio: relation between significant inhibitory effects in vitro/ex vivo and the drug concentration in DES (7.5 mg/ml in the ISAR drug-eluting stent platform. Part I of the study investigated in cytoflow studies the effect of SRL (0.01–1000 ng/ml on TNF-α induced expression of intercellular adhesion molecule 1 (ICAM-1 in human coronary endothelial cells (HCAEC and human coronary smooth muscle cells (HCMSMC. Part II of the study analysed the effect of SRL (0.01–1000 ng/ml on cell migration of HCMSMC. In part III, IV, and V of the study ex vivo angioplasty (9 bar was carried out in a human organ culture model (HOC-model. SRL (50 ng/ml was added for a period of 21 days, after 21 and 56 days cell proliferation, apoptosis, and neointimal hyperplasia was studied. Results Expression of ICAM-1 was significantly inhibited both in HCAEC (SRL ≥ 0.01 ng/ml and HCMSMC (SRL ≥ 10 ng/ml. SRL in concentrations ≥ 0.1 ng/ml significantly inhibited migration of HCMSMC. Cell proliferation and neointimal hyperplasia was inhibited at day 21 and day 56, significance (p Conclusion SI/MPL-ratio's ≤ 1 (ICAM-1 expression, cell migration characterize inhibitory effects of SRL that can be theoretically expected both after systemic and local high dose administration, a SI/MPL-ratio of 7.81 (cell proliferation represents an effect that was achieved with drug concentrations 7.81-times the MPL. SI/DES-ratio's between 10

  15. Ex vivo evaluation of a microneedle array device for transdermal application.

    Science.gov (United States)

    Indermun, Sunaina; Choonara, Yahya E; Kumar, Pradeep; du Toit, Lisa C; Modi, Girish; van Vuuren, Sandy; Luttge, Regina; Pillay, Viness

    2015-12-30

    A new approach of transdermal drug delivery is the use of microneedles. This promising technique offers the potential to be broadly used for drug administration as it enables the dramatic increase in permeation of medicaments across the stratum corneum. The potential of microneedles has evolved to spawn a plethora of potential transdermal applications. In order to advance the microneedle capabilities and possibly revolutionize advanced drug delivery, this study introduces a novel transdermal electro-modulated hydrogel-microneedle array (EMH-MNA) device composed of a nano-porous, embeddable ceramic microneedle array as well as an optimized EMH for the electro-responsive delivery of indomethacin through the skin. The ex vivo permeation as well as drug release experiments were performed on porcine skin tissue to ascertain the electro-responsive capabilities of the device. In addition, the microbial permeation ability of the microneedles across the viable epidermis in both microneedle-punctured skin as well as hypodermic needle-punctured skin was determined. Ex vivo evaluation of the EMH-MNA device across porcine skin demonstrated that without electro-stimulation, significantly less drug release was obtained (±0.4540mg) as compared to electro-stimulation (±2.93mg).

  16. Ex Vivo Produced Oral Mucosa Equivalent by Using the Direct Explant Cell Culture Technique

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    Kamile Öztürk

    2012-09-01

    Full Text Available Objective: The aim of this study is the histological and immunohistochemical evaluation of ex vivo produced oral mucosal equivalents using keratinocytes cultured by direct explant technique.Material and Methods: Oral mucosa tissue samples were obtained from the keratinized gingival tissues of 14 healthy human subjects. Human oral mucosa keratinocytes from an oral mucosa biopsy specimen were dissociated by the explant technique. Once a sufficient population of keratinocytes was reached, they were seeded onto the type IV collagen coated “AlloDerm” and taken for histological and immunohistochemical examinations at 11 days postseeding of the keratinocytes on the cadaveric human dermal matrix.Results: Histopathologically and immunohistochemically, 12 out of 14 successful ex vivo produced oral mucosa equivalents (EVPOME that consisted of a stratified epidermis on a dermal matrix have been developed with keratinocytes cultured by the explant technique.Conclusion: The technical handling involved in the direct explant method at the beginning of the process has fewer steps than the enzymatic method and use of the direct explant technique protocol for culturing of human oral mucosa keratinocyte may be more adequate for EVPOME production.

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

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    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

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

    Science.gov (United States)

    Tsang, Jovian J; Atkins, Harold L

    2015-01-01

    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.

  19. 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.

  20. Optimal Temperature of Graft Preservation after ex Vivo Gene Transfer in Lung Isografts

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    Sano,Yoshifumi

    2008-10-01

    Full Text Available The aim of this study was to determine the optimal temperature of graft preservation after ex vivo gene transfer to rat lung isografts. Left lungs were harvested and infused with cationic lipid/LacZ-DNA complex via the pulmonary artery, and the grafts were stored for 4h. The grafts (n=7 were allocated into groups IンIV according to the storage temperature:4℃, 10℃, 16℃, and 23℃, respectively. Forty-eight h after orthotopic transplantation, the arterial blood gas was analyzed and the peak airway pressure (PAP and the level of LacZ protein production in the grafts were measured by reverse transcription polymerase chain reaction. After reperfusion, the grafts were stained with hematoxylin and eosin. The grafts in groups III and IV showed more deterioration as evidenced by decreased arterial oxygen tension, increased PAP, and predominant infiltration of inflammatory cells compared with groups I and II. The level of LacZ production was significantly lower in group I than in groups IIンIV. The optimal temperature of lung graft preservation after ex vivo gene transfer was determined to be 10℃, balancing considerations of lung injury and efficiency of transgene expression.

  1. In vitro and ex vivo activity of Melaleuca alternifolia against protoscoleces of Echinococcus ortleppi.

    Science.gov (United States)

    Monteiro, Danieli Urach; Azevedo, Maria Isabel; Weiblen, Carla; DE Avila Botton, Sônia; Funk, Nadine Lysyk; DE Bona DA Silva, Cristiane; Zanette, Régis Adriel; Schwanz, Thiago Guilherme; DE LA Rue, Mário Luiz

    2017-02-01

    Cystic echinococcosis is a zoonotic disease of difficult diagnosis and treatment. The use of protoscolicidal agents in procedures is of utmost importance for treatment success. This study was aimed at analysing the in vitro and ex vivo activity of Melaleuca alternifolia oil (tea tree oil - TTO), its nanoemulsion formulation (NE-TTO) and its major component (terpinen-4-ol) against Echinococcus ortleppi protoscoleces obtained from cattle. Concentrations of 2·5, 5 and 10 mg mL-1 of TTO, 10 mg mL-1 of NE-TTO and 1, 1·5 and 2 mg mL-1 of terpinen-4-ol were evaluated in vitro against protoscoleces at 5, 10, 15 and 30 min. TTO was also injected directly into hydatid cysts (ex vivo analysis, n = 20) and the viability of protoscoleces was evaluated at 5, 15 and 30 min. The results indicated protoscolicidal effect at all tested formulations and concentrations. Terpinen-4-ol (2 mg mL-1) activity was superior when compared with the highest concentration of TTO. NE-TTO reached a gradual protoscolicidal effect. TTO at 20 mg mL-1 showed 90% protoscolicidal action in hydatid cysts at 5 min. The results showed that TTO affects the viability of E. ortleppi protoscoleces, suggesting a new protoscolicidal option to the treatment of cystic equinococcosis.

  2. 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.

  3. Instrumented urethral catheter and its ex vivo validation in a sheep urethra

    Science.gov (United States)

    Ahmadi, Mahdi; Rajamani, Rajesh; Timm, Gerald; Sezen, Serdar

    2017-03-01

    This paper designs and fabricates an instrumented catheter for instantaneous measurement of distributed urethral pressure profiles. Since the catheter enables a new type of urological measurement, a process for accurate ex vivo validation of the catheter is developed. A flexible sensor strip is first fabricated with nine pressure sensors and integrated electronic pads for an associated sensor IC chip. The flexible sensor strip and associated IC chip are assembled on a 7 Fr Foley catheter. A sheep bladder and urethra are extracted and used in an ex vivo set up for verification of the developed instrumented catheter. The bladder-urethra are suspended in a test rig and pressure cuffs placed to apply known static and dynamic pressures around the urethra. A significant challenge in the performance of the sensor system is the presence of parasitics that introduce large bias and drift errors in the capacitive sensor signals. An algorithm based on use of reference parasitic transducers is used to compensate for the parasitics. Extensive experimental results verify that the developed compensation method works effectively. Results on pressure variation profiles circumferentially around the urethra and longitudinally along the urethra are presented. The developed instrumented catheter will be useful in improved urodynamics to more accurately diagnose the source of urinary incontinence in patients.

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

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    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.

  5. 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.

  6. An ex vivo porcine nasal mucosa explants model to study MRSA colonization.

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    Pawel Tulinski

    Full Text Available Staphylococcus aureus is an opportunistic pathogen able to colonize the upper respiratory tract and skin surfaces in mammals. Methicillin-resistant S. aureus ST398 is prevalent in pigs in Europe and North America. However, the mechanism of successful pig colonization by MRSA ST398 is poorly understood. To study MRSA colonization in pigs, an ex vivo model consisting of porcine nasal mucosa explants cultured at an air-liquid interface was evaluated. In cultured mucosa explants from the surfaces of the ventral turbinates and septum of the pig nose no changes in cell morphology and viability were observed up to 72 h. MRSA colonization on the explants was evaluated followed for three MRSA ST398 isolates for 180 minutes. The explants were incubated with 3×10(8 CFU/ml in PBS for 2 h to allow bacteria to adhere to the explants surface. Next the explants were washed and in the first 30 minutes post adhering time, a decline in the number of CFU was observed for all MRSA. Subsequently, the isolates showed either: bacterial growth, no growth, or a further reduction in bacterial numbers. The MRSA were either localized as clusters between the cilia or as single bacteria on the cilia surface. No morphological changes in the epithelium layer were observed during the incubation with MRSA. We conclude that porcine nasal mucosa explants are a valuable ex vivo model to unravel the interaction of MRSA with nasal tissue.

  7. 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.

  8. 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.

  9. In vitro and ex vivo microbial leakage assessment in endodontics: A literature review

    Science.gov (United States)

    Savadkouhi, Sohrab Tour; Bakhtiar, Hengameh; Ardestani, Safoura Emami

    2016-01-01

    The aim of this study was to perform a literature review of published in-vitro and ex-vivo studies, which evaluated microbial leakage in endodontics in the past 10 years. A comprehensive electronic literature search was carried out in PubMed database for English articles published from 2005 to 2016 using the keywords “endodontics,” “in vitro,” “ex vivo,” “microbial leakage,” “microbial penetration,” “saliva,” “Enterococcus faecalis,” “E. faecalis,” “endodontic sealers,” “temporary filling material,” “apical plug,” “mineral trioxide aggregate,” and “MTA.” The keywords were combined using Boolean operators AND/OR. Based on our search strategy, 33 relevant articles were included in the study. There are three main methods for assessment of bacterial microleakage, namely, (A) the dual-chamber leakage model, (B) detection of bacteria using a scanning electron microscope (SEM), and (C) polymerase chain reaction. All bacterial leakage models have some limitations and may yield different results compared to other microleakage evaluation techniques (i.e., dye penetration, fluid filtration, or electrochemical tests). The results of SEM correlated with those of microbial leakage test in most studies. Microbial leakage test using saliva better simulates the clinical setting for assessment of the leakage of single or mixed bacterial species. PMID:28032041

  10. 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.

  11. 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.

  12. Automated Segmentation of in Vivo and Ex Vivo Mouse Brain Magnetic Resonance Images

    Directory of Open Access Journals (Sweden)

    Alize E.H. Scheenstra

    2009-01-01

    Full Text Available Segmentation of magnetic resonance imaging (MRI data is required for many applications, such as the comparison of different structures or time points, and for annotation purposes. Currently, the gold standard for automated image segmentation is nonlinear atlas-based segmentation. However, these methods are either not sufficient or highly time consuming for mouse brains, owing to the low signal to noise ratio and low contrast between structures compared with other applications. We present a novel generic approach to reduce processing time for segmentation of various structures of mouse brains, in vivo and ex vivo. The segmentation consists of a rough affine registration to a template followed by a clustering approach to refine the rough segmentation near the edges. Compared with manual segmentations, the presented segmentation method has an average kappa index of 0.7 for 7 of 12 structures in in vivo MRI and 11 of 12 structures in ex vivo MRI. Furthermore, we found that these results were equal to the performance of a nonlinear segmentation method, but with the advantage of being 8 times faster. The presented automatic segmentation method is quick and intuitive and can be used for image registration, volume quantification of structures, and annotation.

  13. 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

  14. Passiflora manicata (Juss.) aqueous leaf extract protects against reactive oxygen species and protein glycation in vitro and ex vivo models.

    Science.gov (United States)

    da Silva Morrone, Maurilio; de Assis, Adriano Martimbianco; da Rocha, Ricardo Fagundes; Gasparotto, Juciano; Gazola, Andressa Córneo; Costa, Geison Modesti; Zucolotto, Silvana Maria; Castellanos, Leonardo H; Ramos, Freddy A; Schenkel, Eloir Paulo; Reginatto, Flávio Henrique; Gelain, Daniel Pens; Moreira, José C F

    2013-10-01

    The leaf extracts of many species of genus Passiflora have been extensively investigated for their biological activities on several rat tissues, but mainly in the central nervous system and liver. They posses anxiolytic-like, sedative effects and antioxidant properties. Evidences suggest a key role of C-glycosylflavonoids in the biological activities of Passiflora extracts. Some species (such as P. manicata) of the genus are still poorly investigated for their chemical and biological activity. In this work, we aim to investigate both antioxidant and antiglycation properties of aqueous extract of P. manicata leaves (PMLE) in vitro and ex vivo models. Crude extract showed the C-glycosylflavonoid isovitexin as the major compound. Isoorientin and vitexin were also identified. In TRAP/TAR assay, PMLE showed a significant antioxidant activity. PMLE at concentrations of 10 and 100 μg mL⁻¹ significantly decreasing LDH leakage in rat liver slices. Antioxidant effect also was observed by decreased in oxidative damage markers in slices hence hydrogen peroxide was added as oxidative stress inductor. PMLE inhibited protein glycation at all concentrations tested. In summary, P. manicata aqueous leaf extract possess protective properties against reactive oxygen species and also protein glycation, and could be considered a new source of natural antioxidants.

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

    Science.gov (United States)

    Darvin, M. E.; Richter, H.; Zhu, Y. J.; Meinke, M. C.; Knorr, F.; Gonchukov, S. A.; Koenig, K.; Lademann, J.

    2014-07-01

    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.

  16. 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)

  17. Optical properties of normal and thermally coagulated chicken liver tissue measured ex-vivo with diffuse reflectance

    Science.gov (United States)

    Hafeez-Ullah; Atif, M.; Firdous, S.; Mehmood, M. S.; Hamza, M. Y.; Imran, M.; Hussain, G.; Ikram, M.

    2011-02-01

    The purpose of the present study is to determine the optical properties of normal and thermally coagulated chicken liver at 720, 740, 770, 810, 825 and 840 nm wavelengths of laser irradiation. So, we were able to evaluate these optical properties (absorption and scattering coefficients) with ex-vivo study using Kubelka Munk Model (KMM) from the radial dependence of the diffuse reflectance with femtosecond pulsed laser in near IR region. These coefficients were significantly increased with coagulation. The penetration depths of the diffused light have been reported to a maximum value of 8.12 ± 0.36 mm in normal liver and 2.49 ± 0.17 mm in coagulated liver at 840 nm showing increasing behavior towards IR region. The Monte Carlo simulation was used to check the theoretical validation of measured optical properties of the tissue that showed a good match with our experimental results. We believe that these differences in optical properties will be helpful for the understanding arid optimal use of laser applications in medicine and differential diagnosis of tissues by using different optical methods. Especially for the investigation of biological tissue for photodynamic therapy (PDT), the knowledge of the specific optical properties and their thermo-induced changes is important.

  18. 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.

  19. Electrochemical fecal pellet sensor for simultaneous real-time ex vivo detection of colonic serotonin signalling and motility

    Science.gov (United States)

    Morris, Rachel; Fagan-Murphy, Aidan; MacEachern, Sarah J.; Covill, Derek; Patel, Bhavik Anil

    2016-03-01

    Various investigations have focused on understanding the relationship between mucosal serotonin (5-HT) and colonic motility, however contradictory studies have questioned the importance of this intestinal transmitter. Here we described the fabrication and use of a fecal pellet electrochemical sensor that can be used to simultaneously detect the release of luminal 5-HT and colonic motility. Fecal pellet sensor devices were fabricated using carbon nanotube composite electrodes that were housed in 3D printed components in order to generate a device that had shape and size that mimicked a natural fecal pellet. Devices were fabricated where varying regions of the pellet contained the electrode. Devices showed that they were stable and sensitive for ex vivo detection of 5-HT, and no differences in the fecal pellet velocity was observed when compared to natural fecal pellets. The onset of mucosal 5-HT was observed prior to the movement of the fecal pellet. The release of mucosal 5-HT occurred oral to the fecal pellet and was linked to the contraction of the bowel wall that drove pellet propulsion. Taken, together these findings provide new insights into the role of mucosal 5-HT and suggest that the transmitter acts as a key initiator of fecal pellet propulsion.

  20. 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.

  1. 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.

  2. Diminished monocytic HLA-DR expression and ex vivo cytokine secretion capacity in patients with glioblastoma: effect of tumor extirpation.

    Science.gov (United States)

    Woiciechowsky, C; Asadullah, K; Nestler, D; Schöning, B; Glöckner, F; Döcke, W D; Volk, H D

    1998-04-15

    Severe immunodysregulation on lymphocyte level has been described in patients with glioblastoma and is likely involved into its unfavorable prognosis. Although the major importance of monocytic cells for immunoregulation is well established, only very limited data exist regarding the monocyte status in glioblastoma patients. Here we demonstrate a markedly diminished monocytic HLA-DR expression and ex vivo cytokine secretion capacity (TNF-alpha, IL-1beta, IL-10) as signs for monocyte deactivation in glioblastoma patients but not in patients with astrocytoma. As known in immunocompromised patients from other reasons, monocyte deactivation indicate global immunodepression associated with an enhanced risk of infectious complications. Interestingly, tumor resection resulted in partial recovery from the monocytic deactivation. This suggests that the glioblastoma itself contributed to this phenomenon. However, IL-10 and the active forms of transforming growth factor-beta2 and -beta1, which are produced by glioblastoma cells and known to inhibit monocyte function, were not detectable in plasma in our patients. Moreover, low levels of the adrenocorticotropic hormone and cortisol excluded hypothalamo-pituitary-adrenal axis involvement. So, further investigations are necessary to clarify the mechanism. The demonstrated severe glioblastoma-associated monocytic deactivation may contribute to its unfavorable prognosis. Therefore, monocytes may represent target cells for new adjuvant immunotherapies in glioblastoma.

  3. 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.

  4. Comparative study of abdominal and thoracic aortic aneurysms: their pathogenesis and a gingival fibroblasts-based ex vivo treatment.

    Science.gov (United States)

    Cherifi, Hafida; Gogly, Bruno; Loison-Robert, Ludwig-Stanislas; Couty, Ludovic; Ferré, François Côme; Nassif, Ali; Lafont, Antoine; Fournier, Benjamin Pj

    2015-01-01

    Aortic aneurysms (AAs) consist of slow proteolysis and loss of both collagen and elastin matrix in the aorta wall, leading to wall dilation, weakening and rupture in well-advanced lesions. This can occur in both abdominal aorta (Abdominal Aortic Aneurysm: AAA) and thoracic aorta (Thoracic Aortic Aneurysm: TAA). To date, no non-surgical therapy has been proposed to slow or stop AA progression. Previously published preclinical studies from our team using an aneurysm rabbit model showed a promising concept for treatment of AAs with gingival fibroblast (GFs) which are readily available cells. In this study, we investigated the possible tissue repair of human AAAs and TAAs using ex vivo models co-cultured with GFs. Histological analysis showed that TAA and AAA are two distinct pathologies. Both lesions presented destruction of the aorta wall, highly evidenced in AAA samples. The results have confirmed the presence of the bacterial Porphyromonas gingivalis (Pg) protein in all AAA samples, but not in TAA samples, indicating the possible role of an infectious factor in the developing and progression of AAA lesions compared to TAA. The co-culture of GFs with AA lesions shows increased expression of TIMP-1, the inhibitor of the aneurysm severity marker MMP-9. Our study indicates that GFs might ameliorate aorta wall reestablishment in both AA types by their regenerative and immunomodulatory capacities. It also demonstrates the possible infectious cause of AAA compared with TAA that may explain their different behavior.

  5. The effect of tendon surface treatment on cell attachment for potential enhancement of tendon graft healing: an ex vivo model.

    Science.gov (United States)

    Hashimoto, Takahiro; Sun, Yu-Long; An, Kai-Nan; Amadio, Peter C; Zhao, Chunfeng

    2012-12-01

    For both tendon allografts and autografts, the surface, initially optimized for gliding, may not be ideal to facilitate tissue integration for graft healing to host tendon or bone. As a prelude to studying tendon-bone integration, we investigated the effect of surface treatments with trypsin or mechanical abrasion on cell attachment to the tendon surface in a canine ex vivo intrasynovial tendon tissue culture model. Intrasynovial tendon allograft surfaces were seeded with cells after the following treatments: (1) no treatment, (2) mechanical abrasion, (3) trypsin, and (4) abrasion and trypsin. The area covered by cells was determined using confocal laser microscopy at one and two weeks. Results were compared to untreated extrasynovial tendon. Additional tendons were characterized with scanning electron microscopy. Tendons with trypsin treatment had significantly more surface coverage with cells than the other groups, after both one and two weeks of culture. In terms of the cellular shape and size, cells on tendons with trypsin treatment spread more and were more polygonal in shape, whereas tendons with mechanical abrasion with/without trypsin treatment contained smaller, more spindle-like cells. Surface roughening can affect cell behavior with topographical stimulation. Trypsin surface digestion exposes a mesh-like structure on the tendon surface, which could enhance cell adherence and, possibly, tendon/bone healing.

  6. Enniatin-containing solutions for oromucosal use: Quality-by-design ex-vivo transmucosal risk assessment of composition variability.

    Science.gov (United States)

    Taevernier, Lien; Detroyer, Sven; Veryser, Lieselotte; De Spiegeleer, Bart

    2015-08-01

    Fusafungine, a mixture of the cyclic hexadepsipeptides enniatins, is currently on the market for the treatment of upper respiratory tract diseases because of its bacteriostatic and anti-inflammatory effects. In this study, a quality-by-design risk assessment was performed with two objectives: (i) investigate whether enniatins are able to permeate the mucosa and reach blood circulation, as the summary of product characteristics indicates this is not the case, and if so, to quantify their transmucosal kinetics and (ii) study the influence of excipient concentration variability on mucosal permeation. First, the concentration of the two main excipients isopropyl myristate and ethanol, known penetration enhancers, in several marketed samples was determined using GC-FID. Then, the transmucosal kinetics of the enniatins were quantitatively evaluated for different dose solutions, using porcine buccal mucosa in an ex-vivo in-vitro Franz diffusion cell set-up, with UHPLC-MS/MS bioanalytics. This study demonstrated that enniatins are capable of permeating the mucosa. However, no risk of a significant different transmucosal permeability with varying excipient concentrations was detected.

  7. Optimization of an ex vivo wound healing model in the adult human skin: Functional evaluation using photodynamic therapy.

    Science.gov (United States)

    Mendoza-Garcia, Jenifer; Sebastian, Anil; Alonso-Rasgado, Teresa; Bayat, Ardeshir

    2015-09-01

    Limited utility of in vitro tests and animal models of human repair, create a demand for alternative models of cutaneous healing capable of functional testing. The adult human skin Wound Healing Organ Culture (WHOC) provides a useful model, to study repair and enable evaluation of therapies such as the photodynamic therapy (PDT). Thus, the aim here was to identify the optimal WHOC model and to evaluate the role of PDT in repair. Wound geometry, system of support, and growth media, cellular and matrix biomarkers were investigated in WHOC models. Subsequently, cellular activity, extracellular matrix remodeling, and oxidative stress plus gene and protein levels of makers of wound repair measured the effect of PDT on the optimized WHOC. WHOCs embedded in collagen and supplemented DMEM were better organized showing stratified epidermis and compact dermis with developing neo-epidermis. Post-PDT, the advancing reepithelialization tongue was 3.5 folds longer, and was highly proliferative with CK-14 plus p16 increased (p < 0.05) compared to controls. The neo-epidermis was fully differentiated forming neo-collagen. Proliferating nuclear antigen, p16, COLI, COLIII, MMP3, MMP19, and α-SMA were significantly more expressed (p < 0.05) in dermis surrounding the healing wound. In conclusion, an optimal model of WHOC treated with PDT shows increased reepithelialization and extracellular matrix reconstruction and remodeling, supporting evidence toward development of an optimal ex vivo wound healing model.

  8. 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.

  9. Accuracy of combined PET/CT in image-guided interventions of liver lesions: An ex-vivo study

    Institute of Scientific and Technical Information of China (English)

    Patrick Veit; Christiane Kuehle; Thomas Beyer; Hilmar Kuehl; Andreas Bockisch; Gerald Antoch

    2006-01-01

    AIM: Positioning of interventional devices in liver lesions is a challenging task if only CT is available. We investigated the potential benefit of combined PET/CT images for localization of interventional devices in interventional liver studies.METHODS: Thirty lesions each of hyperdense, isodense and hypodense attenuation compared to normal liver parenchyma were injected into 15 ex-vivo pig livers. All lesions were composed of the same amounts of gelatine containing 0.5 MBq of 18F-FDG. Following lesion insertion, an interventional needle was placed in each lesion under CT-guidance solely. After that, a PET/CT study was performed. The localization of the needle within the lesion was assessed for CT alone and PET/CT and the root mean square (RMS) was calculated. Results were compared with macroscopic measurements after lesion dissection serving as the standard of reference. RESULTS: In hypo- and isodense lesions PET/CT proved more accurate in defining the position of the interventional device when compared with CT alone. The mean RMS for CT and PET/CT differed significantly in isodense and hypodense lesions. No significant difference was found for hyperdense lesions.CONCLUSION: Combined FDG-PET/CT imaging provides more accurate information than CT alone concerning the needle position in FDG-PET positive liver lesions.Therefore combined PET/CT might be potentially beneficial not only for localization of an interventional device,but may also be beneficial for guidance in interventional liver procedures.

  10. 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

  11. 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.

  12. 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.)

  13. 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.

  14. 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.

  15. Computer-aided diagnosis in phase contrast imaging X-ray computed tomography for quantitative characterization of ex vivo human patellar cartilage.

    Science.gov (United States)

    Nagarajan, Mahesh B; Coan, Paola; Huber, Markus B; Diemoz, Paul C; Glaser, Christian; Wismuller, Axel

    2013-10-01

    Visualization of ex vivo human patellar cartilage matrix through the phase contrast imaging X-ray computed tomography (PCI-CT) has been previously demonstrated. Such studies revealed osteoarthritis-induced changes to chondrocyte organization in the radial zone. This study investigates the application of texture analysis to characterizing such chondrocyte patterns in the presence and absence of osteoarthritic damage. Texture features derived from Minkowski functionals (MF) and gray-level co-occurrence matrices (GLCM) were extracted from 842 regions of interest (ROI) annotated on PCI-CT images of ex vivo human patellar cartilage specimens. These texture features were subsequently used in a machine learning task with support vector regression to classify ROIs as healthy or osteoarthritic; classification performance was evaluated using the area under the receiver operating characteristic curve (AUC). The best classification performance was observed with the MF features perimeter (AUC: 0.94 ±0.08 ) and "Euler characteristic" (AUC: 0.94 ±0.07 ), and GLCM-derived feature "Correlation" (AUC: 0.93 ±0.07). These results suggest that such texture features can provide a detailed characterization of the chondrocyte organization in the cartilage matrix, enabling classification of cartilage as healthy or osteoarthritic with high accuracy.

  16. In Vivo/Ex Vivo MRI-Based 3D Non-Newtonian FSI Models for Human Atherosclerotic Plaques Compared with Fluid/Wall-Only Models.

    Science.gov (United States)

    Yang, Chun; Tang, Dalin; Yuan, Chun; Hatsukami, Thomas S; Zheng, Jie; Woodard, Pamela K

    2007-01-01

    It has been recognized that fluid-structure interactions (FSI) play an important role in cardiovascular disease initiation and development. However, in vivo MRI multi-component FSI models for human carotid atherosclerotic plaques with bifurcation and quantitative comparisons of FSI models with fluid-only or structure-only models are currently lacking in the literature. A 3D non-Newtonian multi-component FSI model based on in vivo/ex vivo MRI images for human atherosclerotic plaques was introduced to investigate flow and plaque stress/strain behaviors which may be related to plaque progression and rupture. Both artery wall and plaque components were assumed to be hyperelastic, isotropic, incompressible and homogeneous. Blood flow was assumed to be laminar, non-Newtonian, viscous and incompressible. In vivo/ex vivo MRI images were acquired using histologically-validated multi-spectral MRI protocols. The 3D FSI models were solved and results were compared with those from a Newtonian FSI model and wall-only/fluid-only models. A 145% difference in maximum principal stresses (Stress-P(1)) between the FSI and wall-only models and 40% difference in flow maximum shear stress (MSS) between the FSI and fluid-only models were found at the throat of the plaque using a severe plaque sample (70% severity by diameter). Flow maximum shear stress (MSS) from the rigid wall model is much higher (20-40% in maximum MSS values, 100-150% in stagnation region) than those from FSI models.

  17. 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.

  18. 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

    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......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...

  19. 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.

  20. Steerable intravitreal inserts for drug delivery: in vitro and ex vivo mobility experiments.

    Science.gov (United States)

    Bergeles, Christos; Kummer, Michael P; Kratochvil, Bradley E; Framme, Carsten; Nelson, Bradley J

    2011-01-01

    The progress of wet age-related macular degeneration can now be controlled by intravitreal drug injection. This approach requires repeated injections, which could be avoided by delivering the drug to the retina. Intraocular implants are a promising solution for drug delivery near the retina. Currently, their accurate placement is challenging, and they can only be removed after a vitrectomy. In this paper, we introduce an approach for minimally invasive retinal drug delivery using magnetic intraocular inserts. We briefly discuss the electromagnetic-control system for magnetic implants and then focus on evaluating their ability to move in the vitreous humor. The mobility of magnetic intraocular implants is estimated in vitro with synthesized vitreous humors, and ex vivo with experiments on cadaver porcine eyes. Preliminary results show that with such magnetic implants a vitrectomy can be avoided.

  1. Enhanced ex vivo intestinal absorption of olmesartan medoxomil nanosuspension: Preparation by combinative technology.

    Science.gov (United States)

    Attari, Zenab; Bhandari, Amita; Jagadish, P C; Lewis, Shaila

    2016-01-01

    The purpose of this study was to develop nanosuspension based on combinative technology to enhance the intestinal absorption of Olmesartan medoxomil (OLM), a potent antihypertensive agent with limited oral bioavailability. Two combinative approaches were employed and then characterized. In vitro intestinal absorption of OLM nanosuspension and plain OLM was studied using non-everted rat intestinal sac model. Optimal OLM nanosuspension was prepared by a combination of ball milling and probe sonication using stabilizer, Poloxamer 407. The formula exhibited particle size of 469.9 nm and zeta potential of -19.1 mV, which was subjected to ex vivo studies. The flux and apparent permeability coefficient in intestine from OLM nanosuspension was higher than the plain drug, thereby suggesting better drug delivery.

  2. Random walks with shape prior for cochlea segmentation in ex vivo μCT

    DEFF Research Database (Denmark)

    Ruiz Pujadas, Esmeralda; Kjer, Hans Martin; Piella, Gemma;

    2016-01-01

    Purpose Cochlear implantation is a safe and effective surgical procedure to restore hearing in deaf patients. However, the level of restoration achieved may vary due to differences in anatomy, implant type and surgical access. In order to reduce the variability of the surgical outcomes, we...... previously proposed the use of a high-resolution model built from μCT images and then adapted to patient-specific clinical CT scans. As the accuracy of the model is dependent on the precision of the original segmentation, it is extremely important to have accurate μCT segmentation algorithms. Methods We...... propose a new framework for cochlea segmentation in ex vivo μCT images using random walks where a distance-based shape prior is combined with a region term estimated by a Gaussian mixture model. The prior is also weighted by a confidence map to adjust its influence according to the strength of the image...

  3. Ex-vivo partial nephrectomy after living donor nephrectomy: Surgical technique for expanding kidney donor pool

    Directory of Open Access Journals (Sweden)

    Yaw A Nyame

    2017-01-01

    Full Text Available Renal transplantation has profound improvements in mortality, morbidity, and overall quality of life compared to renal replacement therapy. This report aims to illustrate the use of ex-vivo partial nephrectomy in a patient with a renal angiomyolipoma prior to living donor transplantation. The surgical outcomes of the donor nephrectomy and recipient transplantation are reported with 2 years of follow-up. Both the donor and recipient are healthy and without any significant comorbidities. In conclusion, urologic techniques such as partial nephrectomy can be used to expand the living donor pool in carefully selected and well informed transplant recipients. Our experience demonstrated a safe and positive outcome for both the recipient and donor, and is consistent with other reported outcomes in the literature.

  4. Temperature profile of ex-vivo organs during radio frequency thermal ablation by fiber Bragg gratings

    Science.gov (United States)

    Palumbo, Giovanna; Iadicicco, Agostino; Tosi, Daniele; Verze, Paolo; Carlomagno, Nicola; Tammaro, Vincenzo; Ippolito, Juliet; Campopiano, Stefania

    2016-11-01

    We report on the integration of fiber optic sensors with commercial medical instrumentation for temperature monitoring during radio frequency ablation for tumor treatment. A suitable configuration with five fiber Bragg grating sensors bonded to a bipolar radio frequency (RF) probe has been developed to monitor the area under treatment. A series of experiments were conducted on ex-vivo animal kidney and liver and the results confirm that we were able to make a multipoint measurement and to develop a real-time temperature profile of the area, with a temperature resolution of 0.1°C and a spatial resolution of 5 mm during a series of different and consecutive RF discharges.

  5. Optical spectroscopy for differentiation of liver tissue under distinct stages of fibrosis: an ex vivo study

    Science.gov (United States)

    Fabila, D. A.; Hernández, L. F.; de la Rosa, J.; Stolik, S.; Arroyo-Camarena, U. D.; López-Vancell, M. D.; Escobedo, G.

    2013-11-01

    Liver fibrosis is the decisive step towards the development of cirrhosis; its early detection affects crucially the diagnosis of liver disease, its prognosis and therapeutic decision making. Nowadays, several techniques are employed to this task. However, they have the limitation in estimating different stages of the pathology. In this paper we present a preliminary study to evaluate if optical spectroscopy can be employed as an auxiliary tool of diagnosis of biopsies of human liver tissue to differentiate the fibrosis stages. Ex vivo fluorescence and diffuse reflectance spectra were acquired from biopsies using a portable fiber-optic system. Empirical discrimination algorithms based on fluorescence intensity ratio at 500 nm and 680 nm as well as diffuse reflectance intensity at 650 nm were developed. Sensitivity and specificity of around 80% and 85% were respectively achieved. The obtained results show that combined use of fluorescence and diffuse reflectance spectroscopy could represent a novel and useful tool in the early evaluation of liver fibrosis.

  6. Release of rosmarinic acid from semisolid formulations and its penetration through human skin ex vivo

    Directory of Open Access Journals (Sweden)

    Stelmakienė Ada

    2015-06-01

    Full Text Available The aim of this study was to evaluate the release of rosmarinic acid (RA from the experimental topical formulations with the Melissa officinalis L. extract and to evaluate its penetration through undamaged human skin ex vivo. The results of the in vitro release study showed that higher amounts of RA were released from the emulsion vehicle when lemon balm extract was added in its dry form. An inverse correlation was detected between the released amount of RA and the consistency index of the formulation. Different penetration of RA into the skin may be influenced by the characteristics of the vehicle as well as by the form of the extract. The results of penetration assessment showed that the intensity of RA penetration was influenced by its lipophilic properties: RA was accumulating in the epidermis, while the dermis served as a barrier, impeding its deeper penetration.

  7. 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 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.

  8. CD64-directed microtubule associated protein tau kills leukemic blasts ex vivo.

    Science.gov (United States)

    Mladenov, Radoslav; Hristodorov, Dmitrij; Cremer, Christian; Gresch, Gerrit; Grieger, Elena; Schenke, Lea; Klose, Diana; Amoury, Manal; Woitok, Mira; Jost, Edgar; Brümmendorf, Tim H; Fendel, Rolf; Fischer, Rainer; Stein, Christoph; Thepen, Theo; Barth, Stefan

    2016-10-11

    Fc gamma receptor I (FcγRI, CD64) is a well-known target antigen for passive immunotherapy against acute myeloid leukemia and chronic myelomonocytic leukemia. We recently reported the preclinical immunotherapeutic potential of microtubule associated protein tau (MAP) against a variety of cancer types including breast carcinoma and Hodgkin's lymphoma. Here we demonstrate that the CD64-directed human cytolytic fusion protein H22(scFv)-MAP kills ex vivo 15-50% of CD64+ leukemic blasts derived from seven myeloid leukemia patients. Furthermore, in contrast to the nonspecific cytostatic agent paclitaxel, H22(scFv)-MAP showed no cytotoxicity towards healthy CD64+ PBMC-derived cells and macrophages. The targeted delivery of this microtubule stabilizing agent therefore offers a promising new strategy for specific treatment of CD64+ leukemia.

  9. 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...... allergen concentrations. At each step basophil activation (CD193(+) CD63(+) ) was monitored with and without (background activation) allergen challenge at optimal concentration. The sequential desensitization protocol was compared to a single-dose desensitization protocols with threshold and subthreshold...... allergen concentrations. Incubation intervals and allergen concentrations were varied in order to optimise the protocol. RESULTS: Sequential desensitization effectively reduced basophil response. The single-dose subthreshold protocol and single-dose threshold protocols did not reduce basophil activation...

  10. 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.

  11. Ex Vivo Expansion or Manipulation of Stem Cells to Improve Outcome of Umbilical Cord Blood Transplantation.

    Science.gov (United States)

    Horwitz, Mitchell E

    2016-02-01

    The outcome of umbilical cord blood transplantation for adult patients with hematologic malignancies now rivals that of matched unrelated donor transplantation. However, delayed hematopoietic and immunologic recovery remains a source of significant morbidity and mortality. Multiple strategies are now being studied to overcome these limitations. One strategy involves ex vivo expansion of the umbilical cord blood unit prior to transplantation. A second strategy involves exposure of the umbilical cord blood graft to compounds aimed at improving homing and engraftment following transplantation. Such a strategy may also address the problem of slow hematopoietic recovery as well as the increased risk of graft failure. Many of these strategies are now being tested in late phase multi-center clinical trials. If proven cost-effective and efficacious, they may alter the landscape of donor options for allogeneic stem cell transplantation.

  12. Surveying the Delivery Methods of CRISPR/Cas9 for ex vivo Mammalian Cell Engineering.

    Science.gov (United States)

    Kelton, William J; Pesch, Theresa; Matile, Stefan; Reddy, Sai T

    2016-01-01

    The simplicity of the CRISPR/Cas9 technology has been transformative in making targeted genome editing accessible for laboratories around the world. However, due to the sheer volume of literature generated in the past five years, determining the best format and delivery method of CRISPR/Cas9 components can be challenging. Here, we provide a brief overview of the progress that has been made in the ex vivo genome editing of mammalian cells and summarize the key advances made for improving efficiency and delivery of CRISPR/Cas9 in DNA, RNA, and protein form. In particular, we highlight the delivery of Cas9 components to human cells for advanced genome editing applications such as large gene insertion.

  13. Measurement of histamine release from human lung tissue ex vivo by microdialysis technique

    DEFF Research Database (Denmark)

    Nissen, Dan; Petersen, Lars Jelstrup; Nolte, H;

    1998-01-01

    OBJECTIVE AND DESIGN: Currently no method is available for measurement of mediator release from intact human lung. In this study, a microdialysis technique was used to measure histamine release from mast cells in human lung tissue ex vivo. MATERIAL: Microdialysis fibers of 216 microm were inserted...... into lung tissue and perfused with Krebs Ringer buffer at a rate of 3 microl/min. After a 15 min period of steady-state perfusion, anti-IgE and vehicle were injected into the lung tissue above individual fibers. Samples from each fibre were collected for 20 min at 2 min intervals. Histamine was assayed...... fluorometrically. RESULTS: Anti-IgE concentrations of 40-40,000 U/ml dose-dependently released histamine, significant histamine release being demonstrated with anti-IgE concentrations of 400 U/ml and greater. The kinetics of histamine release showed peak values 2-8 min after the injection. Great individual...

  14. 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.

  15. 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.

  16. Fiber optic microneedles for transdermal light delivery: ex vivo porcine skin penetration experiments.

    Science.gov (United States)

    Kosoglu, Mehmet A; Hood, Robert L; Chen, Ye; Xu, Yong; Rylander, Marissa Nichole; Rylander, Christopher G

    2010-09-01

    Shallow light penetration in tissue has been a technical barrier to the development of light-based methods for in vivo diagnosis and treatment of epithelial carcinomas. This problem can potentially be solved by utilizing minimally invasive probes to deliver light directly to target areas. To develop this solution, fiber optic microneedles capable of delivering light for either imaging or therapy were manufactured by tapering step-index silica-based optical fibers employing a melt-drawing process. Some of the microneedles were manufactured to have sharper tips by changing the heat source during the melt-drawing process. All of the microneedles were individually inserted into ex vivo pig skin samples to demonstrate the feasibility of their application in human tissues. The force on each microneedle was measured during insertion in order to determine the effects of sharper tips on the peak force and the steadiness of the increase in force. Skin penetration experiments showed that sharp fiber optic microneedles that are 3 mm long penetrate through 2 mm of ex vivo pig skin specimens. These sharp microneedles had a minimum average diameter of 73 mum and a maximum tip diameter of 8 mum. Flat microneedles, which had larger tip diameters, required a minimum average diameter of 125 mum in order to penetrate through pig skin samples. Force versus displacement plots showed that a sharp tip on a fiber optic microneedle decreased the skin's resistance during insertion. Also, the force acting on a sharp microneedle increased more steadily compared with a microneedle with a flat tip. However, many of the sharp microneedles sustained damage during skin penetration. Two designs that did not accrue damage were identified and will provide a basis of more robust microneedles. Developing resilient microneedles with smaller diameters will lead to transformative, novel modes of transdermal imaging and treatment that are less invasive and less painful for the patient.

  17. Hepatic ablation with multiple interstitial ultrasound applicators: initial ex vivo and computational studies

    Science.gov (United States)

    Prakash, Punit; Salgaonkar, Vasant A.; Burdette, E. Clif; Diederich, Chris J.

    2011-03-01

    Radiofrequency (RF) ablation has emerged as an effective method for treating liver tumors under 3 cm in diameter. Multiple applicator devices and techniques - using RF, microwave and other modalities - are under development for thermal ablation of large and irregularly-shaped liver tumors. Interstitial ultrasound (IUS) applicators, comprised of linear arrays of independently powered tubular transducers, enable 3D control of the spatial power deposition profile and simultaneous ablation with multiple applicators. We evaluated IUS applicator configurations (parallel, converging and diverging implants) suitable for percutaneous and laparascopic placement with experiments in ex vivo bovine tissue and computational models. Ex vivo ablation zones measured 4.6+/-0.5 x 4.2+/-0.5 × 3.3+/-0.5 cm3 and 5.6+/-0.5 × 4.9+/-0.5 x 2.8+/-0.3 cm3 using three parallel applicators spaced 2 and 3 cm apart, respectively, and 4.0+/-0.3 × 3.2+/-0.4 × 2.9+/-0.2 cm3 using two parallel applicators spaced 2 cm apart. Computational models indicate in vivo ablation zones up to 4.5 × 4.4 × 5.5 cm3 and 5.7 × 4.8 × 5.2 cm3, using three applicators spaced 2 and 3 cm apart, respectively. Converging and diverging implant patterns can also be employed for conformal ablation of irregularly-shaped tumor margins by tailoring power levels along each device. Simultaneously powered interstitial ultrasound devices can create tailored ablation zones comparable to currently available RF devices and similarly sized microwave antennas.

  18. Characterization and ex vivo Expansion of Human Placenta-Derived Natural Killer Cells for Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Xiaokui eZhang

    2013-05-01

    Full Text Available Recent clinical studies suggest that adoptive transfer of donor-derived natural killer (NK cells may improve clinical outcome in hematological malignancies and some solid tumors by direct antitumor effects as well as by reduction of graft versus host disease (GVHD. NK cells have also been shown to enhance transplant engraftment during allogeneic hematopoietic stem cell transplantation (HSCT for hematological malignancies. The limited ex vivo expansion potential of NK cells from peripheral blood (PB or umbilical cord blood (UCB has however restricted their therapeutic potential. Here we define methods to efficiently generate NK cells from donor matched, full-term human placenta perfusate (termed Human Placenta-Derived Stem Cell, HPDSC and UCB. Following isolation from cryopreserved donor-matched HPDSC and UCB units, CD56+CD3- placenta-derived NK cells, termed pNK cells, were expanded in culture for up to 3 weeks to yield an average of 1.2 billion cells per donor that were >80% CD56+CD3-, comparable to doses previously utilized in clinical applications. Ex vivo-expanded pNK cells exhibited a marked increase in anti-tumor cytolytic activity coinciding with the significantly increased expression of NKG2D, NKp46 and NKp44 (p < 0.001, p < 0.001, and p < 0.05, respectively. Strong cytolytic activity was observed against a wide range of tumor cell lines in vitro. pNK cells display a distinct microRNA (miRNA expression profile, immunophenotype and greater antitumor capacity in vitro compared to PB NK cells used in recent clinical trials. With further development, pNK may represent a novel and effective cellular immunotherapy for patients with high clinical needs and few other therapeutic options.

  19. An "ex vivo model" contributing to the diagnosis and evaluation of new drugs in cystic fibrosis.

    Science.gov (United States)

    Di Lullo, A M; Scorza, M; Amato, F; Comegna, M; Raia, V; Maiuri, L; Ilardi, G; Cantone, E; Castaldo, G; Iengo, M

    2016-11-29

    Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. About 2000 mutations have been described so far. We setup an ex vivo model of human nasal epithelial cells (HNECs) to study CF patients testing the effect of novel mutations and molecular therapies. We performed the sampling (by brushing), followed by culture and analysis of HNECs using a series of molecular techniques. We performed 50 brushings from CF patients and controls. Using cultured cells, we: i) demonstrated the widely heterogeneous CFTR expression in patients and in controls; ii) defined the splicing effect of a CFTR mutation; iii) assessed the CFTR gating activity in patients bearing different mutations; iv) demonstrated that butyrate significantly enhances CFTR expression. Based on our data, we can conclude: 1) HNEC brushing is performed without anaesthesia and is well tolerated in all CF patients (children and adults); 2) HNECs can be preserved for up to 48 hours before culture allowings multicenter studies; 3) HNECs culture can be considered a suitable model to study the molecular effects of new CFTR gene mutations and/or uncertain meaning specific mutations of carriers; 4) an ex vivo model of HNECs may be used to evaluate, before human use, the effect of new drugs on patients' cells bearing specific CFTR mutations; 5) the methodology is adequate for a quantitative measurement, by fluorescence, of the CFTR gating activity of the HNECs from patients with different genotypes identifying: a) CF patients bearing two severe mutations with an activity < 10% (compared to controls - 100%); b) CF patients bearing at least a mild mutation with an activity of 10-20%; c) CF carriers (heterozygous subjects) with an activity between 40-70%.

  20. 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.

  1. A numerical analysis model for interpretation of flow cytometric studies of ex vivo phagocytosis.

    Directory of Open Access Journals (Sweden)

    Ted S Strom

    Full Text Available The study of ex vivo phagocytosis via flow cytometry requires that one distinguish experimentally between uptake and adsorption of fluorescently labeled targets by phagocytes. Removal of the latter quantity from the analysis is the most common means of analyzing such data. Because the probability of phagocytosis is a function of the probability of adsorption, and because partially quenched fluorescence after uptake often overlaps with that of negative controls, this approach is suboptimal at best. Here, we describe a numerical analysis model which overcomes these limitations. We posit that the random adsorption of targets to macrophages, and subsequent phagocytosis, is a function of three parameters: the ratio of targets to macrophages (m, the mean fluorescence intensity imparted to the phagocyte by the internalized target (alpha, and the probability of phagocytosis per adsorbed target (p. The potential values of these parameters define a parameter space and their values at any point in parameter space can be used to predict the fraction of adsorption(+ and [adsorption(-, phagocytosis(+] cells that might be observed experimentally. By systematically evaluating the points in parameter space for the latter two values and comparing them to experimental data, the model arrives at sets of parameter values that optimally predict such data. Using activated THP-1 cells as macrophages and platelets as targets, we validate the model by demonstrating that it can distinguish between the effects of experimental changes in m, alpha, and p. Finally, we use the model to demonstrate that platelets from a congenitally thrombocytopenic WAS patient show an increased probability of ex vivo phagocytosis. This finding correlates with other evidence that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. Our numerical analysis method represents a useful and innovative approach to multivariate analysis.

  2. 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.

  3. Cytokine combinations on the potential for ex vivo expansion of murine hematopoietic stem cells.

    Science.gov (United States)

    Lui, Wing Chi; Chan, Yuen Fan; Chan, Li Chong; Ng, Ray Kit

    2014-08-01

    Hematopoietic stem cell (HSC) is a rare cell population, which is capable of self-renewal and differentiation to all blood lineages. The clinical potential of HSCs for treating hematological disorders has led to the use of cytokine stimulation for ex vivo expansion. However, little is known about the molecular features of the HSC populations expanded under different cytokine combinations. We studied the expansion of murine HSCs cultured with six different cytokine combinations under serum-containing or serum-free conditions for 14days. We found that all the cytokine combinations promoted expansion of murine HSCs. Although SCF/IL-3/IL-6 induced the highest expansion of the immunophenotypic Lineage(-)Sca-1(+)c-Kit(+) (LSK) cells at day 14, over 90% of them were FcεRIα(+) mast cells. In contrast, the serum-free medium with SCF/Flt3-L/IL-11 effectively promoted the expansion of LSK/FcεRIα(-) HSCs by over 50-fold. HSCs expanded by SCF/Flt3-L/IL-11 combination formed compact hematopoietic colonies and demonstrated a higher degree of multipotency compared to the HSCs cultured with other cytokine combinations. Surprisingly, despite the same LSK/FcεRIα(-) immunophenotype, HSCs cultured with different cytokine combinations demonstrated differential patterns of hematopoietic gene expression. HSCs cultured with SCF/Flt3-L/IL-11 maintained a transcription profile resembling that of freshly isolated HSCs. We propose that serum-free medium supplemented with SCF/Flt3-L/IL-11 is the optimal culture condition to maintain the stemness of ex vivo expanded HSCs. This study used molecular characterization of cytokine-expanded murine HSCs to facilitate the selection of cytokine combinations that could induce fully competent HSC for clinical applications.

  4. Rapid evaluation of fresh ex vivo kidney tissue with full-field optical coherence tomography

    Directory of Open Access Journals (Sweden)

    Manu Jain

    2015-01-01

    Full Text Available Background: Full-field optical coherence tomography (FFOCT is a real-time imaging technique that rapidly generates images reminiscent of histology without any tissue processing, warranting its exploration for evaluation of ex vivo kidney tissue. Methods: Fresh tissue sections from tumor and adjacent nonneoplastic kidney (n = 25 nephrectomy specimens; clear cell renal cell carcinoma (CCRCC = 12, papillary RCC (PRCC = 4, chromophobe RCC (ChRCC = 4, papillary urothelial carcinoma (PUC = 1, angiomyolipoma (AML = 2 and cystic nephroma = 2 were imaged with a commercial FFOCT device. Sections were submitted for routine histopathological diagnosis. Results: Glomeruli, tubules, interstitium, and blood vessels were identified in nonneoplastic tissue. In tumor sections, the normal architecture was completely replaced by either sheets of cells/trabeculae or papillary structures. The former pattern was seen predominantly in CCRCC/ChRCC and the latter in PRCC/PUC (as confirmed on H&E. Although the cellular details were not very prominent at this resolution, we could identify unique cytoplasmic signatures in some kidney tumors. For example, the hyper-intense punctate signal in the cytoplasm of CRCC represents glycogen/lipid, large cells with abundant hyper-intense cytoplasm representing histiocytes in PRCC, and signal-void large polygonal cell representing adipocytes in AML. According to a blinded analysis was performed by an uropathologist, all nonneoplastic tissues were differentiated from neoplastic tissues. Further, all benign tumors were called benign and malignant were called malignant. A diagnostic accuracy of 80% was obtained in subtyping the tumors. Conclusion: The ability of FFOCT to reliably differentiate nonneoplastic from neoplastic tissue and identify some tumor types makes it a valuable tool for rapid evaluation of ex vivo kidney tissue e.g. for intraoperative margin assessment and kidney biopsy adequacy. Recently, higher resolution images were

  5. 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.

  6. P-gp activity and inhibition in the different regions of human intestine ex vivo.

    Science.gov (United States)

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

    2017-03-01

    Although intestinal P-glycoprotein (P-gp) has been extensively studied in vitro and in animals, its activity and the consequences of P-gp inhibition for drug disposition and toxicity in humans are still difficult to accurately extrapolate from these studies. Moreover, existing in vitro models do not take into consideration that the intestine is heterogeneous with respect to P-gp expression. Recently, we reported rat precision-cut intestinal slices (PCIS) as a physiological ex vivo model to study the regional gradient of P-gp activity and inhibition. Here we extended the application of PCIS to the human intestine. For this purpose rhodamine 123 (R123) accumulation in the presence or absence of the P-gp inhibitors verapamil, cyclosporine A, quinidine, ketoconazole, PSC833 and CP100356 was measured in PCIS of human duodenum, jejunum, ileum and colon. R123 accumulation in the presence of the P-gp inhibitors appeared to be most enhanced in the ileum compared to the other regions. Moreover, the regional differences in accumulation are in line with published differences in abundance of P-gp. The rank order of the potency of the P-gp inhibitors, reflected by their IC50 , was comparable to that in rat PCIS. However, the increase in accumulation of the P-gp substrate R123 by the inhibitors was larger in human ileum PCIS than in rat PCIS, indicating species difference in P-gp abundance. These data show that human PCIS are an appropriate ex vivo model to study the activity of intestinal P-gp and predict the inhibitory effect of drugs and of transporter-mediated drug-drug interactions in the human intestine. Copyright © 2016 John Wiley & Sons, Ltd.

  7. Gene transfer of integration defective anti-HSV-1 meganuclease to human corneas ex vivo.

    Science.gov (United States)

    Elbadawy, H M; Gailledrat, M; Desseaux, C; Salvalaio, G; Di Iorio, E; Ferrari, B; Bertolin, M; Barbaro, V; Parekh, M; Gayon, R; Munegato, D; Franchin, E; Calistri, A; Palù, G; Parolin, C; Ponzin, D; Ferrari, S

    2014-03-01

    Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.

  8. Tropism and innate host responses of a novel avian influenza A H7N9 virus: an analysis of ex-vivo and in-vitro cultures of the human respiratory tract

    OpenAIRE

    Chan, LY; Fong, JHM; Tao, KP; Chan, MCW; Chan, WY; Mok, KP; Poon, LLM; Nicholls, JM; Guan, Y.; Peiris, JSM; Hui, PY

    2013-01-01

    BACKGROUND: Since March, 2013, an avian-origin influenza A H7N9 virus has caused severe pneumonia in China. The aim of this study was to investigate the pathogenesis of this new virus in human beings. METHODS: We obtained ex-vivo cultures of the human bronchus, lung, nasopharynx, and tonsil and in-vitro cultures of primary human alveolar epithelial cells and peripheral blood monocyte-derived macrophages. We compared virus tropism and induction of proinflammatory cytokine responses ...

  9. 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...

  10. 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...

  11. Alterations in creatine metabolism observed in experimental autoimmune myocarditis using ex vivo proton magic angle spinning MRS

    DEFF Research Database (Denmark)

    Muench, Frédéric; Retel, Joren; Jeuthe, Sarah

    2015-01-01

    of male young Lewis rats with EAM was quantified by performing left ventricular ejection fraction (LVEF) analysis in short-axis cine images throughout the whole heart. Inflammatory cellular infiltrate was assessed by immunohistochemistry. Myocardial tissue was analyzed using ex vivo proton magic angle...

  12. Comparative ex vivo study on humidifying function of three speaking valves with integrated heat and moisture exchanger for tracheotomised patients

    NARCIS (Netherlands)

    van den Boer, C.; Lansaat, L.; Muller, S.H.; van den Brekel, M.W.M.; Hilgers, F.J.M.

    2015-01-01

    Objective Assessment of humidifying function of tracheotomy speaking valves with integrated heat and moisture exchanger. Design Ex vivo measurement of water exchange and storage capacity of three tracheotomy speaking valves: Humidiphon Plus, Spiro and ProTrach DualCare (with two different heat and m

  13. 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...

  14. Polarimetry based partial least square classification of ex vivo healthy and basal cell carcinoma human skin tissues.

    Science.gov (United States)

    Ahmad, Iftikhar; Ahmad, Manzoor; Khan, Karim; Ikram, Masroor

    2016-06-01

    Optical polarimetry was employed for assessment of ex vivo healthy and basal cell carcinoma (BCC) tissue samples from human skin. Polarimetric analyses revealed that depolarization and retardance for healthy tissue group were significantly higher (pleast square (PLS) regression classified the samples with 100% accuracy, sensitivity and specificity. These findings indicate that optical polarimetry together with PLS statistics hold promise for automated pathology classification.

  15. 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

    [³H]CUMI-101 is a 5-HT(1A) partial agonist, which has been evaluated for use as a positron emission tracer in baboon and humans. We sought to evaluate the properties of [³H]CUMI-101 ex vivo in awake rats and determine if [³H]CUMI-101 can measure changes in synaptic levels of serotonin after...

  16. 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

  17. 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...

  18. 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...

  19. 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...

  20. A novel ex vivo rat infection model to study protective immunity against Fasciola hepatica at the gut level

    NARCIS (Netherlands)

    Milligen, van F.J.; Cornelissen, J.B.W.J.; Gaasenbeek, C.P.H.; Bokhout, B.A.

    1998-01-01

    We describe an ex vivo rat infection model to study protective immunity against Fasciola hepatica at the gut level. An exact number of newly excysted juveniles (NEJs) was injected into a gut segment with an intact blood supply and which was still attached to a live anaesthetized rat. NEJs that penet

  1. 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

  2. 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.

  3. Control of circumferential wall stress and luminal shear stress within intact vascular segments perfused ex vivo.

    Science.gov (United States)

    El-Kurdi, Mohammed S; Vipperman, Jeffrey S; Vorp, David A

    2008-10-01

    Proportional, integral, and derivative (PID) controllers have proven to be robust in controlling many applications, and remain the most widely used control system architecture. The purpose of this work was to use this architecture for designing and tuning two PID controllers. The first was used to control the physiologic arterial circumferential wall stress (CWS) and the second to control the physiologic arterial shear stress (SS) imposed on intact vascular segments that were implanted into an ex vivo vascular perfusion system (EVPS). In order to most accurately control the stresses imposed onto vascular segments perfused ex vivo, analytical models were derived to calculate the CWS and SS. The mid-vein-wall CWS was calculated using the classical Lame solution for thick-walled cylinders in combination with the intraluminal pressure and outer diameter measurements. Similarly, the SS was calculated using the Hagen-Poiseuille equation in combination with the flow rate and outer diameter measurements. Performance of each controller was assessed by calculating the root mean square of the error (RMSE) between the desired and measured process variables. The performance experiments were repeated ten times (N=10) and an average RMSE was reported for each controller. RMSE standard deviations were calculated to demonstrate the reproducibility of the results. Sterile methods were utilized for making blood gas and temperature measurements in order to maintain physiologic levels within the EVPS. Physiologic blood gases (pH, pO(2), and pCO(2)) and temperature within the EVPS were very stable and controlled manually. Blood gas and temperature levels were recorded hourly for several (N=9) 24 h perfusion experiments. RMSE values for CWS control (0.427+/-0.027 KPa) indicated that the system was able to generate a physiologic CWS wave form within 0.5% error of the peak desired CWS over each cardiac cycle. RMSE values for SS control (0.005+/-0.0007 dynescm(2)) indicated that the system

  4. Ex vivo piperaquine resistance developed rapidly in Plasmodium falciparum isolates in northern Cambodia compared to Thailand

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    Suwanna Chaorattanakawee

    2016-10-01

    Full Text Available Abstract Background The recent dramatic decline in dihydroartemisinin-piperaquine (DHA-PPQ efficacy in northwestern Cambodia has raised concerns about the rapid spread of piperaquine resistance just as DHA-PPQ is being introduced as first-line therapy in neighbouring countries. Methods Ex vivo parasite susceptibilities were tracked to determine the rate of progression of DHA, PPQ and mefloquine (MQ resistance from sentinel sites on the Thai–Cambodian and Thai–Myanmar borders from 2010 to 2015. Immediate ex vivo (IEV histidine-rich protein 2 (HRP-2 assays were used on fresh patient Plasmodium falciparum isolates to determine drug susceptibility profiles. Results IEV HRP-2 assays detected the precipitous emergence of PPQ resistance in Cambodia beginning in 2013 when 40 % of isolates had an IC90 greater than the upper limit of prior years, and this rate doubled to 80 % by 2015. In contrast, Thai–Myanmar isolates from 2013 to 14 remained PPQ-sensitive, while northeastern Thai isolates appeared to have an intermediate resistance profile. The opposite trend was observed for MQ where Cambodian isolates appeared to have a modest increase in overall sensitivity during the same period, with IC50 declining to median levels comparable to those found in Thailand. A significant association between increased PPQ IC50 and IC90 among Cambodian isolates with DHA-PPQ treatment failure was observed. Nearly all Cambodian and Thai isolates were deemed artemisinin resistant with a >1 % survival rate for DHA in the ring-stage assay (RSA, though there was no correlation among isolates to indicate cross-resistance between PPQ and artemisinins. Conclusions Clinical DHA-PPQ failures appear to be associated with declines in the long-acting partner drug PPQ, though sensitivity appears to remain largely intact for now in western Thailand. Rapid progression of PPQ resistance associated with DHA-PPQ treatment failures in northern Cambodia limits drugs of choice in

  5. Ex-vivo assessment of chronic toxicity of low levels of cadmium on testicular meiotic cells

    Energy Technology Data Exchange (ETDEWEB)

    Geoffroy-Siraudin, Cendrine [Aix-Marseille Univ, UMR CNRS IMBE 7263, FR 3098 ECCOREV, 13005, Marseille (France); Laboratoire de Biologie de la Reproduction, AP-HM, Hôpital de la Conception, 147, Boulevard Baille, 13385 Marseille cedex 5 (France); Perrard, Marie-Hélène [Institut de Génomique Fonctionnelle de Lyon, UMR 5242 CNRS INRA Ecole Normale Supérieure de Lyon 1, 46 allée d' Italie, F-69364 Lyon Cedex 07 (France); Ghalamoun-Slaimi, Rahma [Aix-Marseille Univ, UMR CNRS IMBE 7263, FR 3098 ECCOREV, 13005, Marseille (France); Laboratoire de Biologie de la Reproduction, AP-HM, Hôpital de la Conception, 147, Boulevard Baille, 13385 Marseille cedex 5 (France); Ali, Sazan [Aix-Marseille Univ, UMR CNRS IMBE 7263, FR 3098 ECCOREV, 13005, Marseille (France); Chaspoul, Florence [Aix-Marseille Univ, UMR CNRS IMBE 7263, FR 3098 ECCOREV, 13005, Marseille (France); Unité de Chimie-Physique, Faculté de Pharmacie 13005, Marseille (France); Lanteaume, André [Aix-Marseille Univ, UMR CNRS IMBE 7263, FR 3098 ECCOREV, 13005, Marseille (France); Achard, Vincent [Laboratoire de Biologie de la Reproduction, AP-HM, Hôpital de la Conception, 147, Boulevard Baille, 13385 Marseille cedex 5 (France); Gallice, Philippe [Aix-Marseille Univ, UMR CNRS IMBE 7263, FR 3098 ECCOREV, 13005, Marseille (France); Unité de Chimie-Physique, Faculté de Pharmacie 13005, Marseille (France); Durand, Philippe [Institut de Génomique Fonctionnelle de Lyon, UMR 5242 CNRS INRA Ecole Normale Supérieure de Lyon 1, 46 allée d' Italie, F-69364 Lyon Cedex 07 (France); and others

    2012-08-01

    Using a validated model of culture of rat seminiferous tubules, we assessed the effects of 0.1, 1 and 10 μg/L cadmium (Cd) on spermatogenic cells over a 2‐week culture period. With concentrations of 1 and 10 μg/L in the culture medium, the Cd concentration in the cells, determined by ICP-MS, increased with concentration in the medium and the day of culture. Flow cytometric analysis enabled us to evaluate changes in the number of Sertoli cells and germ cells during the culture period. The number of Sertoli cells did not appear to be affected by Cd. By contrast, spermatogonia and meiotic cells were decreased by 1 and 10 μg/L Cd in a time and dose dependent manner. Stage distribution of the meiotic prophase I and qualitative study of the synaptonemal complexes (SC) at the pachytene stage were performed by immunocytochemistry with an anti SCP3 antibody. Cd caused a time-and-dose-dependent increase of total abnormalities, of fragmented SC and of asynapsis from concentration of 0.1 μg/L. Additionally, we observed a new SC abnormality, the “motheaten” SC. This abnormality is frequently associated with asynapsis and SC widening which increased with both the Cd concentration and the duration of exposure. This abnormality suggests that Cd disrupts the structure and function of proteins involved in pairing and/or meiotic recombination. These results show that Cd induces dose-and-time-dependent alterations of the meiotic process of spermatogenesis ex-vivo, and that the lowest metal concentration, which induces an adverse effect, may vary with the cell parameter studied. -- Highlights: ► Cadmium induces ex-vivo severe time- and dose-dependent germ cell abnormalities. ► Cadmium at very low concentration (0.1 µg/l) induces synaptonemal complex abnormalities. ► The lowest concentration inducing adverse effect varied with the cell parameter studied. ► Cadmium alters proteins involved in pairing and recombination. ► Cadmium leads to achiasmate univalents and

  6. Ex vivo innate immune cytokine signature of enhanced risk of relapsing brucellosis.

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    Kristyn E Feldman

    Full Text Available Brucellosis, a zoonotic infection caused by one of the Gram-negative intracellular bacteria of the Brucella genus, is an ongoing public health problem in Perú. While most patients who receive standard antibiotic treatment recover, 5-40% suffer a brucellosis relapse. In this study, we examined the ex vivo immune cytokine profiles of recovered patients with a history of acute and relapsing brucellosis.Blood was taken from healthy control donors, patients with a history of acute brucellosis, or patients with a history of relapsing brucellosis. Peripheral blood mononuclear cells were isolated and remained in culture without stimulation or were stimulated with a panel of toll-like receptor agonists or heat-killed Brucella melitensis (HKBM isolates. Innate immune cytokine gene expression and protein secretion were measured by quantitative real-time polymerase chain reaction and a multiplex bead-based immunoassay, respectively. Acute and relapse patients demonstrated consistently elevated cytokine gene expression and secretion levels compared to controls. Notably, these include: basal and stimulus-induced expression of GM-CSF, TNF-α, and IFN-γ in response to LPS and HKBM; basal secretion of IL-6, IL-8, and TNF-α; and HKBM or Rev1-induced secretion of IL-1β, IL-2, GM-CSF, IFN-Υ, and TNF-α. Although acute and relapse patients were largely indistinguishable by their cytokine gene expression profiles, we identified a robust cytokine secretion signature that accurately discriminates acute from relapse patients. This signature consists of basal IL-6 secretion, IL-1β, IL-2, and TNF-α secretion in response to LPS and HKBM, and IFN-γ secretion in response to HKBM.This work demonstrates that informative cytokine variations in brucellosis patients can be detected using an ex vivo assay system and used to identify patients with differing infection histories. Targeted diagnosis of this signature may allow for better follow-up care of brucellosis patients

  7. 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

  8. Ex vivo culture of Drosophila pupal testis and single male germ-line cysts: dissection, imaging, and pharmacological treatment.

    Science.gov (United States)

    Gärtner, Stefanie M K; Rathke, Christina; Renkawitz-Pohl, Renate; Awe, Stephan

    2014-09-11

    During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin-the histone-to-protamine switch. Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages. The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

  9. 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.

  10. 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.

  11. Radiolabeled (111)In-FGF-2 Is Suitable for In Vitro/Ex Vivo Evaluations and In Vivo Imaging.

    Science.gov (United States)

    Moscaroli, Alessandra; Jones, Gabriel; Lühmann, Tessa; Meinel, Lorenz; Wälti, Stephanie; Blanc, Alain; Fischer, Eliane; Hilbert, Manuel; Schibli, Roger; Béhé, Martin

    2017-03-06

    Fibroblast growth factor-2 (FGF-2) is a potent modulator of cell growth and regulation, with improper FGF-2 signaling being involved in impaired responses to injury or even cancer. Therefore, the exploitation of FGF-2 as a therapeutic drives the prerequisite for effective insight into drug disposition kinetics. In this article, we present an (111)In-radiolabeled FGF-2 derivative for noninvasive imaging in small animals deploying single photon emission tomography (SPECT). (111)In-FGF-2 is equally well suitable for in vitro and ex vivo investigations as (125)I-FGF-2. Furthermore, (111)In-FGF-2 permits the performance of in vivo imaging, for example for the analysis of FGF-2 containing pharmaceutical formulations in developmental or preclinical stages. (111)In-FGF-2 had affinity for the low-molecular-weight heparin enoxaparin identical to that of unlabeled FGF-2 (Kd: 0.6 ± 0.07 μM and 0.33 ± 0.03 μM, respectively) as assessed by isothermal titration calorimetry. The binding of (111)In-FGF-2 to heparan sulfate proteoglycans (HPSGs) and the biological activity were comparable to those of unlabeled FGF-2, with EC50 values of 12 ± 2 pM and 25 ± 6 pM, respectively. In vivo biodistribution in healthy nude mice indicated a predominant accumulation of (111)In-FGF-2 in filtering organs and minor uptake in the retina and the salivary and pituitary glands, which was confirmed by SPECT imaging. Therefore, (111)In-FGF-2 is a valid tracer for future noninvasive animal imaging of FGF-2 in pharmaceutical development.

  12. Zinc/copper imbalance reflects immune dysfunction in human leishmaniasis: an ex vivo and in vitro study

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    Carvalho Edgar M

    2004-11-01

    Full Text Available Abstract Background The process of elimination of intracellular pathogens, such as Leishmania, requires a Th1 type immune response, whereas a dominant Th2 response leads to exacerbated disease. Experimental human zinc deficiency decreases Th1 but not Th2 immune response. We investigated if zinc and copper levels differ in different clinical forms of leishmaniasis, and if these trace metals might be involved in the immune response towards the parasite. Methods Blood was collected from 31 patients with either localized cutaneous (LCL, mucosal (ML or visceral (VL leishmaniasis, as well as from 25 controls from endemic and non-endemic areas. Anti-Leishmania humoral and cellular immune response were evaluated by quantifying specific plasma IgG, lymphoproliferation and cytokine production, respectively. Plasma levels of Cu and Zn were quantified by atomic absorption spectrophotometry. Results A significant decrease in plasma Zn was observed in all three patient groups (p Leishmania IgG (Spearman r = 0.65, p = 0.0028. Cu/Zn ratios were highest in patients with deficient cellular (VLLCL>ML immune response. Ex vivo production of parasite-induced IFN-γ was negatively correlated to plasma Cu levels in LCL (r = -0.57, p = 0.01. In vitro, increased Cu levels inhibited IFN-γ production. Conclusions 1. Zn deficiency in VL and ML indicate possible therapeutic administration of Zn in these severe forms of leishmaniasis. 2. Plasma Cu positively correlates to humoral immune response across patient groups. 3. Environmentally or genetically determined increases in Cu levels might augment susceptibility to infection with intracellular pathogens, by causing a decrease in IFN-γ production.

  13. Ex vivo generation of interstitial and Langerhans cell-like dendritic cell subset-based vaccines for hematological malignancies.

    Science.gov (United States)

    Hutten, Tim; Thordardottir, Soley; Hobo, Willemijn; Hübel, Jessica; van der Waart, Anniek B; Cany, Jeannette; Dolstra, Harry; Hangalapura, Basav N

    2014-06-01

    Autologous, patient-specific, monocyte-derived dendritic cell (MoDC) vaccines have been successfully applied in the clinical studies so far. However, the routine application of this strategy has been hampered by the difficulties in generating sufficient numbers of DC and the poor DC vaccine quality because of pathology or prior treatment received by the patients. The immunotherapeutic potential of other subsets of DC has not been thoroughly investigated because of their rarity in tissues and difficulties associated with their ex vivo generation. The high expansion and differentiation potential of CD34 hematopoietic progenitor cells (HPC), isolated from umbilical cord blood (UCB), into different DC subsets make them an attractive alternative DC source for cancer immunotherapy. Therefore, the aim of this study was to generate a large number of different DC subsets from CD34 HPC and evaluate their functionality in comparison with MoDC. Our culture protocol generated a clinically relevant number of mature CD1a myeloid DC and CD207 Langerhans cells (LC)-like DC subsets from CD34 HPC with >95% purity. Both DC subsets exhibited a cytokine profile that favors cytotoxic T-cell responses. Furthermore, UCB-DC and UCB-LC demonstrated superior induction of proliferation of both allogeneic as well as viral antigen-specific CD8 T cells, both in vitro and in vivo. Additional studies revealed that UCC-DC and UCB-LC can efficiently expand minor histocompatibility antigen (MiHA) HA-1-specific cytotoxic T cells in the peripheral blood of leukemia patients and prime MiHA HA-1-specific and HA-2-specific cytotoxic T cells in vitro. These preclinical findings support the pharmaceutical development of the described culture protocol for clinical evaluation.

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

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    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.

  15. 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.

  16. Subnormothermic ex vivo liver perfusion is a safe alternative to cold static storage for preserving standard criteria grafts.

    Science.gov (United States)

    Spetzler, Vinzent N; Goldaracena, Nicolas; Echiverri, Juan; Kaths, J Moritz; Louis, Kristine S; Adeyi, Oyedele A; Yip, Paul M; Grant, David R; Selzner, Nazia; Selzner, Markus

    2016-01-01

    We developed a novel technique of subnormothermic ex vivo liver perfusion (SNEVLP) for the storage of liver grafts before transplantation. To test the safety of SNEVLP for the nonextended criteria grafts (standard grafts), we compared it to a control group with minimal cold static storage (CS) time. Heart-beating pig liver retrieval was performed. Grafts were either stored in cold unmodified University of Wisconsin solution (CS-1), in cold University of Wisconsin solution with ex vivo perfusion additives (CS-2), or preserved with a sequence of 3 hours CS and 3 hours SNEVLP (33°C), followed by orthotopic liver transplantation. Liver function tests and histology were investigated. Aspartate aminotransferase (AST) levels during SNEVLP remained stable (54.3 ± 12.6 U/L at 1 hour to 47.0 ± 31.9 U/L at 3 hours). Posttransplantation, SNEVLP versus CS-1 livers had decreased AST levels (peak at day 1, 1081.9 ± 788.5 versus 1546.7 ± 509.3 U/L; P = 0.14; at day 2, 316.7 ± 188.1 versus 948.2 ± 740.9 U/L; P = 0.04) and alkaline phosphatase levels (peak at day 1, 150.4 ± 19.3 versus 203.7 ± 33.6 U/L; P = 0.003). Bilirubin levels were constantly within the physiological range in the SNEVLP group, whereas the CS-1 group presented a large standard deviation, including pathologically increased values. Hyaluronic acid as a marker of endothelial cell (EC) function was markedly improved by SNEVLP during the early posttransplant phase (5 hours posttransplant, 1172.75 ± 598.5 versus 5540.5 ± 2755.4 ng/mL). Peak international normalized ratio was similar between SNEVLP and CS-1 groups after transplantation. Immunohistochemistry for cleaved caspase 3 demonstrated more apoptotic sinusoidal cells in the CS-1 group when compared to SNEVLP grafts 2 hours after reperfusion (19.4 ± 19.5 versus 133.2 ± 48.8 cells/high-power field; P = 0.002). Adding normothermic CS-2 had no impact on liver injury or function after

  17. 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.

  18. 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.

  19. Telomere Attrition Occurs during Ex Vivo Expansion of Human Dental Pulp Stem Cells

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    Jaroslav Mokry

    2010-01-01

    Full Text Available We provide a detailed characteristic of stem cells isolated and expanded from the human dental pulp. Dental pulp stem cells express mesenchymal cell markers STRO-1, vimentin, CD29, CD44, CD73, CD90, CD166, and stem cell markers Sox2, nestin, and nucleostemin. They are multipotent as shown by their osteogenic and chondrogenic potential. We measured relative telomere length in 11 dental pulp stem cell lines at different passages by quantitative real-time PCR. Despite their large proliferative capacity, stable viability, phenotype, and genotype over prolonged cultivation, human dental pulp stem cells suffer from progressive telomere shortening over time they replicate in vitro. Relative telomere length (T/S was inversely correlated with cumulative doubling time. Our findings indicate that excessive ex vivo expansion of adult stem cells should be reduced at minimum to avoid detrimental effects on telomere maintenance and measurement of telomere length should become a standard when certificating the status and replicative age of stem cells prior therapeutic applications.

  20. Compatibility of temporary pacemaker myocardial pacing leads with magnetic resonance imaging: an ex vivo tissue study.

    Science.gov (United States)

    Pfeil, Alexander; Drobnik, Stefanie; Rzanny, Reinhard; Aboud, Anas; Böttcher, Joachim; Schmidt, Peter; Ortmann, Christian; Mall, Gita; Hekmat, Khosro; Brehm, Bernhard; Reichenbach, Juergen; Mayer, Thomas E; Wolf, Gunter; Hansch, Andreas

    2012-02-01

    The presence of temporary myocardial pacing leads is considered a safety contraindication for magnetic resonance imaging (MRI). The aim of this ex vivo tissue study was to measure the heating effects at the tip of the leads using proton magnetic resonance spectroscopy ((1)HMRS) thermometry. The tissue effects were verified by histological analyses. Pig hearts with implanted temporary pacemaker myocardial pacing leads were examined by whole-body MRI at 1.5 Tesla. The tests were performed either by a sequence with high specific absorption rate (SAR) or by standard clinical sequences with lower SAR. Temperature changes were detected via (1)HMRS thermometry, by monitoring the frequency difference between water protons and the reference signals of N-methyl protons of creatine/phosphocreatine (Cr/PCr) and trimethylamine (TMA). Histology was performed using several staining techniques. Standard low-SAR and high-SAR sequences did not cause significant temperature increases in the myocardial tissue surrounding the implanted leads. There were no histopathological signs of thermal damage around the tips of the leads in any of the hearts or in a control implanted heart not subjected to MRI. The present data suggest that temporary pacemaker myocardial pacing leads may be compatible with MR scanning at 1.5 Tesla. However, further in vivo studies and carefully monitored patient studies are needed before final safety recommendations can be made.

  1. Helicobacter pylori-induced gastric pathology: insights from in vivo and ex vivo models

    Science.gov (United States)

    Williams, Jonathan M.

    2017-01-01

    ABSTRACT Gastric colonization with Helicobacter pylori induces diverse human pathological conditions, including superficial gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma and its precursors. The treatment of these conditions often relies on the eradication of H. pylori, an intervention that is increasingly difficult to achieve and that does not prevent disease progression in some contexts. There is, therefore, a pressing need to develop new experimental models of H. pylori-associated gastric pathology to support novel drug development in this field. Here, we review the current status of in vivo and ex vivo models of gastric H. pylori colonization, and of Helicobacter-induced gastric pathology, focusing on models of gastric pathology induced by H. pylori, Helicobacter felis and Helicobacter suis in rodents and large animals. We also discuss the more recent development of gastric organoid cultures from murine and human gastric tissue, as well as from human pluripotent stem cells, and the outcomes of H. pylori infection in these systems. PMID:28151409

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

    Science.gov (United States)

    de Bekker, Charissa; Smith, Philip B; Patterson, Andrew D; Hughes, David P

    2013-01-01

    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.

  3. 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.

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

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    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.

  5. 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.

  6. Ex vivo characterization of normal and adenocarcinoma colon samples by Mueller matrix polarimetry.

    Science.gov (United States)

    Ahmad, Iftikhar; Ahmad, Manzoor; Khan, Karim; Ashraf, Sumara; Ahmad, Shakil; Ikram, Masroor

    2015-05-01

    Mueller matrix polarimetry along with polar decomposition algorithm was employed for the characterization of ex vivo normal and adenocarcinoma human colon tissues by polarized light in the visible spectral range (425-725 nm). Six derived polarization metrics [total diattenuation (DT ), retardance (RT ), depolarization(ΔT ), linear diattenuation (DL), retardance (δ), and depolarization (ΔL)] were compared for normal and adenocarcinoma colon tissue samples. The results show that all six polarimetric properties for adenocarcinoma samples were significantly higher as compared to the normal samples for all wavelengths. The Wilcoxon rank sum test illustrated that total retardance is a good candidate for the discrimination of normal and adenocarcinoma colon samples. Support vector machine classification for normal and adenocarcinoma based on the four polarization properties spectra (ΔT , ΔL, RT ,and δ) yielded 100% accuracy, sensitivity, and specificity, while both DTa nd DL showed 66.6%, 33.3%, and 83.3% accuracy, sensitivity, and specificity, respectively. The combination of polarization analysis and given classification methods provides a framework to distinguish the normal and cancerous tissues.

  7. Improved ex vivo blood compatibility of central venous catheter with noble metal alloy coating.

    Science.gov (United States)

    Vafa Homann, Manijeh; Johansson, Dorota; Wallen, Håkan; Sanchez, Javier

    2016-10-01

    Central line associated bloodstream infections (CLABSIs) are a serious cause of morbidity and mortality induced by the use of central venous catheters (CVCs). Nobel metal alloy (NMA) coating is an advanced surface modification that prevents microbial adhesion and growth on catheters and thereby reduces the risk of infection. In vitro microbiological analyses have shown up to 90% reduction in microbial adhesion on coated CVC compared to uncoated ones. This study aimed to assess the blood compatibility of NMA-coated CVC according to ISO 10993-4. Hemolysis, thrombin-antithrombin (TAT) complex, platelet counts, fibrin deposition, and C3a and SC5b-9 complement activation were analyzed in human blood exposed to the NMA-coated and control CVCs using a Chandler-loop model. NMA-coated CVC did not induce hemolysis and fell in the "nonhemolytic" category according to ASTM F756-00. Significantly lower amounts of TAT were generated and less fibrin was deposited on NMA-coated CVC than on uncoated ones. Slightly higher platelet counts and lower complement markers were observed for NMA-coated CVC compared to uncoated ones. These data suggest that the NMA-coated CVC has better ex vivo blood compatibility compared to uncoated CVC. © 2015 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1359-1365, 2016.

  8. 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.

  9. Ex vivo correlation of the permeability of metoprolol across human and porcine buccal mucosa.

    Science.gov (United States)

    Meng-Lund, Emil; Marxen, Eva; Pedersen, Anne Marie L; Müllertz, Anette; Hyrup, Birgitte; Holm, Rene; Jacobsen, Jette

    2014-07-01

    The pH partition theory proposes a correlation between fraction of unionized drug substance and permeability. The aim of this study was to compare the permeability of metoprolol and mannitol in ex vivo human and porcine buccal mucosa models at varying pH to validate whether the porcine permeability model is predictive for human buccal absorption. Human (n = 9-10) and porcine (n = 6-7) buccal mucosa were mounted in a modified Ussing chamber, and the kinetics of metoprolol and mannitol transport was assessed for a period of 5.5 h with the pH values of donor medium set at 7.4, 8.5, and 9.0. In addition, hematoxylin-eosin and Alcian blue-van Gieson were used as tissue stains to evaluate the histology and the presence of acidic polysaccharides (e.g., mucins), respectively. The permeability of metoprolol was decreased in human buccal mucosa by almost twofold when compared with porcine buccal mucosa with a positive correlation (r(2) = 0.96) between the permeability assessed in porcine and human buccal mucosa. There was no change in the degree of either epithelial swelling or desquamation when treating with the pH 9.0 donor medium for 5.5 h. These data suggest that buccal mucosa from pigs can be used to predict human buccal absorption.

  10. Generation of islet-like cells from mouse gall bladder by direct ex vivo reprogramming.

    Science.gov (United States)

    Hickey, Raymond D; Galivo, Feorillo; Schug, Jonathan; Brehm, Michael A; Haft, Annelise; Wang, Yuhan; Benedetti, Eric; Gu, Guoqiang; Magnuson, Mark A; Shultz, Leonard D; Lagasse, Eric; Greiner, Dale L; Kaestner, Klaus H; Grompe, Markus

    2013-07-01

    Cell replacement is an emerging therapy for type 1 diabetes. Pluripotent stem cells have received a lot of attention as a potential source of transplantable β-cells, but their ability to form teratomas poses significant risks. Here, we evaluated the potential of primary mouse gall bladder epithelial cells (GBCs) as targets for ex vivo genetic reprogramming to the β-cell fate. Conditions for robust expansion and genetic transduction of primary GBCs by adenoviral vectors were developed. Using a GFP reporter for insulin, conditions for reprogramming were then optimized. Global expression analysis by RNA-sequencing was used to quantitatively compare reprogrammed GBCs (rGBCs) to true β-cells, revealing both similarities and differences. Adenoviral-mediated expression of NEUROG3, Pdx1, and MafA in GBCs resulted in robust induction of pancreatic endocrine genes, including Ins1, Ins2, Neurod1, Nkx2-2 and Isl1. Furthermore, expression of GBC-specific genes was repressed, including Sox17 and Hes1. Reprogramming was also enhanced by addition of retinoic acid and inhibition of Notch signaling. Importantly, rGBCs were able to engraft long term in vivo and remained insulin-positive for 15weeks. We conclude that GBCs are a viable source for autologous cell replacement in diabetes, but that complete reprogramming will require further manipulations.

  11. 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.

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

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    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.

  13. Cell population kinetics of collagen scaffolds in ex vivo oral wound repair.

    Directory of Open Access Journals (Sweden)

    Hermann Agis

    Full Text Available Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.

  14. Hybrid System for Ex Vivo Hemorheological and Hemodynamic Analysis: A Feasibility Study.

    Science.gov (United States)

    Yeom, Eunseop; Jun Kang, Yang; Lee, Sang Joon

    2015-06-19

    Precise measurement of biophysical properties is important to understand the relation between these properties and the outbreak of cardiovascular diseases (CVDs). However, a systematic measurement for these biophysical parameters under in vivo conditions is nearly impossible because of complex vessel shape and limited practicality. In vitro measurements can provide more biophysical information, but in vitro exposure changes hemorheological properties. In this study, a hybrid system composed of an ultrasound system and microfluidic device is proposed for monitoring hemorheological and hemodynamic properties under more reasonable experimental conditions. Biophysical properties including RBC aggregation, viscosity, velocity, and pressure of blood flows are simultaneously measured under various conditions to demonstrate the feasibility and performance of this measurement system. The proposed technique is applied to a rat extracorporeal loop which connects the aorta and jugular vein directly. As a result, the proposed system is found to measure biophysical parameters reasonably without blood collection from the rat and provided more detailed information. This hybrid system, combining ultrasound imaging and microfluidic techniques to ex vivo animal models, would be useful for monitoring the variations of biophysical properties induced by chemical agents. It can be used to understand the relation between biophysical parameters and CVDs.

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

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    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.

  16. 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.

  17. Bridging the gap: functional healing of embryonic small intestine ex vivo.

    Science.gov (United States)

    Coletta, Riccardo; Roberts, Neil A; Oltrabella, Francesca; Khalil, Basem A; Morabito, Antonino; Woolf, Adrian S

    2016-02-01

    The ability to grow embryonic organs ex vivo provides an opportunity to follow their differentiation in a controlled environment, with resulting insights into normal development. Additionally, similar strategies can be used to assess effects on organogenesis of physical and chemical manipulations. This study aimed to create an organ culture model with which to test physical manipulations to enhance healing of gut segments, thus generating a single functional organ. Embryonic mouse jejunum was isolated and cut into 2-3 mm tubes, which were placed in pairs, separated by a small gap, on semi-permeable supports. Each pair was linked by a nylon suture threaded through their lumens. After 3 days in organ culture fed by defined serum-free media, the rudiments differentiated to form tubes of smooth muscle surrounding a core of rudimentary villi. Of 34 such pairs, 74% had touching and well aligned proximate ends. Of these joined structures, 80% (59% of the total pairs) had a continuous lumen, as assessed by observing the trajectories of fluorescent dextrans injected into their distal ends. Fused organ pairs formed a single functional unit, as assessed by spontaneous contraction waves propagated along their lengths. In these healed intestines, peripherin(+) neurons formed a nexus in the zone of fusion, linking the rudiment pairs. In future, this system could be used to test whether growth factors enhance fusion. Such results should in turn inform the design of novel treatments for short bowel syndrome, a potentially fatal condition with a currently limited and imperfect range of therapies.

  18. The Transcriptome of Exophiala dermatitidis during Ex-vivo Skin Model Infection

    Science.gov (United States)

    Poyntner, Caroline; Blasi, Barbara; Arcalis, Elsa; Mirastschijski, Ursula; Sterflinger, Katja; Tafer, Hakim

    2016-01-01

    The black yeast Exophiala dermatitidis is a widespread polyextremophile and human pathogen, that is found in extreme natural habitats and man-made environments such as dishwashers. It can cause various diseases ranging from phaeohyphomycosis and systemic infections, with fatality rates reaching 40%. While the number of cases in immunocompromised patients are increasing, knowledge of the infections, virulence factors and host response is still scarce. In this study, for the first time, an artificial infection of an ex-vivo skin model with Exophiala dermatitidis was monitored microscopically and transcriptomically. Results show that Exophiala dermatitidis is able to actively grow and penetrate the skin. The analysis of the genomic and RNA-sequencing data delivers a rich and complex transcriptome where circular RNAs, fusion transcripts, long non-coding RNAs and antisense transcripts are found. Changes in transcription strongly affect pathways related to nutrients acquisition, energy metabolism, cell wall, morphological switch, and known virulence factors. The L-Tyrosine melanin pathway is specifically upregulated during infection. Moreover the production of secondary metabolites, especially alkaloids, is increased. Our study is the first that gives an insight into the complexity of the transcriptome of Exophiala dermatitidis during artificial skin infections and reveals new virulence factors. PMID:27822460

  19. The transcriptome of Exophiala dermatitidis during ex-vivo kin model infection

    Directory of Open Access Journals (Sweden)

    Caroline Poyntner

    2016-10-01

    Full Text Available The black yeast Exophiala dermatitidis is a widespread polyextremophile and humanpathogen, that is found in extreme natural habitats and man-made environments suchas dishwashers. It can cause various diseases ranging from phaehyphomycosis andsystemic infections, with fatality rates reaching 40%. While the number of cases inimmunocompromised patients are increasing, knowledge of the infections, virulencefactors and host response is still scarce. In this study, for the first time, an artificialinfection of an ex-vivo skin model with Exophiala dermatitidis was monitoredmicroscopically and transcriptomically. Results show that Exophiala dermatitidis is ableto actively grow and penetrate the skin. The analysis of the genomic andRNA-sequencing data delivers a rich and complex transcriptome where circular RNAs,fusion transcripts, long non-coding RNAs and antisense transcripts are found. Changesin transcription strongly affect pathways related to nutrients acquisition, energymetabolism, cell wall, morphological switch, and known virulence factors. TheL-Tyrosine melanin pathway is specifically upregulated during infection. Moreover theproduction of secondary metabolites, especially alkaloids, is icreased. Our study is thefirst that gives an insight into the complexity of the transcriptome of Exophialadermatitidis during artificial skin infections and reveals new virulence factors.

  20. Cell population kinetics of collagen scaffolds in ex vivo oral wound repair.

    Science.gov (United States)

    Agis, Hermann; Collins, Amy; Taut, Andrei D; Jin, Qiming; Kruger, Laura; Görlach, Christoph; Giannobile, William V

    2014-01-01

    Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.

  1. Patterns of histological changes following hepatic electrolytic ablation in an ex-vivo perfused model.

    Science.gov (United States)

    Gravante, Gianpiero; Ong, Seok Ling; West, Kevin; McGregor, Angus; Maddern, Guy J; Metcalfe, Matthew S; Lloyd, David M; Dennison, Ashley R

    2012-10-01

    Electrolytic ablation (EA) destroys the liver by releasing toxic radicles and producing modifications in the local pH without increasing the tissue temperature. We assessed the histological changes produced by EA using an ex-vivo perfused model. Five porcine livers were harvested, preserved in ice and reperfused for six hours in an extracorporeal circuit using autologous normothermic blood. One hour after reperfusion EA was performed and liver biopsies collected at the end of the experiments. The main necrotic zone consisted of coagulative necrosis, sinusoidal dilatation and haemorrhage with an unusual morphological pattern. The coagulative necrosis and haemorrhage affected mainly the peripheral area of the lobule with relative sparing of the area surrounding the centrilobular vein. Contrasting with this sinusoidal dilatation appeared to be more prominent in the centrilobular area. EA produces patterns of tissue destruction that have not been observed with the more commonly used thermal techniques. Further studies should obtain more information about the influence of adjacent biliary and vascular structures so that appropriate clinical trials can be designed.

  2. 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...

  3. 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.

  4. Ex vivo imaging of early dental caries within the interproximal space

    Science.gov (United States)

    Choo-Smith, Lin-P'ing; Hewko, Mark D.; Dufour, Marc L.; Fulton, Crystal; Qiu, Pingli; Gauthier, Bruno; Padioleau, Christian; Bisaillon, Charles-Etienne; Dong, Cecilia; Cleghorn, Blaine M.; Lamouche, Guy; Sowa, Michael G.

    2009-02-01

    Optical coherence tomography (OCT) is emerging as a technology that can potentially be used for the detection and monitoring of early dental enamel caries since it can provide high-resolution depth imaging of early lesions. To date, most caries detection optical technologies are well suited for examining caries at facial, lingual, incisal and occlusal surfaces. The approximal surfaces between adjacent teeth are difficult to examine due to lack of visual access and limited space for these new caries detection tools. Using a catheter-style probe developed at the NRC-Industrial Materials Institute, the probe was inserted into the interproximal space to examine the approximal surfaces with OCT imaging at 1310 nm. The probe was rotated continuously and translated axially to generate depth images in a spiral fashion. The probe was used in a mock tooth arch model consisting of extracted human teeth mounted with dental rope wax in their anatomically correct positions. With this ex vivo model, the probe provided images of the approximal surfaces revealing morphological structural details, regions of calculus, and especially regions of early dental caries (white spot lesions). Results were compared with those obtained from OCT imaging of individual samples where the approximal surfaces of extracted teeth are accessible on a lab-bench. Issues regarding access, regions of interest, and factors to be considered in an in vivo setting will be discussed. Future studies are aimed at using the probe in vivo with patient volunteers.

  5. 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.

  6. 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.

  7. A comparative ex vivo drug permeation study of beta-blockers through porcine buccal mucosa.

    Science.gov (United States)

    Amores, Sonia; Lauroba, Jacinto; Calpena, Ana; Colom, Helena; Gimeno, Alvaro; Domenech, José

    2014-07-01

    Apparent permeability coefficients (kp) of a series of beta-blockers: acebutolol, atenolol, labetalol, metoprolol, oxprenolol and propranolol, through porcine buccal mucosa were determined. The aim of the study was to determine the permeation parameters (apparent permeability coefficient, kp; flux, J; and lag time, TL) as a measure of the intrinsic permeability of porcine buccal mucosa to these drugs, in order to predict the efficacy of their possible administration through human buccal mucosa. A positive linear correlation was observed between the apparent permeability coefficient, kpand the partition coefficient, P. Oxprenolol and propranolol are the drugs that presented the highest values of kp: 0.3231×10(2) cm/h and 0.5666×10(2) cm/h, respectively. Multiple linear regression (MLR) using least square estimation was performed on the data set with logkpas dependent variable and the descriptors as predictor variables. The potential systemic capacity after a buccal administration was predicted by estimating the plasma concentrations at steady-stated (Css). Considering the entire process of permeation ex vivo, propranolol and oxprenolol would seem to be the best candidates for administration through the buccal mucosa.

  8. Blind source separation of ex-vivo aorta tissue multispectral images.

    Science.gov (United States)

    Galeano, July; Perez, Sandra; Montoya, Yonatan; Botina, Deivid; Garzón, Johnson

    2015-05-01

    Blind Source Separation methods (BSS) aim for the decomposition of a given signal in its main components or source signals. Those techniques have been widely used in the literature for the analysis of biomedical images, in order to extract the main components of an organ or tissue under study. The analysis of skin images for the extraction of melanin and hemoglobin is an example of the use of BSS. This paper presents a proof of concept of the use of source separation of ex-vivo aorta tissue multispectral Images. The images are acquired with an interference filter-based imaging system. The images are processed by means of two algorithms: Independent Components analysis and Non-negative Matrix Factorization. In both cases, it is possible to obtain maps that quantify the concentration of the main chromophores present in aortic tissue. Also, the algorithms allow for spectral absorbance of the main tissue components. Those spectral signatures were compared against the theoretical ones by using correlation coefficients. Those coefficients report values close to 0.9, which is a good estimator of the method's performance. Also, correlation coefficients lead to the identification of the concentration maps according to the evaluated chromophore. The results suggest that Multi/hyper-spectral systems together with image processing techniques is a potential tool for the analysis of cardiovascular tissue.

  9. Mechanically loaded ex vivo bone culture system 'Zetos': Systems and culture preparation

    Directory of Open Access Journals (Sweden)

    C M Davies

    2006-04-01

    Full Text Available This paper introduces the culture preparation of ovine, bovine and human cancellous bone cores to be used in an explants model Zetos. The three dimensional (3D bone cores were prepared and evaluated for all three animals. Bone cells in vivo constantly interact with each other, migratory cells, surrounding extracellular matrix (ECM and interstitial fluid in a microenvironment, which continuously responds to various endogenous and exogenous stimuli. The Zetos system was designed to culture and mechanically load viable cancellous bone explants in their near natural microenvironment. This 3D ex vivo system bridges the current gap between in vitro and in vivo methods. One aim of this work was to compare the macro and micro-architecture of ovine, bovine and human cancellous bone tissue in preparation for culture within the Zetos system in order to determine the optimal source of experimental material. A second aim was to optimise the preparations of the bone cores as well as develop techniques involved during tissue maintenance. Bone core response was visualised using histological and immunohistochemical methods. The results demonstrate that cancellous bone explants vary greatly in trabecular density and bone volume depending on species, age and location. Sheep and human samples displayed the greatest variation between bones cores when compared to bovine. Even cores taken from the same animal possessed very different characteristics. The histology demonstrated normal bone and cell structure after the core preparation. Immunohistochemistry results demonstrated antigen retention after preparation methods.

  10. Classical and adaptive control of ex vivo skeletal muscle contractions using Functional Electrical Stimulation (FES)

    Science.gov (United States)

    Shoemaker, Adam; Grange, Robert W.; Abaid, Nicole; Leonessa, Alexander

    2017-01-01

    Functional Electrical Stimulation is a promising approach to treat patients by stimulating the peripheral nerves and their corresponding motor neurons using electrical current. This technique helps maintain muscle mass and promote blood flow in the absence of a functioning nervous system. The goal of this work is to control muscle contractions from FES via three different algorithms and assess the most appropriate controller providing effective stimulation of the muscle. An open-loop system and a closed-loop system with three types of model-free feedback controllers were assessed for tracking control of skeletal muscle contractions: a Proportional-Integral (PI) controller, a Model Reference Adaptive Control algorithm, and an Adaptive Augmented PI system. Furthermore, a mathematical model of a muscle-mass-spring system was implemented in simulation to test the open-loop case and closed-loop controllers. These simulations were carried out and then validated through experiments ex vivo. The experiments included muscle contractions following four distinct trajectories: a step, sine, ramp, and square wave. Overall, the closed-loop controllers followed the stimulation trajectories set for all the simulated and tested muscles. When comparing the experimental outcomes of each controller, we concluded that the Adaptive Augmented PI algorithm provided the best closed-loop performance for speed of convergence and disturbance rejection. PMID:28273101

  11. An ex vivo study of the correlation between acoustic emission and microvascular damage.

    Science.gov (United States)

    Samuel, Stanley; Cooper, Michol A; Bull, Joseph L; Fowlkes, J Brian; Miller, Douglas L

    2009-09-01

    The objective of this study was to conduct an ex vivo examination of correlation between acoustic emission and tissue damage. Intravital microscopy was employed in conjunction with ultrasound exposure in cremaster muscle of male Wistar rats. Definity microbubbles were administered intravenously through the tail vein (80microL.kg(-1).min(-1)infusion rate) with the aid of a syringe pump. For the pulse repetition frequency (PRF) study, exposures were performed at four locations of the cremaster at a PRF of 1000, 500, 100 and 10Hz (one location per PRF per rat). The 100-pulse exposures were implemented at a peak rarefactional pressure (P(r)) of 2MPa, frequency of 2.25MHz with 46 cycle pulses. For the pressure amplitude threshold study, 100-pulse exposures (46 cycle pulses) were conducted at various peak rarefactional pressures from 0.5MPa to 2MPa at a frequency of 2.25MHz and PRF of 100Hz. Photomicrographs were captured before and 2-min postexposure. On a pulse-to-pulse basis, the 10Hz acoustic emission was considerably higher and more sustained than those at other PRFs (1000, 500, and 100Hz) (pCRIPS; R(2)=0.75). No visible damage was present at P(r) or =1.0MPa and it increased with increasing acoustic pressure.

  12. 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.

  13. 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.

  14. [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.

  15. 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.

  16. 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

    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...... decrease. Accordingly, a 2-fold higher oxygen delivery and oxygen uptake per unit muscle mass are seen in vivo during 1-leg exercise compared to 2-leg cycling indicating a significant limitation of the circulation during exercise with a large muscle mass. This analysis also reveals that mitochondrial......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...

  17. 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.

  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. PLGA/DPPC/trimethylchitosan spray-dried microparticles for the nasal delivery of ropinirole hydrochloride: in vitro, ex vivo and cytocompatibility assessment

    Energy Technology Data Exchange (ETDEWEB)

    Karavasili, Christina [Laboratory of Pharmaceutical Technology, Department of Pharmacy, Aristotle University of Thessaloniki, 54124 (Greece); Bouropoulos, Nikolaos [Department of Materials Science, University of Patras, 26504 Rio, Patras (Greece); Foundation for Research and Technology, Hellas-Institute of Chemical Engineering and High Temperature, P.O. Box 1414, 26504 Patras (Greece); Sygellou, Lamprini [Foundation for Research and Technology, Hellas-Institute of Chemical Engineering and High Temperature, P.O. Box 1414, 26504 Patras (Greece); Amanatiadou, Elsa P.; Vizirianakis, Ioannis S. [Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, GR-54124 Thessaloniki (Greece); Fatouros, Dimitrios G., E-mail: dfatouro@pharm.auth.gr [Laboratory of Pharmaceutical Technology, Department of Pharmacy, Aristotle University of Thessaloniki, 54124 (Greece)

    2016-02-01

    In the present study we investigated polymer-lipid microparticles loaded with ropinirole hydrochloride (RH) for nasal delivery. RH microparticles were further evaluated by means of scanning electron microscopy (SEM), ζ-potential measurements, Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and x-ray diffraction (XRD). In vitro release studies were performed in simulated nasal electrolyte solution (SNES) pH 5.5 at 35 °C. Ex vivo permeation studies were conducted across sheep nasal mucosa. Cytocompatibility was tested in cultured human airway epithelial cells (Calu-3). SEM studies revealed spheroid microparticles in the range of 2.09 μm to 2.41 μm. The presence of trimethylchitosan (TMC) induced a slight shift towards less negative ζ-potential values. Surface chemistry (XPS) revealed the presence of dipalmitoylphospatidylcholine (DPPC) and poly(lactic-co-glycolic acid) (PLGA) onto microparticles' surface, further corroborating the FT-IR and XRD findings. In vitro release studies showed that the microparticle composition can partly modulate the release of RH. Ex vivo studies demonstrated a 2.35-folded enhancement of RH permeation when RH was co-formulated with TMC of low molecular weight, compared to the control. All formulations tested were found to be non-toxic to cells. The results suggest that polymer-lipid microparticles may be a promising carrier for the nasal delivery of RH. - Highlights: • Development of microparticles comprising PLGA/DPPC/TMC for nasal drug delivery. • Physicochemical characterization showed that DPPC dominated microparticles' surface. • Microparticles enhanced permeation of ropinirole across sheep nasal epithelium. • The cytotoxicity assay with Calu-3 cells demonstrated satisfactory cell viability.

  20. UV laser ablation to measure cell and tissue-generated forces in the zebrafish embryo in vivo and ex vivo.

    Science.gov (United States)

    Smutny, Michael; Behrndt, Martin; Campinho, Pedro; Ruprecht, Verena; Heisenberg, Carl-Philipp

    2015-01-01

    Mechanically coupled cells can generate forces driving cell and tissue morphogenesis during development. Visualization and measuring of these forces is of major importance to better understand the complexity of the biomechanic processes that shape cells and tissues. Here, we describe how UV laser ablation can be utilized to quantitatively assess mechanical tension in different tissues of the developing zebrafish and in cultures of primary germ layer progenitor cells ex vivo.

  1. Ex vivo cutaneous penetration of econazole nitrate from SLN incorporated in hydrophilic gels

    OpenAIRE

    Canu, Giacomina; Sanna, Vanna Annunziata; Gavini, Elisabetta; Cossu, Massimo; Rassu, Giovanna; Giunchedi, Paolo

    2006-01-01

    Solid lipid nanoparticles (SLN) are considered as innovative drug carrier for topical application. The small size of the lipid particles ensures close contact to the stratum corneum and can increase the amount of the drug penetrating into the skin and it allows controlled drug release. The aim of this study was to develop controlled release gels containing SLN dispersions loaded with Econazole Nitrate (ECN) and to evaluate ex vivo skin penetration of the drug released from t...

  2. 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.

  3. 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

  4. Anti-inflammatory activity of Punica granatum L. (Pomegranate) rind extracts applied topically to ex vivo skin.

    Science.gov (United States)

    Houston, David M J; Bugert, Joachim; Denyer, Stephen P; Heard, Charles M

    2017-03-01

    Coadministered pomegranate rind extract (PRE) and zinc (II) produces a potent virucidal activity against Herpes simplex virus (HSV); however, HSV infections are also associated with localised inflammation and pain. Here, the objective was to determine the anti-inflammatory activity and relative depth penetration of PRE, total pomegranate tannins (TPT) and zinc (II) in skin, ex vivo. PRE, TPT and ZnSO4 were dosed onto freshly excised ex vivo porcine skin mounted in Franz diffusion cells and analysed for COX-2, as a marker for modulation of the arachidonic acid inflammation pathway, by Western blotting and immunohistochemistry. Tape stripping was carried out to construct relative depth profiles. Topical application of PRE to ex vivo skin downregulated expression of COX-2, which was significant after just 6h, and maintained for up to 24h. This was achieved with intact stratum corneum, proving that punicalagin penetrated skin, further supported by the depth profiling data. When PRE and ZnSO4 were applied together, statistically equal downregulation of COX-2 was observed when compared to the application of PRE alone; no effect followed the application of ZnSO4 alone. TPT downregulated COX-2 less than PRE, indicating that tannins alone may not be entirely responsible for the anti-inflammatory activity of PRE. Punicalagin was found throughout the skin, in particular the lower regions, indicating appendageal delivery as a significant route to the viable epidermis. Topical application of TPT and PRE had significant anti-inflammatory effects in ex vivo skin, confirming that PRE penetrates the skin and modulates COX-2 regulation in the viable epidermis. Pomegranates have potential as a novel approach in ameliorating the inflammation and pain associated with a range of skin conditions, including cold sores and herpetic stromal keratitis.

  5. 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

  6. 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.

  7. In vitro and ex vivo evaluation of cyclic aminoalkyl benzilates as potential emission tomography ligands for the muscarinic receptor.

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    Otto, C A; Mulholland, G K; Perry, S E; Combs, R; Sherman, P S; Fisher, S J

    1989-01-01

    A series of muscarinic antagonists were screened as potential receptor imaging agents. (+)2 alpha-tropanyl benzilate (TRB), N-methyl-4-piperidyl benzilate (NMPB) and several analogs amenable to labeling with positron emitting isotopes were evaluated for muscarinic binding to mouse brain tissue in vitro and ex vivo using [3H]quinuclidinyl benzilate as the probe. The in vitro assay directly compared the innate binding affinities of the compounds. The rank order of binding (IC50) was TRB (0.7 nm), QNB (0.8 nm), scopolamine (1.3 nm) and NMPB (1.6 nm). The ex vivo assay was used to gain information regarding the pharmacokinetics and brain penetration of the compounds in live animals. Ex vivo results demonstrated that TRB was rapidly taken up into the brain and was equipotent with QNB in occupying muscarinic binding sites at early time points, but TRB binding decreased twice as fast over time as QNB binding. The results suggest TRB would be a good candidate for radiolabeling and further study.

  8. Ex Vivo and In Silico Feasibility Study of Monitoring Electric Field Distribution in Tissue during Electroporation Based Treatments

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    Kranjc, Matej; Bajd, Franci; Sersa, Igor; Woo, Eung Je; Miklavcic, Damijan

    2012-01-01

    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. PMID:23029212

  9. 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.

  10. 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.

  11. Analysis of Endothelial Adherence of Bartonella henselae and Acinetobacter baumannii Using a Dynamic Human Ex Vivo Infection Model.

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    Weidensdorfer, Marko; Chae, Ju Ik; Makobe, Celestine; Stahl, Julia; Averhoff, Beate; Müller, Volker; Schürmann, Christoph; Brandes, Ralf P; Wilharm, Gottfried; Ballhorn, Wibke; Christ, Sara; Linke, Dirk; Fischer, Doris; Göttig, Stephan; Kempf, Volkhard A J

    2015-12-28

    Bacterial adherence determines the virulence of many human-pathogenic bacteria. Experimental approaches elucidating this early infection event in greater detail have been performed using mainly methods of cellular microbiology. However, in vitro infections of cell monolayers reflect the in vivo situation only partially, and animal infection models are not available for many human-pathogenic bacteria. Therefore, ex vivo infection of human organs might represent an attractive method to overcome these limitations. We infected whole human umbilical cords ex vivo with Bartonella henselae or Acinetobacter baumannii under dynamic flow conditions mimicking the in vivo infection situation of human endothelium. For this purpose, methods for quantifying endothelium-adherent wild-type and trimeric autotransporter adhesin (TAA)-deficient bacteria were set up. Data revealed that (i) A. baumannii binds in a TAA-dependent manner to endothelial cells, (ii) this organ infection model led to highly reproducible adherence rates, and furthermore, (iii) this model allowed to dissect the biological function of TAAs in the natural course of human infections. These findings indicate that infection models using ex vivo human tissue samples ("organ microbiology") might be a valuable tool in analyzing bacterial pathogenicity with the capacity to replace animal infection models at least partially.

  12. How to Recondition Ex Vivo Initially Rejected Donor Lungs for Clinical Transplantation: Clinical Experience from Lund University Hospital

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    Sandra Lindstedt

    2011-01-01

    Full Text Available A major problem in clinical lung transplantation is the shortage of donor lungs. Only about 20% of donor lungs are accepted for transplantation. We have recently reported the results of the first six double lung transplantations performed with donor lungs reconditioned ex vivo that had been deemed unsuitable for transplantation by the Scandiatransplant, Eurotransplant, and UK Transplant organizations because the arterial oxygen pressure was less than 40 kPa. The three-month survival of patients undergoing transplant with these lungs was 100%. One patient died due to sepsis after 95 days, and one due to rejection after 9 months. Four recipients are still alive and well 24 months after transplantation, with no signs of bronchiolitis obliterans syndrome. The donor lungs were reconditioned ex vivo in an extracorporeal membrane oxygenation circuit using STEEN solution mixed with erythrocytes, to dehydrate edematous lung tissue. Functional evaluation was performed with deoxygenated perfusate at different inspired fractions of oxygen. The arterial oxygen pressure was significantly improved in this model. This ex vivo evaluation model is thus a valuable addition to the armamentarium in increasing the number of acceptable lungs in a donor population with inferior arterial oxygen pressure values, thereby, increasing the lung donor pool for transplantation. In the following paper we present our clinical experience from the first six patients in the world. We also present the technique we used in detail with flowchart.

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

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    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.

  14. Ex vivo stimulation of whole blood as a means to determine glucocorticoid sensitivity

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    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

  15. Ex vivo and in vivo diffusion of ropivacaine through spinal meninges: influence of absorption enhancers.

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    Brandhonneur, Nolwenn; Dollo, Gilles; Ratajczak-Enselme, Maja; Deniau, Anne Laure; Chevanne, François; Estèbe, Jean Pierre; Legrand, Alain; Le Corre, Pascal

    2011-02-14

    Following epidural administration, cerebrospinal fluid bioavailability of local anesthetics is low, one major limiting factor being diffusion across the arachnoid mater barrier. The aim of this study was to evaluate the influence of absorption enhancers on the meningeal permeability of epidurally administered ropivacaine. Five enhancers known for their ability to increase drug permeability via transcellular and/or paracellular pathways, i.e. palmitoyl carnitine, ethylenediaminetetraacetic acid, sodium caprate, dodecylphosphocholine and pentylglycerol, were tested ex vivo on fresh specimen of meninges removed from cervical to lumbar level of rabbit spine following laminectomy and placed in diffusion chambers. Among them, sodium caprate lead to the best permeability improvement for both marker and drug (440% and 112% for mannitol and ropivacaine, respectively) and was therefore selected for in vivo study in a sheep model using microdialysis technique to evaluate epidural and intrathecal ropivacaine concentrations following epidural administration. Resulting dialysate and plasma concentrations were used to calculate pharmacokinetic parameters. Following sodium caprate pre-treatment, ropivacaine intrathecal maximal concentration (Cmax) was 1.6 times higher (78 ± 16 μg ml(-1) vs 129 ± 26 μg ml(-1), p<0.05) but the influence of the absorption enhancer was only effective the first 30 min following ropivacaine injection, as seen with the significantly increase of intrathecal AUC(0-30 min) (1629 ± 437 μg min ml(-1) vs 2477 ± 559 μg min ml(-1), p<0.05) resulting in a bioavailable fraction 130% higher 30 min after ropivavaine administration. Co-administration of local anesthetics with sodium caprate seems to allow a transient and reversible improvement of transmeningeal passage into intrathecal space.

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

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    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.

  17. 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.

  18. 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.

  19. 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.

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

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    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.

  1. 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.

  2. Rapid infrared laser sealing and cutting of porcine renal vessels, ex vivo

    Science.gov (United States)

    Giglio, Nicholas C.; Hutchens, Thomas C.; Perkins, William C.; Latimer, Cassandra; Ward, Arlen; Nau, William H.; Fried, Nathaniel M.

    2014-03-01

    Suture ligation with subsequent cutting of blood vessels to maintain hemostasis during surgery is time consuming and skill intensive. Energy-based, electrosurgical and ultrasonic devices are often used to replace sutures and mechanical clips to provide rapid hemostasis, and decrease surgical time. Some of these devices may create undesirably large collateral zones of thermal damage and tissue necrosis, or require separate mechanical blades for cutting. Infrared lasers are currently being explored as alternative energy sources for vessel sealing applications. In a previous study, a 1470-nm laser was used to seal vessels of 1-6 mm in diameter in 5 s, yielding burst pressures of ~ 500 mmHg. The purpose of this study was to provide faster sealing, incorporate transection of the sealed vessels, and increase the burst pressure. A 110-Watt, 1470-nm laser beam was transmitted through a fiber and beam shaping optics, producing a linear beam 3.0 mm by 9.5 mm for sealing, and 1.1 mm by 9.6 mm for cutting (FWHM). A twostep process sealed then transected ex vivo porcine renal vessels (1-8.5 mm diameter) in a bench top setup. Seal and cut times were 1.0 s each. A standard burst pressure system measured resulting seal strength, and gross and histologic thermal damage measurements were also recorded. All blood vessels tested (n = 30) were sealed and cut, with total irradiation times of 2.0 s, mean burst pressures > 1000 mmHg (compared to normal systolic blood pressure of 120 mmHg), and combined seal/collateral thermal coagulation zones of 2-3 mm. The results of this study demonstrated that an optical-based system is capable of precisely sealing and cutting a wide range of porcine renal vessel sizes, and with further development, may provide an alternative to radiofrequency and ultrasound-based vessel sealing devices.

  3. Ozone therapy as an adjuvant for endondontic protocols: microbiological – ex vivo study and citotoxicity analyses

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    NOGALES, Carlos Goes; FERREIRA, Marina Beloti; MONTEMOR, Antonio Fernando; RODRIGUES, Maria Filomena de Andrade; Lage-MARQUES, José Luiz; ANTONIAZZI, João Humberto

    2016-01-01

    ABSTRACT Objectives This study evaluated the antimicrobial efficacy of ozone therapy in teeth contaminated with Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus using a mono-species biofilm model. Parallel to this, the study aimed to evaluate the cytotoxicity of ozone for human gingival fibroblasts. Material and Methods: One hundred and eighty single-root teeth were contaminated with a mono-species biofilm of Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus. Groups were formed: Group I – control; Group II – standard protocol; Group III – standard protocol + ozone gas at 40 µg/mL; and Group IV – standard protocol + aqueous ozone at 8 µg/mL. In parallel, human gingival fibroblasts were submitted to the MTT test. Cells were plated, then ozone was applied as follows: Group I (control) – broth medium; Group II – aqueous ozone at 2 µg/mL; Group III – aqueous ozone at 5 µg/mL; and Group IV – aqueous ozone at 8 µg/mL. Data were submitted to the Kruskal Wallis test and Bonferroni post hoc analyses to assess microbiology and cytotoxicity, respectively (p<0.05%). Results The results revealed antimicrobial efficacy by Group IV with no CFU count. The cytotoxicity assay showed Groups III and IV to be the most aggressive, providing a decrease in cell viability at hour 0 from 100% to 77.3% and 68.6%, respectively. Such a decrease in cell viability was reverted, and after 72 hours Groups III and IV provided the greatest increase in cell viability, being statistically different from Groups I and II. Conclusion According to the applied methodology and the limitations of this study, it was possible to conclude that ozone therapy improved the decontamination of the root canal ex vivo. Ozone was toxic to the cells on first contact, but cell viability was recovered. Thus, these findings suggest that ozone might be useful to improve root canal results. PMID:28076466

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Ex vivo expansion protocol for human tumor specific T cells for adoptive T cell therapy.

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    Rasmussen, Anne-Marie; Borelli, Gabriel; Hoel, Hanna Julie; Lislerud, Kari; Gaudernack, Gustav; Kvalheim, Gunnar; Aarvak, Tanja

    2010-04-15

    Adoptive T cell therapy is a promising treatment strategy for patients with different types of cancer. The methods used for generation of high numbers of tumor specific T cells usually require long-term ex vivo culture, which frequently lead to generation of terminally differentiated effector cells, demonstrating low persistence in vivo. Therefore, optimization of protocols for generation of T cells for adoptive cell therapy is warranted. The aim of this work was to develop a protocol for expansion of antigen-specific T cells using Dynabeads CD3/CD28 to obtain T cells expressing markers important for in vivo persistence and survival. To achieve high numbers of antigen-specific T cells following expansion, we have tested the effect of depleting regulatory T cells using Dynabeads CD25 and including a pre-stimulation step with peptide prior to the non-specific expansion with Dynabeads. Our data demonstrate that virus- and tumor specific T cells can be expanded to high numbers using Dynabeads CD3/CD28 following optimization of the culture conditions. The expansion protocol presented here results in enrichment of antigen-specific CD8(+) T cells with an early/intermediate memory phenotype. This is observed even when the antigen-specific CD8(+) T cells demonstrated a terminal effector phenotype prior to expansion. This protocol thus results in expanded T cells with a phenotypic profile which may increase the chance of retaining long-term persistence following adoptive transfer. Based on these data we have developed a cGMP protocol for expansion of tumor specific T cells for adoptive T cell therapy.

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

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    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.

  8. 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.

  9. Endodontic filling removal procedure: an ex vivo comparative study between two rotary techniques

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    Monica Sampaio do Vale

    2013-12-01

    Full Text Available In this study, we compared the ex vivo removal capacity of two endodontic rotary techniques and determined whether there was a significant quantitative difference in residual material when comparing root thirds. Forty extracted molars were used. The palatal roots were selected, and the canals were prepared using a step-back technique and filled using a lateral condensation technique with gutta-percha points and Endofill sealer. After two weeks of storage in a 0.9% saline solution at 37ºC in an oven, the specimens were divided into 2 groups of 20, with group 1 samples subjected to Gates-Glidden drills and group 2 samples subjected to the ProTaper retreatment System. Hedstroem files and eucalyptol solvent were used in both groups to complete the removal procedure. Then, the roots thirds were radiographed and the images were submitted to the NIH ImageJ program to measure the residual filling material in mm. Each root third was related to the total area of the root canals. The data were analyzed using Student's t test. There was a statistically significant difference between the two techniques as more filling material was removed by technique 2 (ProTaper than technique 1 (Gates-Glidden drills, p < 0.05. The apical third had a greater amount of residual filling material than the cervical and middle thirds, and the difference was statistically significant (p < 0.05. None of the selected techniques removed all filling material, and the material was most difficult to remove from the apical third. The ProTaper files removed more material than the Gates-Glidden drills

  10. Ex-vivo assessment of chronic toxicity of low levels of cadmium on testicular meiotic cells.

    Science.gov (United States)

    Geoffroy-Siraudin, Cendrine; Perrard, Marie-Hélène; Ghalamoun-Slaimi, Rahma; Ali, Sazan; Chaspoul, Florence; Lanteaume, André; Achard, Vincent; Gallice, Philippe; Durand, Philippe; Guichaoua, Marie-Roberte

    2012-08-01

    Using a validated model of culture of rat seminiferous tubules, we assessed the effects of 0.1, 1 and 10 μg/L cadmium (Cd) on spermatogenic cells over a 2-week culture period. With concentrations of 1 and 10 μg/L in the culture medium, the Cd concentration in the cells, determined by ICP-MS, increased with concentration in the medium and the day of culture. Flow cytometric analysis enabled us to evaluate changes in the number of Sertoli cells and germ cells during the culture period. The number of Sertoli cells did not appear to be affected by Cd. By contrast, spermatogonia and meiotic cells were decreased by 1 and 10 μg/L Cd in a time and dose dependent manner. Stage distribution of the meiotic prophase I and qualitative study of the synaptonemal complexes (SC) at the pachytene stage were performed by immunocytochemistry with an anti SCP3 antibody. Cd caused a time-and-dose-dependent increase of total abnormalities, of fragmented SC and of asynapsis from concentration of 0.1 μg/L. Additionally, we observed a new SC abnormality, the "motheaten" SC. This abnormality is frequently associated with asynapsis and SC widening which increased with both the Cd concentration and the duration of exposure. This abnormality suggests that Cd disrupts the structure and function of proteins involved in pairing and/or meiotic recombination. These results show that Cd induces dose-and-time-dependent alterations of the meiotic process of spermatogenesis ex-vivo, and that the lowest metal concentration, which induces an adverse effect, may vary with the cell parameter studied.

  11. Prenatal alcohol exposure alters expression of neurogenesis-related genes in an ex vivo cell culture model.

    Science.gov (United States)

    Tyler, Christina R; Allan, Andrea M

    2014-08-01

    Prenatal alcohol exposure can lead to long-lasting changes in functional and genetic programs of the brain, which may underlie behavioral alterations seen in Fetal Alcohol Spectrum Disorder (FASD). Aberrant fetal programming during gestational alcohol exposure is a possible mechanism by which alcohol imparts teratogenic effects on the brain; however, current methods used to investigate the effects of alcohol on development often rely on either direct application of alcohol in vitro or acute high doses in vivo. In this study, we used our established moderate prenatal alcohol exposure (PAE) model, resulting in maternal blood alcohol content of approximately 20 mM, and subsequent ex vivo cell culture to assess expression of genes related to neurogenesis. Proliferating and differentiating neural progenitor cell culture conditions were established from telencephalic tissue derived from embryonic day (E) 15-17 tissue exposed to alcohol via maternal drinking throughout pregnancy. Gene expression analysis on mRNA derived in vitro was performed using a microarray, and quantitative PCR was conducted for genes to validate the microarray. Student's t tests were performed for statistical comparison of each exposure under each culture condition using a 95% confidence interval. Eleven percent of genes on the array had significantly altered mRNA expression in the prenatal alcohol-exposed neural progenitor culture under proliferating conditions. These include reduced expression of Adora2a, Cxcl1, Dlg4, Hes1, Nptx1, and Vegfa and increased expression of Fgf13, Ndn, and Sox3; bioinformatics analysis indicated that these genes are involved in cell growth and proliferation. Decreased levels of Dnmt1 and Dnmt3a were also found under proliferating conditions. Under differentiating conditions, 7.3% of genes had decreased mRNA expression; these include Cdk5rap3, Gdnf, Hey2, Heyl, Pard6b, and Ptn, which are associated with survival and differentiation as indicated by bioinformatics analysis

  12. Ex Vivo Host and Parasite Response to Antileishmanial Drugs and Immunomodulators

    Science.gov (United States)

    McMahon-Pratt, Diane; Saravia, Nancy Gore

    2015-01-01

    antimoniate. However, pentoxifylline diminished secretion of TNF-α, IFN-γ and IL-13, cytokines associated with the outcome of infection by species of the Viannia subgenus. Exposure to CpG diminished the leishmanicidal effect of meglumine antimoniate, but not miltefosine, and significantly reduced secretion of IL -10, alone and in combination with either antileishmanial drug. IL-13 increased in response to CpG plus miltefosine. Conclusions and Significance Human PBMCs allow integrated ex vivo assessment of antileishmanial treatments, providing information on host and parasite determinants of therapeutic response that may be used to tailor therapeutic strategies to optimize clinical resolution. PMID:26024228

  13. 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

  14. Lyophilized phytosomal nanocarriers as platforms for enhanced diosmin delivery: optimization and ex vivo permeation

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    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

  15. A novel human ex vivo model for the analysis of molecular events during lung cancer chemotherapy

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    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

  16. Biomimetic molecules lower catabolic expression and prevent chondroitin sulfate degradation in an osteoarthritic ex vivo model.

    Science.gov (United States)

    Sharma, Shaili; Vazquez-Portalatin, Nelda; Calve, Sarah; Panitch, Alyssa

    2016-02-08

    Aggrecan, the major proteoglycan in cartilage, serves to protect cartilage tissue from damage and degradation during the progression of osteoarthritis (OA). In cartilage extracellular matrix (ECM) aggrecan exists in an aggregate composed of several aggrecan molecules that bind to a single filament of hyaluronan. Each molecule of aggrecan is composed of a protein core and glycosaminoglycan sides chains, the latter of which provides cartilage with the ability to retain water and resist compressive loads. During the progression of OA, loss of aggrecan is considered to occur first, after which other cartilage matrix components become extremely susceptible to degradation. Proteolytic cleavage of the protein core of aggrecan by enzymes such as aggrecanases, prevent its binding to HA and lower cartilage mechanical strength. Here we present the use of HA-binding or collagen type II-binding molecules that functionally mimic aggrecan but lack known cleavage sites, protecting the molecule from proteolytic degradation. These molecules synthesized with chondroitin sulfate backbones conjugated to hyaluronan- or collagen type II- binding peptides, are capable of diffusing through a cartilage explant and adhering to the ECM of this tissue. The objective of this study was to test the functional efficacy of these molecules in an ex vivo osteoarthritic model to discern the optimal molecule for further studies. Different variations of chondroitin sulfate conjugated to the binding peptides were diffused through aggrecan depleted explants and assessed for their ability to enhance compressive stiffness, prevent CS degradation, and modulate catabolic (MMP-13 and ADAMTS-5) and anabolic (aggrecan and collagen type II) gene expression. A pilot in vivo study assessed the ability to retain the molecule within the joint space of an osteoarthritic guinea pig model. The results indicate chondroitin sulfate conjugated to hyaluronan-binding peptides is able to significantly restore equilibrium

  17. Accuracy of five electronic foramen locators with different operating systems: an ex vivo study

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    Bruno Carvalho de Vasconcelos

    2013-04-01

    Full Text Available Objective: The aim of this study was to evaluate, ex vivo, the precision of five electronic root canal length measurement devices (ERCLMDs with different operating systems: the Root ZX, Mini Apex Locator, Propex II, iPex, and RomiApex A-15, and the possible influence of the positioning of the instrument tips short of the apical foramen. Material and Methods: Forty-two mandibular bicuspids had their real canal lengths (RL previously determined. Electronic measurements were performed 1.0 mm short of the apical foramen (-1.0, followed by measurements at the apical foramen (0.0. The data resulting from the comparison of the ERCLMD measurements and the RL were evaluated by the Wilcoxon and Friedman tests at a significance level of 5%. Results: Considering the measurements performed at 0.0 and -1.0, the precision rates for the ERCLMDs were: 73.5% and 47.1% (Root ZX, 73.5% and 55.9% (Mini Apex Locator, 67.6% and 41.1% (Propex II, 61.7% and 44.1% (iPex, and 79.4% and 44.1% (RomiApex A-15, respectively, considering ±0.5 mm of tolerance. Regarding the mean discrepancies, no differences were observed at 0.0; however, in the measurements at -1.0, the iPex, a multi-frequency ERCLMD, had significantly more discrepant readings short of the apical foramen than the other devices, except for the Propex II, which had intermediate results. When the ERCLMDs measurements at -1.0 were compared with those at 0.0, the Propex II, iPex and RomiApex A-15 presented significantly higher discrepancies in their readings. Conclusions: Under the conditions of the present study, all the ERCLMDs provided acceptable measurements at the 0.0 position. However, at the -1.0 position, the ERCLMDs had a lower precision, with statistically significant differences for the Propex II, iPex, and RomiApex A-15.

  18. Ex vivo and in vivo evaluation of an ultrasonic device for precise dissection, coagulation, and transection

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    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

  19. Effective in vivo and ex vivo gene transfer to intestinal mucosa by VSV-G-pseudotyped lentiviral vectors

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    Kasahara Noriyuki

    2010-05-01

    Full Text Available Abstract Background Gene transfer to the gastrointestinal (GI mucosa is a therapeutic strategy which could prove particularly advantageous for treatment of various hereditary and acquired intestinal disorders, including inflammatory bowel disease (IBD, GI infections, and cancer. Methods We evaluated vesicular stomatitis virus glycoprotein envelope (VSV-G-pseudotyped lentiviral vectors (LV for efficacy of gene transfer to both murine rectosigmoid colon in vivo and human colon explants ex vivo. LV encoding beta-galactosidase (LV-β-Gal or firefly-luciferase (LV-fLuc reporter genes were administered by intrarectal instillation in mice, or applied topically for ex vivo transduction of human colorectal explant tissues from normal individuals. Macroscopic and histological evaluations were performed to assess any tissue damage or inflammation. Transduction efficiency and systemic biodistribution were evaluated by real-time quantitative PCR. LV-fLuc expression was evaluated by ex vivo bioluminescence imaging. LV-β-Gal expression and identity of transduced cell types were examined by histochemical and immunofluorescence staining. Results Imaging studies showed positive fLuc signals in murine distal colon; β-Gal-positive cells were found in both murine and human intestinal tissue. In the murine model, β-Gal-positive epithelial and lamina propria cells were found to express cytokeratin, CD45, and CD4. LV-transduced β-Gal-positive cells were also seen in human colorectal explants, consisting mainly of CD45, CD4, and CD11c-positive cells confined to the LP. Conclusions We have demonstrated the feasibility of LV-mediated gene transfer into colonic mucosa. We also identified differential patterns of mucosal gene transfer dependent on whether murine or human tissue was used. Within the limitations of the study, the LV did not appear to induce mucosal damage and were not distributed beyond the distal colon.

  20. Effect of anti-CD52 antibody alemtuzumab on ex-vivo culture of umbilical cord blood stem cells

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    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.

  1. Microwave ablation: results in ex vivo and in vivo porcine livers with 2450-MHz cooled-shaft antenna

    Institute of Scientific and Technical Information of China (English)

    ZHOU Qi; JIN Xing; JIAO De-chao; ZHANG Fu-jun; ZHANG Liang; HAN Xin-wei; DUAN Guang-feng; LI Chuan-xing; HAN Jian-jun

    2011-01-01

    Background Imaging-guided thermal ablation using different energy sources continues to gain favor as a minimally invasive technique for the treatment of primary and metastatic hepatic malignant tumors.This study aimed to evaluate the performance of microwave ablation with 2450-MHz internally cooled-shaft antenna in ex vivo and in vivo porcine livers.Methods All studies were animal care and ethics committee approved.Microwave ablation was performed using a noncooled or cooled-shaft antenna in 23 ex vivo (92 ablations) and eight in vivo (36 ablations) porcine livers.Diameters of the coagulation zone were observed on gross specimens.The coagulation diameters achieved in different microwave ablation parameter groups were compared.Curve estimation analysis was performed to characterize the relationship between applied power and treatment duration and coagulation diameter (including short-axis and long-axis diameter).Results Coagulation zones were elliptical and an arrowed-shaped carbonization zone around the shaft was observed in all groups.But the antenna track was also coagulated in the noncooled-shaft antenna groups.In ex vivo livers,the short-axis diameter correlated with the power output in a quadratic curve fashion (R2=0.95) by fixing ablation duration to 10 minutes,and correlated with the ablation duration in a logarithmic curve fashion (R2=0.98) by fixing power output to 80 W.The short-axis reached a relative plateau within 25 minutes.In in vivo livers,short-axis diameter correlated with the coagulation duration in a sigmoidal curve fashion (60 W group R2=0.76,80 W group R2=0.87),with a relative plateau achieved within 10 minutes for power settings of 60 W and 80 W.Conclusions The internally cooled microwave antenna may be advantageous to minimize collateral damage.The short-axis diameter enlargement has a plateau by fixing power output.

  2. TGF-b Downregulation by RNAi Technique in ex Vivo-Expanded HSCs on 3D DBM Scaffold

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    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.

  3. Genetic polymorphisms in catalase and CYP1B1 determine DNA adduct formation by benzo(a)pyrene ex vivo.

    Science.gov (United States)

    Schults, Marten A; Chiu, Roland K; Nagle, Peter W; Wilms, Lonneke C; Kleinjans, Jos C; van Schooten, Frederik J; Godschalk, Roger W

    2013-03-01

    Genetic polymorphisms can partially explain the large inter-individual variation in DNA adduct levels following exposure to polycyclic aromatic hydrocarbons. Effects of genetic polymorphisms on DNA adduct formation are difficult to assess in human studies because exposure misclassification attenuates underlying relationships. Conversely, ex vivo studies offer the advantage of controlled exposure settings, allowing the possibility to better elucidate genotype-phenotype relationships and gene-gene interactions. Therefore, we exposed lymphocytes of 168 non-smoking volunteers ex vivo to the environmental pollutant benzo(a)pyrene (BaP) and BaP-related DNA adducts were quantified. Thirty-four genetic polymorphisms were assessed in genes involved in carcinogen metabolism, oxidative stress and DNA repair. Polymorphisms in catalase (CAT, rs1001179) and cytochrome P450 1B1 (CYP1B1, rs1800440) were significantly associated with DNA adduct levels, especially when combined. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) analysis in a subset of 30 subjects revealed that expression of catalase correlated strongly with expression of CYP1B1 (R = 0.92, P CYP1B1 and how they simultaneously affect BaP-related DNA adduct levels, catalase expression was transiently knocked down in the human lung epithelial cell line A549. Although catalase knockdown did not immediately change CYP1B1 gene expression, recovery of catalase expression 8 h after the knockdown coincided with a 2.2-fold increased expression of CYP1B1 (P polymorphism in the promoter region of CAT may determine the amount and activity of catalase, which may subsequently regulate the expression of CYP1B1. As a result, both genetic polymorphisms modulate DNA adduct levels in lymphocytes by BaP ex vivo.

  4. Formulation and in Vitro, ex Vivo and in Vivo Evaluation of Elastic Liposomes for Transdermal Delivery of Ketorolac Tromethamine

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    Néstor Mendoza

    2011-12-01

    Full Text Available The objective of the current study was to formulate ketorolac tromethamine-loaded elastic liposomes and evaluate their in vitro drug release and their ex vivo and in vivo transdermal delivery. Ketorolac tromethamine (KT, which is a potent analgesic, was formulated in elastic liposomes using Tween 80 as an edge activator. The elastic vesicles were prepared by film hydration after optimizing the sonication time and number of extrusions. The vesicles exhibited an entrapment efficiency of 73 ± 11%, vesicle size of 127.8 ± 3.4 nm and a zeta potential of −12 mV. In vitro drug release was analyzed from liposomes and an aqueous solution, using Franz diffusion cells and a cellophane dialysis membrane with molecular weight cut-off of 8000 Da. Ex vivo permeation of KT across pig ear skin was studied using a Franz diffusion cell, with phosphate buffer (pH 7.4 at 32 °C as receptor solution. An in vivo drug permeation study was conducted on healthy human volunteers using a tape-stripping technique. The in vitro results showed (i a delayed release when KT was included in elastic liposomes, compared to an aqueous solution of the drug; (ii a flux of 0.278 mg/cm2h and a lag time of about 10 h for ex vivo permeation studies, which may indicate that KT remains in the skin (with the possibility of exerting a local effect before reaching the receptor medium; (iii a good correlation between the total amount permeated, the penetration distance (both determined by tape stripping and transepidermal water loss (TEWL measured during the in vivo permeation studies. Elastic liposomes have the potential to transport the drug through the skin, keep their size and drug charge, and release the drug into deep skin layers. Therefore, elastic liposomes hold promise for the effective topical delivery of KT.

  5. Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

    Science.gov (United States)

    Grafmüller, Stefanie; Manser, Pius; Krug, Harald F.; Wick, Peter; von Mandach, Ursula

    2013-01-01

    Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles. Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results. The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data. PMID:23851364

  6. Determination of the transport rate of xenobiotics and nanomaterials across the placenta using the ex vivo human placental perfusion model.

    Science.gov (United States)

    Grafmüller, Stefanie; Manser, Pius; Krug, Harald F; Wick, Peter; von Mandach, Ursula

    2013-06-18

    Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ (1). Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 (2) and continuously modified by Schneider et al. in 1972 (3), can serve as an excellent model to study the transfer of xenobiotics or particles. Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results. The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

  7. A head-to-head hands-on comparison of ERCP mechanical simulator (EMS) and Ex-vivo Porcine Stomach Model (PSM).

    Science.gov (United States)

    Leung, Joseph W; Wang, Dong; Hu, Bing; Lim, Brian; Leung, Felix W

    2011-07-01

    BACKGROUND: ERCP mechanical simulator (EMS) and ex-vivo porcine stomach model (PSM) have been described. No direct comparison was reported on endoscopists' perception regarding their efficacy for ERCP training OBJECTIVE: Comparative assessment of EMS and PSM. DESIGN: Questionnaire survey before and after practice. SETTING: Hands-on practice workshops. SUBJECTS: 22 endoscopists with prior experience in 111±225 (mean±SD) ERCP. INTERVENTIONS: Participants performed scope insertion, selective bile duct cannulation with guide wire and insertion of a single biliary stent. Simulated fluoroscopy with external pin-hole camera (EMS), or with additional transillumination (PSM) was used to monitor exchange of accessories. MAIN OUTCOME MEASURE: Participants rated their understanding and confidence before and after hands-on practice, and credibility of each simulator for ERCP training. Comparative efficacy of EMS and PSM for ERCP education was scored (1=not, 10=very) based on pre and post practice surveys: realism (tissue pliability, papilla anatomy, visual/cannulation realism, wire manipulation, simulated fluoroscopy, overall experience); usefulness (assessment of results, supplementing clinical experience, easy for trainees to learn new skills) and application (overall ease of use, prepare trainees to use real instrument and ease of incorporation into training). RESULTS: Before hands-on practice, both EMS and PSM received high scores. After practice, there was a significantly greater increase in confidence score for EMS than PSM (pPSM credible options for improving understanding and supplementing clinical ERCP training. EMS is more useful for basic learning.

  8. 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

  9. 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.

  10. 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.

  11. Human tumor-derived genomic DNA transduced into a recipient cell induces tumor-specific immune responses ex vivo

    OpenAIRE

    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...

  12. 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...

  13. 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.

  14. 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.

  15. 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.

  16. 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....

  17. 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

  18. 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

  19. 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

  20. 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

  1. Antithrombin III, but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting.

    Science.gov (United States)

    Kellner, Patrick; Nestler, Frank; Leimert, Anja; Bucher, Michael; Czeslick, Elke; Sablotzki, Armin; Raspè, Christoph

    2014-12-01

    In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting. Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27±7years) were pre-incubated with clinically relevant concentrations of AT-III (n=11) and C1-INH (n=12), then stimulated with 0.2 ng/mL lipopolysaccharide (LPS) for 3h. After phenotyping CD14⁺ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n=6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes. This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14⁺ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14⁺ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs. Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1

  2. Assessment of penetration potential of pH responsive double walled biodegradable nanogels coated with eucalyptus oil for the controlled delivery of 5-fluorouracil: In vitro and ex vivo studies.

    Science.gov (United States)

    Sahu, Prashant; Kashaw, Sushil K; Jain, Sanyog; Sau, Samaresh; Iyer, Arun K

    2017-03-17

    Penetration enhancers coated biodegradable polymeric nanogels loaded with cytotoxic drugs applied via the topical route, can be a promising strategy for improving the chemotherapeutic efficiency of skin cancers. The major objective of proposed research was to investigate the in vitro and ex vivo chemotherapeutic potential of double walled PLGA-chitosan biodegradable nanogel entrapped with 5-fluororuacil (5-FU) coated with eucalyptus oil, topically applied onto the skin. 5-FU was first entrapped in PLGA core by solvent evaporation technique followed by coating with cationic chitosan for ionic interaction with anionic skin cancer cell membrane. A surface coating of eucalyptus oil (1%) was employed to improve the penetration efficacy of the nanogel into stratum corneum. The surface modified biodegradable double walled nanogel was characterized for particle size, charge and thermal properties followed by pH dependent in vitro analysis. Human keratinocyte (HaCaT) cell line was employed for the bio- and cyto-compatibility testing prior to the hemolysis assay and coagulation assessment. A porcine skin ex vivo screening was performed for assessing the penetration potential of the nanogels. DLS and TEM revealed a particle size of 170nm for the double walled nanogels. The nanogels also exhibited high thermal stability as analyzed by thermogravimetry (TG) and differential thermal analysis (DTA). The drug entrapment efficacy was about ~45%. The drug release showed sustained release pattern noted up to 24h. The low hemolysis of 3.31% with short prothrombin time (PT) and activated partial thromboplastin time (APTT) of 13.5 and 33s respectively, revealed high biocompatibility of the nanogels. The cellular uptake and localization was assessed by confocal microscopy. The cytotoxicity (MTT assay) on HaCaT cell line demonstrated high cytocompatibilty of the nanogels. An ex vivo evaluation using porcine skin displayed efficient and steady state flux of 5-FU from the biodegradable

  3. 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

  4. 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

  5. Spt5 accumulation at variable genes distinguishes somatic hypermutation in germinal center B cells from ex vivo-activated cells.

    Science.gov (United States)

    Maul, Robert W; Cao, Zheng; Venkataraman, Lakshmi; Giorgetti, Carol A; Press, Joan L; Denizot, Yves; Du, Hansen; Sen, Ranjan; Gearhart, Patricia J

    2014-10-20

    Variable (V) genes of immunoglobulins undergo somatic hypermutation by activation-induced deaminase (AID) to generate amino acid substitutions that encode antibodies with increased affinity for antigen. Hypermutation is restricted to germinal center B cells and cannot be recapitulated in ex vivo-activated splenic cells, even though the latter express high levels of AID. This suggests that there is a specific feature of antigen activation in germinal centers that recruits AID to V genes which is absent in mitogen-activated cultured cells. Using two Igh knock-in mouse models, we found that RNA polymerase II accumulates in V regions in B cells after both types of stimulation for an extended distance of 1.2 kb from the TATA box. The paused polymerases generate abundant single-strand DNA targets for AID. However, there is a distinct accumulation of the initiating form of polymerase, along with the transcription cofactor Spt5 and AID, in the V region from germinal center cells, which is totally absent in cultured cells. These data support a model where mutations are prevalent in germinal center cells, but not in ex vivo cells, because the initiating form of polymerase is retained, which affects Spt5 and AID recruitment.

  6. 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

    2014-10-24

    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.

  7. THE CELLS WITH MYCOBACTERIA IN GRANULOMATOUS AGGREGATES FROM MICE WITH LATENT TUBERCULOUS INFECTION IN EX VIVO CULTURE

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    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. 

  8. Freeze Dried Quetiapine-Nicotinamide Binary Solid Dispersions: A New Strategy for Improving Physicochemical Properties and Ex Vivo Diffusion

    Science.gov (United States)

    Al-Remawi, Mayyas Mohammad Ahmad

    2016-01-01

    Improving the physicochemical properties and oral bioavailability of quetiapine fumarate (QF) enabling enhanced antipsychotic attributes are the main aims of this research. The freeze dried solid dispersion strategy was adopted using nicotinamide (NIC) as highly soluble coformer. The prepared dispersions were characterized using scanning electron microscopy (SEM) differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). Static disc intrinsic dissolution rate and ex vivo diffusion through intestinal tissues were conducted and compared to pure quetiapine fumarate. The results demonstrated a highly soluble coamorphous system formed between quetiapine fumarate and nicotinamide at 1 : 3 molar ratio through H-bonding interactions. The results showed >14-fold increase in solubility of QF from the prepared dispersions. Increased intrinsic dissolution rate (from 0.28 to 0.603 mg cm−2 min−1) and faster flux rate through duodenum (from 0.027 to 0.041 mg cm−2 h−1) and jejunum (0.027 to 0.036 mg cm−2 h−1) were obtained. The prepared coamorphous dispersion proved to be effective in improving the drug solubility and dissolution rate and ex vivo diffusion. Therefore, binary coamorphous dispersions could be a promising solution to modify the physicochemical properties, raise oral bioavailability, and change the biopharmaceutics classification (BCS) of some active pharmaceutical ingredients. PMID:28042494

  9. 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.

  10. Evaluation of nitric oxide scavenging activity, in vitro and ex vivo, of selected medicinal plants traditionally used in inflammatory diseases.

    Science.gov (United States)

    Basu, Subhalakshmi; Hazra, Banasri

    2006-10-01

    Steroidal and non-steroidal antiinflammatory drugs, despite their various side effects, are in great demand worldwide. Alternatively, herbal formulations provide relief to a large percentage of the population suffering from inflammatory diseases. Therefore, such practices need to be rationalized through a mechanistic approach. Thus, four traditional medicinal plants, namely Ventilago madraspatana Gaertn., Rubia cordifolia Linn., Lantana camara Linn. and Morinda citrifolia Linn. were selected for a study on the inhibition of nitric oxide (NO*), a key mediator in the phenomenon of inflammation, signifying the presence of effective antiinflammatory constituents therein. Plant samples were extracted with different solvents for evaluation of their inhibitory activity on NO* produced in vitro from sodium nitroprusside, and in LPS-activated murine peritoneal macrophages, ex vivo. Further, the inhibition of NO* synthesis was correlated with the reduction of iNOS protein expression through Western blot. Notable NO* scavenging activity was exhibited in vitro by some extracts of V. madraspatana, R. cordifolia and L. camara (IC(50) < 0.2 mg/mL). Most of them showed marked inhibition (60%-80%), ex vivo, at a dose of 80 microg/mL without appreciable cytotoxic effect on the cultured macrophages. Immunoblot analysis confirmed that the modulatory effect of the samples had occurred through suppression of iNOS protein.

  11. 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.

  12. 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.

  13. 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.

  14. Acceleration of Enterococcus faecalis biofilm formation by aggregation substance expression in an ex vivo model of cardiac valve colonization.

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    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.

  15. 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.

  16. 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.

  17. 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

  18. Ex Vivo and In Situ Evaluation of 'Dispelling-Wind' Chinese Medicine Herb-Drugs on Intestinal Absorption of Chlorogenic Acid.

    Science.gov (United States)

    Zhai, Lixiang; Shi, Jun; Xu, Weitong; Heinrich, Michael; Wang, Jianying; Deng, Wenji

    2015-12-01

    This study aims to investigate the additive or synergistic effects and mechanism of intestinal absorption of extracts from two commonly used 'dispelling-wind' TCM botanical drugs [roots of Angelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav. (RAD) and Saposhnikovia divaricata (Turcz.) Schischk. (RSD)] using chlorogenic acid as a marker substance. Ex vivo everted intestinal sac and in situ single pass perfusion methods using rats were employed to investigate the effects of two TCM botanical drugs extracts on the intestinal absorption of chlorogenic acid. Both the extracts of RAD and RSD showed synergistic properties on the intestinal absorption of chlorogenic acid. The verapamil (a P-gp inhibitor) and intestinal dysbacteriosis model induced by norfloxacin increased the P(app) and K(a) of intestinal absorption of chlorogenic acid. These synergistic effects on intestinal absorption in a rat model can be correlated with the inhibition of P-gp and regulation of gut microbiota. This experimental approach has helped to better understand changes in the absorption of chlorogenic acid under different conditions.

  19. 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.

  20. 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

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    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.

  1. 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.

  2. 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.

  3. Ex vivo susceptibility of Plasmodium falciparum isolates from Dakar, Senegal, to seven standard anti-malarial drugs

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    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.

  4. Pulsed ultrasound enhances the delivery of nitric oxide from bubble liposomes to ex vivo porcine carotid tissue

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    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

  5. 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

  6. Supersonic Shear Wave Imaging to Assess Arterial Nonlinear Behavior and Anisotropy: Proof of Principle via Ex Vivo Testing of the Horse Aorta

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    D. A. Shcherbakova

    2014-09-01

    Full Text Available Supersonic shear wave imaging (SSI is a noninvasive, ultrasound-based technique to quantify the mechanical properties of bulk tissues by measuring the propagation speed of shear waves (SW induced in the tissue with an ultrasound transducer. The technique has been successfully validated in liver and breast (tumor diagnostics and is potentially useful for the assessment of the stiffness of arteries. However, SW propagation in arteries is subjected to different wave phenomena potentially affecting the measurement accuracy. Therefore, we assessed SSI in a less complex ex vivo setup, that is, a thick-walled and rectangular slab of an excised equine aorta. Dynamic uniaxial mechanical testing was performed during the SSI measurements, to dispose of a reference material assessment. An ultrasound probe was fixed in an angle position controller with respect to the tissue to investigate the effect of arterial anisotropy on SSI results. Results indicated that SSI was able to pick up stretch-induced stiffening of the aorta. SW velocities were significantly higher along the specimen's circumferential direction than in the axial direction, consistent with the circumferential orientation of collagen fibers. Hence, we established a first step in studying SW propagation in anisotropic tissues to gain more insight into the feasibility of SSI-based measurements in arteries.

  7. Human Islet Amyloid Polypeptide Transgenic Mice: In Vivo and Ex Vivo Models for the Role of hIAPP in Type 2 Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    J. W. M. Höppener

    2008-01-01

    Full Text Available Human islet amyloid polypeptide (hIAPP, a pancreatic islet protein of 37 amino acids, is the main component of islet amyloid, seen at autopsy in patients with type 2 diabetes mellitus (DM2. To investigate the roles of hIAPP and islet amyloid in DM2, we generated transgenic mice expressing hIAPP in their islet beta cells. In this study, we found that after a long-term, high-fat diet challenge islet amyloid was observed in only 4 of 19 hIAPP transgenic mice. hIAPP transgenic females exhibited severe glucose intolerance, which was associated with a downregulation of GLUT-2 mRNA expression. In isolated islets from hIAPP males cultured for 3 weeks on high-glucose medium, the percentage of amyloid containing islets increased from 5.5% to 70%. This ex vivo system will allow a more rapid, convenient, and specific study of factors influencing islet amyloidosis as well as of therapeutic strategies to interfere with this pathological process.

  8. 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.)

  9. IL-3 or IL-7 increases ex vivo gene transfer efficiency in ADA-SCID BM CD34+ cells while maintaining in vivo lymphoid potential.

    Science.gov (United States)

    Ficara, Francesca; Superchi, Daniela B; Hernández, Raisa Jofra; Mocchetti, Cristina; Carballido-Perrig, Nicole; Andolfi, Grazia; Deola, Sara; Colombo, Augusto; Bordignon, Claudio; Carballido, José M; Roncarolo, Maria Grazia; Aiuti, Alessandro

    2004-12-01

    To improve maintenance and gene transfer of human lymphoid progenitors for clinical use in gene therapy of adenosine deaminase (ADA)-deficient SCID we investigated several gene transfer protocols using various stem cell-enriched sources. The lymphoid differentiation potential was measured by an in vitro clonal assay for B/NK cells and in the in vivo SCID-hu mouse model. Ex vivo culture with the cytokines TPO, FLT3-ligand, and SCF (T/F/S) plus IL-3 or IL-7 substantially increased the yield of transduced bone marrow (BM) CD34(+) cells purified from ADA-SCID patients or healthy donors, compared to T/F/S alone. Moreover, the use of IL-3 or IL-7 significantly improved the maintenance of in vitro B cell progenitors from ADA-SCID BM cells and allowed the efficient transduction of B and NK cell progenitors. Under these optimized conditions transduced CD34(+) cells were efficiently engrafted into SCID-hu mice and gave rise to B and T cell progeny, demonstrating the maintenance of in vivo lymphoid reconstitution capacity. The protocol based on the T/F/S + IL-3 combination was included in a gene therapy clinical trial for ADA-SCID, resulting in long-term engraftment of stem/progenitor cells. Remarkably, gene-corrected BM CD34(+) cells obtained from one patient 4 and 11 months after gene therapy were capable of repopulating the lymphoid compartment of SCID-hu hosts.

  10. Protective Mechanisms of S. lycopersicum Aqueous Fraction (Nucleosides and Flavonoids on Platelet Activation and Thrombus Formation: In Vitro, Ex Vivo and In Vivo Studies

    Directory of Open Access Journals (Sweden)

    Eduardo Fuentes

    2013-01-01

    Full Text Available The purpose of this research was to investigate mechanisms of antiplatelet action of bioactive principle from S. lycopersicum. Aqueous fraction had a high content of nucleosides (adenosine, guanosine, and adenosine 5′-monophosphate by HPLC analysis. Also aqueous fraction presented flavonoids content. Aqueous fraction inhibited platelet activation by 15 ± 6% (P<0.05. Fully spread of human platelets on collagen in the presence of aqueous fraction was inhibited from 15 ± 1 to 9 ± 1 μm2 (P<0.001. After incubation of whole blood with aqueous fraction, the platelet coverage was inhibited by 55 ± 12% (P<0.001. Platelet ATP secretion and aggregation were significantly inhibited by the aqueous fraction. At the same concentrations that aqueous fraction inhibits platelet aggregation, levels of sCD40L significantly decreased and the intraplatelet cAMP levels increased. In addition, SQ22536, an adenylate cyclase inhibitor, attenuated the effect of aqueous fraction toward ADP-induced platelet aggregation and intraplatelet level of cAMP. Platelet aggregation ex vivo (human study and thrombosis formation in vivo (murine model were inhibited by aqueous fraction. Finally, aqueous fraction may be used as a functional ingredient adding antiplatelet activities (nucleosides and flavonoids to processed foods.

  11. In vitro and ex vivo evaluation of silica-coated super paramagnetic iron oxide nanoparticles (SPION) as biomedical photoacoustic contrast agent

    Science.gov (United States)

    Alwi, Rudolf; Telenkov, Sergey A.; Mandelis, Andreas; Leshuk, Timothy; Gu, Frank; Oladepo, Sulayman; Michaelian, Kirk; Dickie, Kristopher

    2013-03-01

    The employment of contrast agents in photoacoustic imaging has gained significant attention within the past few years for their biomedical applications. In this study, the use of silica-coated superparamagnetic iron oxide (Fe3O4) nanoparticles (SPION) was investigated as a contrast agent in biomedical photoacoustic imaging. SPIONs have been widely used as Food-and-Drug-Administration (FDA)-approved contrast agents for magnetic resonance imaging (MRI) and are known to have an excellent safety profile. Using our frequency-domain photoacoustic correlation technique ("the photoacoustic radar") with modulated laser excitation, we examined the effects of nanoparticle size, concentration and biological medium (e.g. serum, sheep blood) on its photoacoustic response in turbid media (intralipid solution). Maximum detection depth and minimum measurable SPION concentration were determined experimentally. The detection was performed using a single element transducer. The nanoparticle-induced optical contrast ex vivo in dense muscular tissues (avian pectus) was evaluated using a phased array photoacoustic probe and the strong potential of silicacoated SPION as a possible photoacoustic contrast agent was demonstrated. This study opens the way for future clinical applications of nanoparticle-enhanced photoacoustic imaging in cancer therapy.

  12. 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.

  13. 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.

  14. Short- and long-term effects of MDMA ("ecstasy") on synaptosomal and vesicular uptake of neurotransmitters in vitro and ex vivo.

    Science.gov (United States)

    Bogen, Inger Lise; Haug, Kristin Huse; Myhre, Oddvar; Fonnum, Frode

    2003-01-01

    3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") is a commonly abused drug which has been shown to be neurotoxic to serotonergic neurons in many species. The exact mechanism responsible for the neurotoxicity of MDMA is, however, poorly understood. In this study, the effects of MDMA on the synaptosomal and vesicular uptake of neurotransmitters were investigated. Our results show that MDMA (0.5-20 microM) reduces both synaptosomal and vesicular uptake of serotonin and dopamine in a dose dependent manner in vitro, while the uptake of glutamate and gamma-aminobutyric acid (GABA) remains unaffected. Ex vivo experiments support the importance of the monoamines, with predominant dopaminergic inhibition at short-term exposure (3 x 15 mg/kg; 2-h intervals), and exclusively serotonergic inhibition at long-term exposure (2 x 10 mg/kg per day; 4 days). This study also compares MDMA and the structurally related antidepressant paroxetine, in an attempt to reveal possible cellular mechanisms for the serotonergic toxicity of MDMA. One important difference between paroxetine and MDMA is that only MDMA has the capability of inhibiting vesicular uptake of monoamines at doses used. We suggest that inhibition of the vesicular monoamine transporter-2, and a following increase in cytoplasmatic monoamine concentrations, might be crucial for the neurotoxic effect of MDMA.

  15. Ex vivo detection of primary leukemia cells resistant to granule cytotoxin-induced cell death: a rapid isolation method to study granzyme-B-mediated cell death.

    Science.gov (United States)

    Grüllich, Carsten; Friske, Viktoria; Finke, Jürgen

    2008-09-01

    Cytotoxic T lymphocytes and natural killer cells (CTL/NK) induce cell death in leukemia cells by the granzyme B (grB)-dependent granule cytotoxin (GC) pathway. Resistance to GC may be involved in immune evasion of leukemia cells. The delivery of active grB into the cytoplasma is dependent on the presence of perforin (PFN) and grB complexes. We developed a rapid method for the isolation of GC to investigate GC-mediated cell death in primary leukemia cells. We isolated GC containing grB, grB complexes and PFN by detergent free hypotonic lysis of the human NK cell leukemia line YT. The GC induce grB-mediated, caspase-dependent apoptosis in live cells. The human leukemia cell lines KG-1, U937, K562 (myeloid leukemia), Jurkat, Daudi, and BV173 (lymphoblastic leukemia) treated with GC internalized grB and underwent cell death. In primary leukemia cells analyzed ex vivo, we found GC-resistant leukemia cells in three out of seven patients with acute myeloid leukemia and one out of six patients with acute lymphoblastic leukemia. We conclude that our method is fast (approximately 1 h) and yields active GC that induce grB-dependent cell death. Furthermore, resistance to GC can be observed in acute leukemias and may be an important mechanism contributing to leukemia cell immune evasion.

  16. First ex vivo study demonstrating that {sup 99m}Tc-NTP 15-5 radiotracer binds to human articular cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Cachin, Florent; Culot, Damien [Jean Perrin Cancer Centre, Nuclear Medicine Department, Clermont-Ferrand (France); Universite d' Auvergne, UMR 990 INSERM, Clermont-Ferrand (France); Boisgard, Stephane [Gabriel Montpied University Hospital, Orthopaedic Surgery, Clermont-Ferrand (France); Universite d' Auvergne, UMR 990 INSERM, Clermont-Ferrand (France); Vidal, Aurelien; Auzeloux, Philippe; Madelmont, Jean-Claude; Chezal, Jean-Michel; Miot-Noirault, Elisabeth [Universite d' Auvergne, UMR 990 INSERM, Clermont-Ferrand (France); Filaire, Marc [Universite d' Auvergne, Anatomy Laboratory, Clermont-Ferrand (France); Askienazy, Serge [Cyclopharma Laboratoire, Saint-Beauzire (France)

    2011-11-15

    Preclinical data pointed to {sup 99m}Tc-NTP 15-5 as a good candidate for single photon emission computed tomography (SPECT) imaging of cartilaginous disease. We set out to investigate and quantify {sup 99m}Tc-NTP 15-5 ex vivo uptake by human articular cartilage relative to bone {sup 99m}Tc-hydroxymethylene diphosphonate (HMDP) radiotracer. Three osteoarthritic human tibial plateaux and four tibiofemoral joints were incubated with {sup 99m}Tc-NTP 15-5 and {sup 99m}Tc-HMDP for 2 h. Affinity of tracers for cartilage was determined by visual analysis of SPECT/CT acquisitions and measurement of cartilage to cortical bone uptake ratios. Cartilage to cortical bone uptake ratios were 3.90 {+-} 2.35 and 0.76 {+-} 0.24, respectively, for {sup 99m}Tc-NTP 15-5 and {sup 99m}Tc-HMDP radiotracers. Visual analysis of fused SPECT/CT slices showed selective, intense {sup 99m}Tc-NTP 15-5 accumulation in articular cartilage, whereas {sup 99m}Tc-HMDP binding was low. Interestingly, a cartilage defect visualized on CT was clearly associated with focal decreased uptake of {sup 99m}Tc-NTP 15-5. The tracer {sup 99m}Tc-NTP 15-5 is of major interest for human cartilage molecular imaging and could find clinical applications in osteoarthritis staging and monitoring. (orig.)

  17. The StcE metalloprotease of enterohaemorrhagic Escherichia coli reduces the inner mucus layer and promotes adherence to human colonic epithelium ex vivo.

    Science.gov (United States)

    Hews, Claire L; Tran, Seav-Ly; Wegmann, Udo; Brett, Bernard; Walsham, Alistair D S; Kavanaugh, Devon; Ward, Nicole J; Juge, Nathalie; Schüller, Stephanie

    2017-01-05

    Enterohaemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen and tightly adheres to human colonic epithelium by forming attaching/effacing lesions. To reach the epithelial surface, EHEC must penetrate the thick mucus layer protecting the colonic epithelium. In this study, we investigated how EHEC interacts with the intestinal mucus layer using mucin-producing LS174T colon carcinoma cells and human colonic mucosal biopsies. The level of EHEC binding and attaching/effacing lesion formation in LS174T cells was higher compared to mucin-deficient colon carcinoma cell lines, and initial adherence was independent of the presence of flagellin, Escherichia coli common pilus, or long polar fimbriae. Although EHEC infection did not affect gene expression of secreted mucins, it resulted in reduced MUC2 glycoprotein levels. This effect was dependent on the catalytic activity of the secreted metalloprotease StcE, which reduced the inner mucus layer and thereby promoted EHEC access and binding to the epithelium in vitro and ex vivo. Given the lack of efficient therapies against EHEC infection, StcE may represent a suitable target for future treatment and prevention strategies.

  18. Adaptive Thermal Therapy using Planar Ultrasound Transducers with Real-time MR Temperature Feedback: Demonstration in Gel Phantoms and Ex-vivo Tissues

    Science.gov (United States)

    Tang, Kee; Choy, Vanessa; Chopra, Rajiv; Bronskill, Michael

    2007-05-01

    MRI-guided transurethral ultrasound therapy offers a minimally invasive approach for the treatment of localized prostate cancer. The main goal of this study was to evaluate active temperature feedback on a clinical 1.5T MR imager to control conformal thermal therapy. MR thermometry was performed during heating in both thermal gel phantoms and ex-vivo tissue with a single-element transurethral heating applicator. The applicator rotation rate and power were controlled based on MRI-temperature measurements. The influence of a cooling gradient (to simulate cooling of the rectum or urethra) was also investigated in gel phantoms. The 55°C isotherm generated during heating closely matched the targeted prostate shape, with an average distance error of 0.9 mm ± 0.4 mm in turkey breasts, 1.3 mm ± 0.5 mm in gel phantoms without rectal cooling and 1.4 mm ± 0.6 mm in gel phantoms with rectal cooling. Accurate, MRI-guided, active feedback has been successfully demonstrated experimentally and has the capability to adjust for unpredictable and varying tissue properties during the treatment.

  19. 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.

  20. Effects of human hair on trans-cranial focused ultrasound efficacy in an ex-vivo cadaver model

    Science.gov (United States)

    Hananel, Arik; Snell, John W.; Kassell, Neal F.; Eames, Matthew D. C.

    2012-11-01

    Current practice before a trans-cranial MR guided Focused ultrasound procedure is shaving the patient head on treatment day. Here we present an initial attempt to evaluate the feasibility of trans-cranial FUS, in an unshaved, ex-vivo cadaver skull. We have sonicated using 220kHz and 710kHz head transducers, a cadaver skull filled with tissue mimicking phantom and covered with a wig made of human hair to evaluate feasibility of acoustic energy transfer in a full size model. Heating at focal point was measured using MR proton resonance shift thermometry. Results showed negligible effect of hair in 220kHz, and an 18% drop in temperature elevation when using 710kHz.

  1. 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.

  2. 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.

  3. 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.

  4. Enhanced functional integration of human photoreceptor precursors into human and rodent retina in an ex vivo retinal explant model system.

    Science.gov (United States)

    Yanai, Anat; Laver, Christopher R J; Gregory-Evans, Cheryl Y; Liu, Ran R; Gregory-Evans, Kevin

    2015-06-01

    Retinal disease is the major cause of irreversible blindness in developed countries. Transplantation of photoreceptor precursor cells (PPCs) derived from human embryonic stem cells (hESCs) is a promising and widely applicable approach for the treatment of these blinding conditions. Previously, it has been shown that after transplantation into the degenerating retina, the percentage of PPCs that undergo functional integration is low. The factors that inhibit PPC engraftment remain largely unknown, in part, because so many adverse factors could be at play during in vivo experiments. To advance our knowledge in overcoming potential adverse effects and optimize PPC transplantation, we have developed a novel ex vivo system. Harvested neural retina was placed directly on top of cultured retinal pigment epithelial (RPE) cells from a number of different sources. To mimic PPC transplantation into the subretinal space, hESC-derived PPCs were inserted between the retinal explant and underlying RPE. Explants cocultured with hESC-derived RPE maintained normal gross morphology and viability for up to 2 weeks, whereas the explants cultured on ARPE19 and RPE-J failed by 7 days. Furthermore, the proportion of PPCs expressing ribbon synapse-specific proteins BASSOON and RIBEYE was significantly higher when cocultured with hESC-derived RPE (20% and 10%, respectively), than when cocultured with ARPE19 (only 6% and 2%, respectively). In the presence of the synaptogenic factor thrombospondin-1 (TSP-1), the proportion of BASSOON-positive and RIBEYE-positive PPCs cocultured with hESC-derived RPE increased to ∼30% and 15%, respectively. These data demonstrate the utility of an ex vivo model system to define factors, such as TSP-1, which could influence integration efficiency in future in vivo experiments in models of retinal degeneration.

  5. 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

  6. 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.

  7. Experimental study on ex vivo retrovirus-mediated aFGF gene transfer therapy in traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    WANG Qing-hua; LI Bang-yin; XU Ru-xiang; LIU Chuan; ZOU Yu-xi; WANG Hui-xin

    2001-01-01

    Objective: To explore the effects of ex vivo retrovirus-mediated gene transfer therapy with acidic fibroblast growth factor (aFGF) in the management of traumatic brain injury. Methods: PLXSN-SPaFGF, a recombinant retroviral vector expressing biologically active aFGF was constructed and transfected into cultured embryonic astroglial cells which were injected into the surrounding areas of the contusion in the rat left parietal cortex. From 3 d to 1 month after the implantation, the survival of and aFGF gene expression in the implanted astroglial cells were examined, and neuronal apoptosis and rat motor function impairment evaluated. Results: The implanted aFGF-transduced astroglial cells survived and expressed aFGF mRNA and protein evidently at 3 d after grafting. The number of and aFGF gene expression in the astroglial cells increased remarkebly 7 d and decreased to some extent 1 month after the implantation. There were significant aFGF mRNA and protein expression in the neurons surrounding the contusion at 7 d that decreased to relatively low levels 1 month after the implantation of aFGF-transdued astrocytes. Diminished neuronal apoptosis (P<0.05) and significantly improved in the previously impaired motor function (P<0.05) of the rats were observed from 7 d to 1 month after the implantation. Conclusion: This experiment successfully conducted ex vivo aFGF gene transfer therapy in traumatic brain injury which proved to be effective in rescuing injured nerve cell from death and enhancing recovery of neurological deficiency.

  8. 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.

  9. 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 

  10. Increased survival of normal cells during laser photodynamic therapy: implications for ex vivo autologous bone marrow purging

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    Gulliya, K.S.; Matthews, J.L.; Fay, J.W.; Dowben, R.M.

    1988-01-01

    Laser light-induced, dye-mediated photolysis of leukemic cells was tested in an in vitro model for its efficacy in eliminating occult tumor cells for ex vivo autologous bone marrow purging. Merocyanine 540 (MC540) was mixed with acute promyelocytic leukemia (HL-60) cells in the presence of human albumin. This cell-dye mixture was irradiated with 514 nm argon laser light. Results show that in the presence of 0.1%, 0.25% and 0.5% albumin, laser light doses of 62.4 J/cm/sup 2/, 93.6 J/cm/sup 2/ and 109.2 J/cm/sup 2/, respectively, were required for a 5 log reduction in the survival of leukemic cells. Under identical conditions, 80% to 84% of the normal bone marrow cells and 41% of the granulocyte-macrophage colony forming cells survived. The number of surviving stromal cells was reduced (1+) compared to the untreated control (4+). Mixing of irradiated bone marrow cells with equal number of HL-60 cells did not interfere with the killing of HL-60 cells treated with MC540 and laser light. The non-specific cytotoxicity of laser light alone was less than 6% for normal bone marrow cells. These results suggest that the concentration of human albumin plays an important role in laser light-induced phototoxicity. This laser light-induced selective photolysis of leukemic cells can be used in ex vivo purging of tumor cell-contaminated bone marrow grafts to achieve very high survival rates of normal bone marrow cells and granulocyte-macrophage colony forming cells.

  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. Endothelial targeting with C1-inhibitor reduces complement activation in vitro and during ex vivo reperfusion of pig liver.

    Science.gov (United States)

    Bergamaschini, L; Gobbo, G; Gatti, S; Caccamo, L; Prato, P; Maggioni, M; Braidotti, P; Di Stefano, R; Fassati, L R

    2001-12-01

    Tissue damage during cold storage and reperfusion remains a major obstacle to wider use of transplantation. Vascular endothelial cells and complement activation are thought to be involved in the inflammatory reactions following reperfusion, so endothelial targeting of complement inhibitors is of great interest. Using an in vitro model of human umbilical vein endothelial cells (HUVEC) cold storage and an animal model of ex vivo liver reperfusion after cold ischaemia, we assessed the effect of C1-INH on cell functions and liver damage. We found that in vitro C1-INH bound to HUVEC in a manner depending on the duration of cold storage. Cell-bound C1-INH was functionally active since retained the ability to inhibit exogenous C1s. To assess the ability of cell-bound C1-INH to prevent complement activation during organ reperfusion, we added C1-INH to the preservation solution in an animal model of extracorporeal liver reperfusion. Ex vivo liver reperfusion after 8 h of cold ischaemia resulted in plasma C3 activation and reduction of total serum haemolytic activity, and at tissue level deposition of C3 associated with variable level of inflammatory cell infiltration and tissue damage. These findings were reduced when livers were stored in preservation solution containing C1-INH. Immunohistochemical analysis of C1-INH-treated livers showed immunoreactivity localized on the sinusoidal pole of the liver trabeculae, linked to sinusoidal endothelium, so it is likely that the protective effect was due to C1-INH retained by the livers. These results suggest that adding C1-INH to the preservation solution may be useful to reduce complement activation and tissue injury during the reperfusion of an ischaemic liver.

  13. Preserved ex vivo inflammatory status in decidual cells from women with preterm labor and subclinical intrauterine infection.

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    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.

  14. Cryopreservation of in vitro matured oocytes after ex vivo oocyte retrieval from gynecologic cancer patients undergoing radical surgery

    Science.gov (United States)

    Park, Chan Woo; Lee, Sun Hee; Yang, Kwang Moon; Lee, In Ho; Lim, Kyung Teak; Lee, Ki Heon

    2016-01-01

    Objective The aim of this study was to report a case series of in vitro matured (IVM) oocyte freezing in gynecologic cancer patients undergoing radical surgery under time constraints as an option for fertility preservation (FP). Methods Case series report. University-based in vitro fertilization center. Six gynecologic cancer patients who were scheduled to undergo radical surgery the next day were referred for FP. The patients had endometrial (n=2), ovarian (n=3), and double primary endometrial and ovarian (n=1) cancer. Ex vivo retrieval of immature oocytes from macroscopically normal ovarian tissue was followed by mature oocyte freezing after IVM or embryo freezing with intracytoplasmic sperm injection. Results A total of 53 oocytes were retrieved from five patients, with a mean of 10.6 oocytes per patient. After IVM, a total of 36 mature oocytes were obtained, demonstrating a 67.9% maturation rate. With regard to the ovarian cancer patients, seven IVM oocytes were frozen from patient 3, who had stage IC cancer, whereas one IVM oocyte was frozen from patient 4, who had stage IV cancer despite being of a similar age. With regard to the endometrial cancer patients, 15 IVM oocytes from patient 1 were frozen. Five embryos were frozen after the fertilization of IVM oocytes from patient 6. Conclusion Immature oocytes can be successfully retrieved ex vivo from macroscopically normal ovarian tissue before radical surgery. IVM oocyte freezing provides a possible FP option in patients with advanced-stage endometrial or ovarian cancer without the risk of cancer cell spillage or time delays. PMID:27358831

  15. 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

  16. 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.

  17. A novel approach for computer-assisted template-guided autotransplantation of teeth with custom 3d designed/printed surgical tooling. An ex vivo proof of concept

    NARCIS (Netherlands)

    Anssari Moin, D.; Derksen, W.; Verweij, J.P.; van Merkesteyn, R.; Wismeijer, D.

    2016-01-01

    Purpose: The aim of this study was to introduce a novel method for accurate autotransplantation with computer-assisted guided templates and assembled custom-designed surgical tooling and to test the feasibility and accuracy of this method ex vivo. Materials and Methods: A partially edentulous human

  18. Comparison of the Ex Vivo Expansion of UCB-Derived CD34+ in 3D DBM/MBA Scaffolds with USSC as a Feeder Layer

    Directory of Open Access Journals (Sweden)

    Zahra Sadat Hashemi

    2013-10-01

    Because of acid extraction in the DBM production process, mineral materials were removed and the protein background that was more flexible was presented. Therefore these results suggest that USSC-DBM can be a suitable ex vivo mimicry niche by intensifying of surface/volume ratio and supporting the stem cell differentiation and expansion.

  19. 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.

  20. Effect of temperature and fixation on the optical properties of atherosclerotic tissue: a validation study of an ex-vivo whole heart cadaveric model

    NARCIS (Netherlands)

    Gnanadesigan, M.; Van Soest, G.; White, S.; Scoltock, S.; Ughi, G.J.; Baumbach, A.; Van der Steen, A.F.W.; Regar, E.; Johnson, T.W.

    2014-01-01

    Atherosclerotic plaque composition can be imaged using the optical attenuation coefficient derived from intravascular optical coherence tomography (OCT) data. The relation between optical properties and tissue type has been established on autopsy tissues. In this study, we validate an ex-vivo model

  1. Astaxanthin, a Carotenoid, Stimulates Immune Responses by Enhancing IFN-γ and IL-2 Secretion in Primary Cultured Lymphocytes in Vitro and ex Vivo.

    Science.gov (United States)

    Lin, Kuan-Hung; Lin, Kao-Chang; Lu, Wan-Jung; Thomas, Philip-Aloysius; Jayakumar, Thanasekaran; Sheu, Joen-Rong

    2015-12-29

    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 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.

  2. 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.

  3. The kiwi fruit peptide kissper displays anti-inflammatory and anti-oxidant effects in in-vitro and ex-vivo human intestinal models.

    Science.gov (United States)

    Ciacci, C; Russo, I; Bucci, C; Iovino, P; Pellegrini, L; Giangrieco, I; Tamburrini, M; Ciardiello, M A

    2014-03-01

    Literature reports describe kiwi fruit as a food with significant effects on human health, including anti-oxidant and anti-inflammatory activity. Fresh fruit or raw kiwi fruit extracts have been used so far to investigate these effects, but the molecule(s) responsible for these health-promoting activities have not yet been identified. Kissper is a kiwi fruit peptide displaying pore-forming activity in synthetic lipid bilayers, the composition of which is similar to that found in intestinal cells. The objective of this study was to investigate the kissper influence on intestinal inflammation using cultured cells and ex-vivo tissues from healthy subjects and Crohn's disease (CD) patients. The anti-oxidant and anti-inflammatory properties of kissper were tested on Caco-2 cells and on the colonic mucosa from 23 patients with CD, by challenging with the lipopolysaccharide from Escherichia coli (EC-LPS) and monitoring the appropriate markers by Western blot and immunofluorescence. EC-LPS challenge determined an increase in the intracellular concentration of calcium and reactive oxygen species (ROS). The peptide kissper was highly effective in preventing the increase of LPS-induced ROS levels in both the Caco-2 cells and CD colonic mucosa. Moreover, it controls the calcium increase, p65-nuclear factor (NF)-kB induction and transglutaminase 2 (TG2) activation inflammatory response in Caco-2 cells and CD colonic mucosa. Kissper efficiently counteracts the oxidative stress and inflammatory response in valuable model systems consisting of intestinal cells and CD colonic mucosa. This study reports the first evidence supporting a possible correlation between some beneficial effects of kiwi fruit and a specific protein molecule rather than generic nutrients.

  4. Alginate Microencapsulation for Oral Immunisation of Finfish: Release Characteristics, Ex Vivo Intestinal Uptake and In Vivo Administration in Atlantic Salmon, Salmo salar L.

    Science.gov (United States)

    Ghosh, Bikramjit; Nowak, Barbara F; Bridle, Andrew R

    2015-12-01

    This study examined the feasibility of alginate microcapsules manufactured using a low-impact technology and reagents to protect orally delivered immunogens for use as immunoprophylactics for fish. Physical characteristics and protein release kinetics of the microcapsules were examined at different pH and temperature levels using a microencapsulated model protein, bovine serum albumin (BSA). Impact of the microencapsulation process on contents was determined by analysing change in bioactivity of microencapsulated lysozyme. Feasibility of the method for oral immunoprophylaxis of finfish was assessed using FITC-labelled microcapsules. These were applied to distal intestinal explants of Atlantic salmon (Salmo salar) to investigate uptake ex vivo. Systemic distribution of microcapsules was investigated by oral administration of FITC-labelled microcapsules to Atlantic salmon fry by incorporating into feed. The microcapsules produced were structurally robust and retained surface integrity, with a modal size distribution of 250-750 nm and a tendency to aggregate. Entrapment efficiency of microencapsulation was 51.2 % for BSA and 43.2 % in the case of lysozyme. Microcapsules demonstrated controlled release of protein, which increased with increasing pH or temperature, and the process had no significant negative effect on bioactivity of lysozyme. Uptake of fluorescent-labelled microcapsules was clearly demonstrated by intestinal explants over a 24-h period. Evidence of microcapsules was found in the intestine, spleen, kidney and liver of fry following oral administration. Amenability of the microcapsules to intestinal uptake and distribution reinforced the strong potential for use of this microencapsulation method in oral immunoprophylaxis of finfish using sensitive immunogenic substances.

  5. Confocal Raman microscopy and multivariate statistical analysis for determination of different penetration abilities of caffeine and propylene glycol applied simultaneously in a mixture on porcine skin ex vivo.

    Science.gov (United States)

    Mujica Ascencio, Saul; Choe, ChunSik; Meinke, Martina C; Müller, Rainer H; Maksimov, George V; Wigger-Alberti, Walter; Lademann, Juergen; Darvin, Maxim E

    2016-07-01

    Propylene glycol is one of the known substances added in cosmetic formulations as a penetration enhancer. Recently, nanocrystals have been employed also to increase the skin penetration of active components. Caffeine is a component with many applications and its penetration into the epidermis is controversially discussed in the literature. In the present study, the penetration ability of two components - caffeine nanocrystals and propylene glycol, applied topically on porcine ear skin in the form of a gel, was investigated ex vivo using two confocal Raman microscopes operated at different excitation wavelengths (785nm and 633nm). Several depth profiles were acquired in the fingerprint region and different spectral ranges, i.e., 526-600cm(-1) and 810-880cm(-1) were chosen for independent analysis of caffeine and propylene glycol penetration into the skin, respectively. Multivariate statistical methods such as principal component analysis (PCA) and linear discriminant analysis (LDA) combined with Student's t-test were employed to calculate the maximum penetration depths of each substance (caffeine and propylene glycol). The results show that propylene glycol penetrates significantly deeper than caffeine (20.7-22.0μm versus 12.3-13.0μm) without any penetration enhancement effect on caffeine. The results confirm that different substances, even if applied onto the skin as a mixture, can penetrate differently. The penetration depths of caffeine and propylene glycol obtained using two different confocal Raman microscopes are comparable showing that both types of microscopes are well suited for such investigations and that multivariate statistical PCA-LDA methods combined with Student's t-test are very useful for analyzing the penetration of different substances into the skin.

  6. 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.

  7. Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in Ex vivo Organ Culture

    Institute of Scientific and Technical Information of China (English)

    Li-Guo Zhu; Min-Shan Feng; Jia-Wen Zhan; Ping Zhang; Jie Yu

    2016-01-01

    Background:The development of mechanically active culture systems helps increase the understanding of the role of mechanical stress in intervertebral disc (IVD) degeneration.Motion segment cultures allow for preservation of the native IVD structure,and adjacent vertebral bodies facilitate the application and control of mechanical loads.The purpose of this study was to establish loading and organ culture methods for rabbit IVD motion segments to study the effect of static load on the whole disc organ.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 (COL2A 1) 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 l;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

  8. Identification of small molecule lead compounds for visceral leishmaniasis using a novel ex vivo splenic explant model system.

    Directory of Open Access Journals (Sweden)

    Yaneth Osorio

    Full Text Available BACKGROUND: New drugs are needed to treat visceral leishmaniasis (VL because the current therapies are toxic, expensive, and parasite resistance may weaken drug efficacy. We established a novel ex vivo splenic explant culture system from hamsters infected with luciferase-transfected Leishmania donovani to screen chemical compounds for anti-leishmanial activity. METHODOLOGY/PRINCIPAL FINDINGS: THIS MODEL HAS ADVANTAGES OVER IN VITRO SYSTEMS IN THAT IT: 1 includes the whole cellular population involved in the host-parasite interaction; 2 is initiated at a stage of infection when the immunosuppressive mechanisms that lead to progressive VL are evident; 3 involves the intracellular form of Leishmania; 4 supports parasite replication that can be easily quantified by detection of parasite-expressed luciferase; 5 is adaptable to a high-throughput screening format; and 6 can be used to identify compounds that have both direct and indirect anti-parasitic activity. The assay showed excellent discrimination between positive (amphotericin B and negative (vehicle controls with a Z' Factor >0.8. A duplicate screen of 4 chemical libraries containing 4,035 compounds identified 202 hits (5.0% with a Z score of <-1.96 (p<0.05. Eighty-four (2.1% of the hits were classified as lead compounds based on the in vitro therapeutic index (ratio of the compound concentration causing 50% cytotoxicity in the HepG(2 cell line to the concentration that caused 50% reduction in the parasite load. Sixty-nine (82% of the lead compounds were previously unknown to have anti-leishmanial activity. The most frequently identified lead compounds were classified as quinoline-containing compounds (14%, alkaloids (10%, aromatics (11%, terpenes (8%, phenothiazines (7% and furans (5%. CONCLUSIONS/SIGNIFICANCE: The ex vivo splenic explant model provides a powerful approach to identify new compounds active against L. donovani within the pathophysiologic environment of the infected spleen

  9. 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.

  10. 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

  11. 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.

  12. Ex vivo-expanded bone marrow stem cells home to the liver and ameliorate functional recovery in a mouse model of acute hepatic injury

    Institute of Scientific and Technical Information of China (English)

    Shi-Zhu Jin; Ming-Zi Han; Bing-Rong Liu; Jun Xu; Fu-Lai Gao; Zong-Jing Hu; Xin-Hong Wang; Feng-Hua Pei; Yu Hong; Hong-Yan Hu

    2012-01-01

    BACKGROUND: Stem cell transplantation provides a theoretical approach for liver regeneration medicine; it may promote liver regeneration  and  self-repair.  However,  the  transplantation  of bone  marrow-mesenchymal  stem  cells  expanded  ex vivo  as  a therapy for liver disease has rarely been investigated. This study aimed  to  explore  whether  bone  marrow  stem  cells  expanded ex vivo home to the liver and foster hepatic recovery after CCl4 injury. METHODS: Bone  marrow  cells  from  BALB/c  mice  were expanded ex vivo by multiple-passage cultivation, characterized by cytoflow immunofluorescence, and pre-labeled with PKH26 before intravenous infusion into animals treated with CCl4. The integration of bone marrow cells into the liver was examined microscopically, and plasma hepatic enzymes were determined biochemically. RESULTS: Cultured  bone  marrow  cells  exhibited  antigenic profiles comparable to those of primary medullary stem cells. Double  immunofluorescence  showed  colocalization  of  these cells with proliferative activity and albumin expression in the liver of CCl4-treated mice. Densitometry showed increased in situ  cell  proliferation  (50±14  vs  20±3  cells/high-power  field, P CONCLUSIONS: Ex vivo-expanded  bone  marrow  cells  are capable  of  relocating  to  and  proliferating  in  the  chemically-injured  liver.  Transplantation  of  these  pluripotent  stem cells appears to improve serum indices of liver function and survival rate in mice after CCl4-induced hepatic damage.

  13. Ex vivo effects of low-dose rivaroxaban on specific coagulation assays and coagulation factor activities in patients under real life conditions.

    Science.gov (United States)

    Mani, Helen; Hesse, Christian; Stratmann, Gertrud; Lindhoff-Last, Edelgard

    2013-01-01

    Global coagulation assays display variable effects at different concentrations of rivaroxaban. The aim of this study is to quantify the ex vivo effects of low-dose rivaroxaban on thrombophilia screening assays and coagulation factor activities based on the administration time, and to show how to mask possible interferences. Plasma samples from 40 patients receiving rivaroxaban 10 mg daily were investigated to measure activities of clotting factor II, V, VII, VIII, IX, XI, XII and XIII; protein C- and protein S-levels; lupus anticoagulants; anticardiolipin IgG and IgM; D-dimer, heparin-platelet factor 4 (HPF4) antibodies and screening tests for von Willebrand disease (VWD). Two hours after rivaroxaban administration, the activities of clotting factors were significantly decreased to different extents, except for factor XIII. Dilution of plasma samples resulted in neutralisation of these interferences. The chromogenic protein C activity assay was not affected by rivaroxaban. Depending on the timing of tablet intake in relation to blood sampling protein S activity was measured falsely high when a clotting assay was used. False-positive results for lupus anticoagulants were observed depending on the assay system used and the administration time of rivaroxaban. ELISA-based assays such as anticardiolipin IgG and IgM, D-dimer, HPF4-antibodies and the turbidimetric assays for VWD were not affected by rivaroxaban. Specific haemostasis clotting tests should be performed directly prior to rivaroxaban intake. Assay optimisation in the presence of rivaroxaban can be achieved by plasma dilution. Immunologic assays are not influenced by rivaroxaban, while chromogenic assays can be used, when they do not depend on factor Xa.

  14. Topical delivery of clobetasol propionate loaded microemulsion based gel for effective treatment of vitiligo: ex vivo permeation and skin irritation studies.

    Science.gov (United States)

    Patel, Hetal K; Barot, Bhavesh S; Parejiya, Punit B; Shelat, Pragna K; Shukla, Arunkumar

    2013-02-01

    The aim of the present investigation was to evaluate microemulsion as a vehicle for dermal drug delivery and to develop microemulsion based gel (MBC) of clobetasol propionate (CP) for the effective treatment of vitiligo. D-Optimal mixture experimental design was adopted to optimize the amount of oil (X(1)), S(mix) (mixture of surfactant and cosurfactant) (X(2)) and water (X(3)) in the microemulsion. The formulations were assessed for globule size (nm) (Y(1)) and solubility of CP in microemulsion (mg/ml) (Y(2)). The microemulsion containing 3% oil, 45% S(mix) and 50% water was selected as the optimized batch (ME). The globule size and solubility of CP in ME were 18.26 nm and 36.42 mg/ml respectively. Transmission electron microscopy showed that ME globules were spherical in shape. Carbopol 934P was used to convert microemulsion containing drug into gel form without affecting its structure. Ex-vivo permeation studies showed that cumulative amount of CP permeated (Q(n)) from ME, MBC and market formulation (MFCP) at 8h after application were 53.6±2.18, 28.43±0.67 and 37.73±0.77 μg cm(-2) respectively. MBC showed greater retention of CP in to skin layers than ME and MFCP. Skin irritation studies showed MBC to be significantly less irritating than MFCP. Photomicrographs and scanning electron micrographs of skin sections treated with MBC showed significant changes in the skin structure, which was attributed to the interaction of microemulsion components with skin resulting in permeation enhancement and retention of CP into skin layers. It was concluded that CP loaded gel could be a promising formulation for effective treatment of vitiligo.

  15. In vivo and ex vivo phagocytic potential of macrophages from progeny of breeder hens kept on ochratoxin A (OTA)-contaminated diet.

    Science.gov (United States)

    Zahoor-ul-Hassan; Khan, Muhammad Zargham; Khan, Ahrar; Javed, Ijaz; Noreen, Mnaza

    2012-01-01

    This study aimed to investigate the phagocytic potential of macrophages in progeny of breeder hens kept on an OTA-contaminated diet. For this purpose, 84 White Leghorn (WL) layer breeder hens (40-weeks-of-age) were divided into seven groups (A-G). Hens in Group A were fed a commercial layer ration while those in Groups B-G were kept on a diet amended with 0.1, 0.5, 1.0, 3.0, 5.0, or 10.0 mg OTA/kg, respectively, for up to 3 weeks (n = 12/treatment group; n = 4/time sub-group/treatment group). Fertile eggs were set for hatching on a weekly basis to get the progeny of each week separately. Hatched chicks (n = 10 from each group) were injected with India ink at day 14-of-age to study the in vivo phagocytosis of carbon particles. At day 30, abdominal macrophages were collected from 15 chicks/group and were used to assess their ex vivo/in vitro phagocytic potential against sheep red blood cells (SRBC) as well as for nitrite production upon challeng