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Sample records for ex-vivo human intestinal

  1. 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.

  2. Dietary emulsifiers directly alter human microbiota composition and gene expression ex vivo potentiating intestinal inflammation.

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    Chassaing, Benoit; Van de Wiele, Tom; De Bodt, Jana; Marzorati, Massimo; Gewirtz, Andrew T

    2017-08-01

    The intestinal microbiota plays a central role in the development of many chronic inflammatory diseases including IBD and metabolic syndrome. Administration of substances that alter microbiota composition, including the synthetic dietary emulsifiers polysorbate 80 (P80) and carboxymethylcellulose (CMC), can promote such inflammatory disorders. However, that inflammation itself impacts microbiota composition has obfuscated defining the extent to which these compounds or other substances act directly upon the microbiota versus acting on host parameters that promote inflammation, which subsequently reshapes the microbiota. We examined the direct impact of CMC and P80 on the microbiota using the mucosal simulator of the human intestinal microbial ecosystem (M-SHIME) model that maintains a complex stable human microbiota in the absence of a live host. This approach revealed that both P80 and CMC acted directly upon human microbiota to increase its proinflammatory potential, as revealed by increased levels of bioactive flagellin. The CMC-induced increase in flagellin was rapid (1 day) and driven by altered microbiota gene expression. In contrast, the P80-induced flagellin increase occurred more slowly and was closely associated with altered species composition. Transfer of both emulsifier-treated M-SHIME microbiotas to germ-free recipient mice recapitulated many of the host and microbial alterations observed in mice directly treated with emulsifiers. These results demonstrate a novel paradigm of deconstructing host-microbiota interactions and indicate that the microbiota can be directly impacted by these commonly used food additives, in a manner that subsequently drives intestinal inflammation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  3. Human and rodent ex vivo model for intestinal fibrosis in inflammatory bowel disease (IBD)

    NARCIS (Netherlands)

    Pham, B.T.; Van Haaften, T.; Oosterhuis, D.; De Graaf, I.A.M.; Olinga, P.

    2013-01-01

    Background: One of the major complications in IBD is intestinal fibrosis (IF). It is the result of the chronic inflammation of intestinal tissue. IF causes narrowing of the intestinal lumen and potential stricture formation. For the study of the cellular and molecular mechanism of IF in IBD adequate

  4. Hydrolytic Fate of 3/15-Acetyldeoxynivalenol in Humans: Specific Deacetylation by the Small Intestine and Liver Revealed Using in Vitro and ex Vivo Approaches

    Directory of Open Access Journals (Sweden)

    El Hassan Ajandouz

    2016-07-01

    Full Text Available In addition to deoxynivalenol (DON, acetylated derivatives, i.e., 3-acetyl and 15-acetyldexynivalenol (or 3/15ADON, are present in cereals leading to exposure to these mycotoxins. Animal and human studies suggest that 3/15ADON are converted into DON after their ingestion through hydrolysis of the acetyl moiety, the site(s of such deacetylation being still uncharacterized. We used in vitro and ex vivo approaches to study the deacetylation of 3/15ADON by enzymes and cells/tissues present on their way from the food matrix to the blood in humans. We found that luminal deacetylation by digestive enzymes and bacteria is limited. Using human cells, tissues and S9 fractions, we were able to demonstrate that small intestine and liver possess strong deacetylation capacity compared to colon and kidneys. Interestingly, in most cases, deacetylation was more efficient for 3ADON than 15ADON. Although we initially thought that carboxylesterases (CES could be responsible for the deacetylation of 3/15ADON, the use of pure human CES1/2 and of CES inhibitor demonstrated that CES are not involved. Taken together, our original model system allowed us to identify the small intestine and the liver as the main site of deacetylation of ingested 3/15ADON in humans.

  5. Ex-vivo MR Volumetry of Human Brain Hemispheres

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    Kotrotsou, Aikaterini; Bennett, David A.; Schneider, Julie A.; Dawe, Robert J.; Golak, Tom; Leurgans, Sue E.; Yu, Lei; Arfanakis, Konstantinos

    2013-01-01

    Purpose The aims of this work were to: a) develop an approach for ex-vivo MR volumetry of human brain hemispheres that does not contaminate the results of histopathological examination, b) longitudinally assess regional brain volumes postmortem, and c) investigate the relationship between MR volumetric measurements performed in-vivo and ex-vivo. Methods An approach for ex-vivo MR volumetry of human brain hemispheres was developed. Five hemispheres from elderly subjects were imaged ex-vivo longitudinally. All datasets were segmented. The longitudinal behavior of volumes measured ex-vivo was assessed. The relationship between in-vivo and ex-vivo volumetric measurements was investigated in seven elderly subjects imaged both ante-mortem and postmortem. Results The presented approach for ex-vivo MR volumetry did not contaminate the results of histopathological examination. For a period of 6 months postmortem, within-subject volume variation across time points was substantially smaller than inter-subject volume variation. A close linear correspondence was detected between in-vivo and ex-vivo volumetric measurements. Conclusion Regional brain volumes measured with the presented approach for ex-vivo MR volumetry remain relatively unchanged for a period of 6 months postmortem. Furthermore, the linear relationship between in-vivo and ex-vivo MR volumetric measurements suggests that the presented approach captures information linked to ante-mortem macrostructural brain characteristics. PMID:23440751

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

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    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. © 2013 British Society for Immunology.

  7. Ultrasound shear wave elastography helps discriminate low-grade from high-grade bowel wall fibrosis in ex vivo human intestinal specimens.

    Science.gov (United States)

    Dillman, Jonathan R; Stidham, Ryan W; Higgins, Peter D R; Moons, David S; Johnson, Laura A; Keshavarzi, Nahid R; Rubin, Jonathan M

    2014-12-01

    To determine whether bowel wall fibrosis can be detected in freshly resected human intestinal specimens based on ultrasound-derived shear wave speed. Seventeen intact (>3-cm) bowel segments (15 small and 2 large intestine) from 12 patients with known or suspected inflammatory bowel disease were procured immediately after surgical resection. Ultrasound shear wave elastography of the bowel wall was performed by two methods (Virtual Touch Quantification [VTQ] and Virtual Touch-IQ [VT-IQ]; Siemens Medical Solutions USA, Inc, Mountain View, CA). Eighteen short-axis shear wave speed measurements were acquired from each specimen: 3 from the 9-, 12-, and 3-o'clock locations for each method. Imaging was performed in two areas for specimens greater than 10 cm in length (separated by ≥5 cm). A gastrointestinal pathologist scored correlative histologic slides for inflammation and fibrosis. Differences in mean shear wave speed between bowel segments with low and high inflammation/fibrosis scores were assessed by a Student t test. Receiver operating characteristic curve analysis was performed. High-fibrosis score (n = 11) bowel segments had a significantly greater mean shear wave speed than low-fibrosis score (n = 6) bowel segments (mean ± SD: VTQ, 1.59 ± 0.37 versus 1.18 ± 0.08 m/s; P= .004; VT-IQ, 1.87 ± 0.44 versus 1.50 ± 0.26 m/s; P= .049). There was no significant difference in mean shear wave speed between high-and low-inflammation score bowel segments (P > .05 for both VTQ and VT-IQ). Receiver operating characteristic curves showed areas under the curve of 0.91 (95% confidence interval, 0.67-0.99) for VTQ and 0.77 (95% confidence interval, 0.51-0.94) for VT-IQ in distinguishing low-from high-fibrosis score bowel segments. Ex vivo bowel wall shear wave speed measurements increase when transmural intestinal fibrosis is present. © 2013 by the American Institute of Ultrasound in Medicine.

  8. Insights into intermediate phases of human intestinal fluids visualized by atomic force microscopy and cryo-transmission electron microscopy ex vivo.

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    Müllertz, Anette; Fatouros, Dimitrios G; Smith, James R; Vertzoni, Maria; Reppas, Christos

    2012-02-06

    The current work aims to study at the ultrastructural level the morphological development of colloidal intermediate phases of human intestinal fluids (HIFs) produced during lipid digestion. HIFs were aspirated near the ligament of Treitz early (30 min), Aspirate(early), and 1 h, Aspirate(1h)(ave,comp), after the administration of a heterogeneous liquid meal into the antrum. The composition of the sample aspirated 1 h after meal administration was similar to the average lumenal composition 1 h after meal administration (Aspirate(1h)(ave,comp)). The colloidal structures of individual aspirates and supernatants of aspirates after ultracentrifugation (micellar phase) were characterized by means of atomic force microscopy (AFM) and cryogenic transmission electron microscopy (Cryo-TEM). AFM revealed domain-like structures in Aspirate(early) and both vesicles and large aggregates Aspirate(1h)(ave,comp). Rough surfaces and domains varying in size were frequently present in the micellar phase of both Aspirate(early) and Aspirate(1h)(ave,comp). Cryo-TEM revealed an abundance of spherical micelles and occasionally presented worm-like micelles coexisting with faceted and less defined vesicles in Aspirate(early) and Aspirate(1h)(ave,comp). In Aspirate(1h)(ave,comp) oil droplets were visualized with bilayers closely located to their surface suggesting lipolytic product phases accumulated on the surface of the oil droplet. In the micellar phase of Aspirate(early), Cryo-TEM revealed the presence of spherical micelles, small vesicles, membrane fragments, oil droplets and plate-like structures. In the micellar phase of Aspirate(1h)(ave,comp) the only difference was the absence of oil droplets. Visualization studies previously performed with biorelevant media revealed structural features with many similarities as presented in the current investigation. The impression of the complexity and diversion of these phases has been reinforced with the excessive variation of structural

  9. Human Milk Oligosaccharides and Synthetic Galactosyloligosaccharides Contain 3′-, 4-, and 6′-Galactosyllactose and Attenuate Inflammation in Human T84, NCM-460, and H4 Cells and Intestinal Tissue Ex Vivo12

    Science.gov (United States)

    Ko, Jae Sung; Leone, Serena; Nanthakumar, N Nanda

    2016-01-01

    Background: The immature intestinal mucosa responds excessively to inflammatory insult, but human milk protects infants from intestinal inflammation. The ability of galactosyllactoses [galactosyloligosaccharides (GOS)], newly found in human milk oligosaccharides (HMOS), to suppress inflammation was not known. Objective: The objective was to test whether GOS can directly attenuate inflammation and to explore the components of immune signaling modulated by GOS. Methods: Galactosyllactose composition was measured in sequential human milk samples from days 1 through 21 of lactation and in random colostrum samples from 38 mothers. Immature [human normal fetal intestinal epithelial cell (H4)] and mature [human metastatic colonic epithelial cell (T84) and human normal colon mucosal epithelial cell (NCM-460)] enterocyte cell lines were treated with the pro-inflammatory molecules tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) or infected with Salmonella or Listeria. The inflammatory response was measured as induction of IL-8, monocyte chemoattractant protein 1 (MCP-1), or macrophage inflammatory protein-3α (MIP-3α) protein by ELISA and mRNA by quantitative reverse transcriptase-polymerase chain reaction. The ability of HMOS or synthetic GOS to attenuate this inflammation was tested in vitro and in immature human intestinal tissue ex vivo. Results: The 3 galactosyllactoses (3′-GL, 4-GL, and 6′-GL) expressed in colostrum rapidly declined over early lactation (P milk, contributing to innate immune modulation. The potential clinical utility of galactosyllactose warrants investigation. PMID:26701795

  10. Human Milk Oligosaccharides and Synthetic Galactosyloligosaccharides Contain 3'-, 4-, and 6'-Galactosyllactose and Attenuate Inflammation in Human T84, NCM-460, and H4 Cells and Intestinal Tissue Ex Vivo.

    Science.gov (United States)

    Newburg, David S; Ko, Jae Sung; Leone, Serena; Nanthakumar, N Nanda

    2016-02-01

    The immature intestinal mucosa responds excessively to inflammatory insult, but human milk protects infants from intestinal inflammation. The ability of galactosyllactoses [galactosyloligosaccharides (GOS)], newly found in human milk oligosaccharides (HMOS), to suppress inflammation was not known. The objective was to test whether GOS can directly attenuate inflammation and to explore the components of immune signaling modulated by GOS. Galactosyllactose composition was measured in sequential human milk samples from days 1 through 21 of lactation and in random colostrum samples from 38 mothers. Immature [human normal fetal intestinal epithelial cell (H4)] and mature [human metastatic colonic epithelial cell (T84) and human normal colon mucosal epithelial cell (NCM-460)] enterocyte cell lines were treated with the pro-inflammatory molecules tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) or infected with Salmonella or Listeria. The inflammatory response was measured as induction of IL-8, monocyte chemoattractant protein 1 (MCP-1), or macrophage inflammatory protein-3α (MIP-3α) protein by ELISA and mRNA by quantitative reverse transcriptase-polymerase chain reaction. The ability of HMOS or synthetic GOS to attenuate this inflammation was tested in vitro and in immature human intestinal tissue ex vivo. The 3 galactosyllactoses (3'-GL, 4-GL, and 6'-GL) expressed in colostrum rapidly declined over early lactation (P milk, contributing to innate immune modulation. The potential clinical utility of galactosyllactose warrants investigation. © 2016 American Society for Nutrition.

  11. Susceptibility of anthocyanins to ex vivo degradation in human saliva

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    Kamonpatana, Kom; Giusti, M. Mónica; Chitchumroonchokchai, Chureeporn; MorenoCruz, Maria; Riedl, Ken M.; Kumar, Purnima; Failla, Mark L.

    2012-01-01

    Some fruits and their anthocyanin-rich extracts have been reported to exhibit chemopreventive activity in the oral cavity. Insights regarding oral metabolism of anthocyanins remain limited. Anthocyanin-rich extracts from blueberry, chokeberry, black raspberry, red grape, and strawberry were incubated ex vivo with human saliva from 14 healthy subjects. All anthocyanins were partially degraded in saliva. Degradation of chokeberry anthocyanins in saliva was temperature dependent and decreased by...

  12. Susceptibility of anthocyanins to ex vivo degradation in human saliva

    Science.gov (United States)

    Kamonpatana, Kom; Giusti, M. Mónica; Chitchumroonchokchai, Chureeporn; MorenoCruz, Maria; Riedl, Ken M.; Kumar, Purnima; Failla, Mark L.

    2013-01-01

    Some fruits and their anthocyanin-rich extracts have been reported to exhibit chemopreventive activity in the oral cavity. Insights regarding oral metabolism of anthocyanins remain limited. Anthocyanin-rich extracts from blueberry, chokeberry, black raspberry, red grape, and strawberry were incubated ex vivo with human saliva from 14 healthy subjects. All anthocyanins were partially degraded in saliva. Degradation of chokeberry anthocyanins in saliva was temperature dependent and decreased by heating saliva to 80 °C and after removal of cells. Glycosides of delphinidin and petunidin were more susceptible to degradation than those of cyanidin, pelargonidin, peonidin and malvidin in both intact and artificial saliva. Stability of di- and tri-saccharide conjugates of anthocyanidins slightly, but significantly, exceeded that of monosaccharide compounds. Ex vivo degradation of anthocyanins in saliva was significantly decreased after oral rinsing with antibacterial chlorhexidine. These results suggest that anthocyanin degradation in the mouth is structure-dependent and largely mediated by oral microbiota. PMID:22868153

  13. Validation and Optimization of an Ex Vivo Assay of Intestinal Mucosal Biopsies in Crohn's Disease

    DEFF Research Database (Denmark)

    Vadstrup, Kasper; Galsgaard, Elisabeth Douglas; Gerwien, Jens

    2016-01-01

    are non-responders. This study demonstrates this version of an ex vivo culture as a valid and robust assay to assess inflammation in mucosal biopsies and test of the efficacy of novel drug candidates and current treatments on individual patients-potentially for a personalized medicine approach....... human explant method to test drug candidates and pathophysiological conditions in CD intestinal biopsies. Mucosal biopsies from 27 CD patients and 6 healthy individuals were collected to validate an explant assay test where the polarized tissue was cultured on a novel metal mesh disk, slightly immersed......Crohn's disease (CD) is a chronic illness demanding better therapeutics. The marketed biologics only benefit some patients or elicit diminishing effect over time. To complement the known methods in drug development and to obtain patient specific drug responses, we optimized and validated a known...

  14. Ischemic small intestine-in vivo versus ex vivo bioimpedance measurements.

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    Strand-Amundsen, Runar J; Reims, Henrik M; Tronstad, Christian; Kalvøy, Håvard; Martinsen, Ørjan G; Høgetveit, Jan O; Ruud, Tom E; Tønnessen, Tor I

    2017-05-01

    Bioimpedance has been used to investigate changes in electrical parameters during ischemia in various tissues. The small intestine is a multi-layered structure, with several distinct tissue types, and ischemia related changes occur at different times in the different intestinal layers. When investigating how the electrical properties in the small intestine is affected by ischemia, some researchers have used ex vivo models while others have used in vivo models. In this study, we compare ischemic time development of electrical parameters in ischemic in vivo versus ex vivo small intestine. Measurements were performed using a two-electrode setup, with a Solartron 1260/1294 impedance gain-phase analyser. Electrodes were placed on the surface of ischemic pig jejunum, applying a voltage and measuring the resulting electrical admittance. In each pig, 4 segments of the jejunum were made ischemic by clamping the mesenteric arteries and veins, resulting in a 30 cm central zone of warm ischemia and edema. The in vivo part of the experiment lasted 10 h, after which 3 pieces of perfused small intestine were resected, stored in Ringer-acetat at 38 °C, and measured during a 10 h ex vivo experiment. Main results and significance: We found significant differences (p  vivo and ex vivo measurements as a function of ischemic time development. We also observed some similarities in the trends. In vivo, we measured an overall decrease in impedance during the duration of the experiment, probably as a result from the formation of edema. Ex vivo, the low frequency impedance increased initially for approximately 3 h before starting to decrease.

  15. Susceptibility of anthocyanins to ex vivo degradation in human saliva.

    Science.gov (United States)

    Kamonpatana, Kom; Giusti, M Mónica; Chitchumroonchokchai, Chureeporn; MorenoCruz, Maria; Riedl, Ken M; Kumar, Purnima; Failla, Mark L

    2012-11-15

    Some fruits and their anthocyanin-rich extracts have been reported to exhibit chemopreventive activity in the oral cavity. Insights regarding oral metabolism of anthocyanins remain limited. Anthocyanin-rich extracts from blueberry, chokeberry, black raspberry, red grape, and strawberry were incubated ex vivo with human saliva from 14 healthy subjects. All anthocyanins were partially degraded in saliva. Degradation of chokeberry anthocyanins in saliva was temperature dependent and decreased by heating saliva to 80 °C and after removal of cells. Glycosides of delphinidin and petunidin were more susceptible to degradation than those of cyanidin, pelargonidin, peonidin and malvidin in both intact and artificial saliva. Stability of di- and tri-saccharide conjugates of anthocyanidins slightly, but significantly, exceeded that of monosaccharide compounds. Ex vivo degradation of anthocyanins in saliva was significantly decreased after oral rinsing with antibacterial chlorhexidine. These results suggest that anthocyanin degradation in the mouth is structure-dependent and largely mediated by oral microbiota. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Histological ex vivo analysis of retrieved human tantalum augmentations.

    Science.gov (United States)

    Breer, Stefan; Hahn, Michael; Kendoff, Daniel; Krause, Matthias; Koehne, Till; Haasper, Carl; Gehrke, Thorsten; Amling, Michael; Gebauer, Matthias

    2012-11-01

    The characteristics of tantalum augment osseointegration in human ex vivo specimens from re-revision procedures are unknown and limited data in this regard is available. The purpose of this study was to investigate the osseointegration pattern into porous tantalum augmentations harvested during re-revision procedures. Between 2007 and 2010 a total of 324 hip and knee revisions with a tantalum augmentation were performed in our institution. Out of this cohort, seven patients (2.2 %) had to be re-revised. To analyse the status of trabecular ingrowth in the retrieved cases (four hips, three knees), all specimens were analysed by contact radiography, subjected to undecalcified processing, histology, thin-section analysis and backscattered electron imaging. Trabecular and vascular ingrowth could be found along the bone-augment-interface in two of seven revised specimens, respectively. The depth of bone ingrowth reached up to 2.6 mm. However, the analysis of the remaining cases revealed no bony ingrowth into trabecular metal. Rather, large parts of the implants were embedded in cement or pores were filled with autologous bone. Although the cause for the missing bony ingrowth seems to be multifactorial, some fundamental conditions, such as the provision of the greatest possible interface between the tantalum implant and the host bone, should be met and thus, bone cement and autologous bone grafts should be used with caution.

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

    Science.gov (United States)

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

    2016-04-01

    Ex vivo lung perfusion (EVLP) is a platform to treat infected donor lungs with antibiotic therapy before lung transplantation. Human donor lungs that were rejected for transplantation because of clinical concern regarding infection were randomly assigned to two groups. In the antibiotic group (n = 8), lungs underwent EVLP for 12 h with high-dose antibiotics (ciprofloxacin 400 mg or azithromycin 500 mg, vancomycin 15 mg/kg, and meropenem 2 g). In the control group (n = 7), lungs underwent EVLP for 12 h without antibiotics. A quantitative decrease in bacterial counts in bronchoalveolar lavage (BAL) was found in all antibiotic-treated cases but in only two control cases. Perfusate endotoxin levels at 12 h were significantly lower in the antibiotic group compared with the control group. EVLP with broad-spectrum antibiotic therapy significantly improved pulmonary oxygenation and compliance and reduced pulmonary vascular resistance. Perfusate endotoxin levels at 12 h were strongly correlated with levels of perfusates tumor necrosis factor α, IL-1β and macrophage inflammatory proteins 1α and 1β at 12 h. In conclusion, EVLP treatment of infected donor lungs with broad-spectrum antibiotics significantly reduced BAL bacterial counts and endotoxin levels and improved donor lung function. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

  19. Sorbitol increases muscle glucose uptake ex vivo and inhibits intestinal glucose absorption ex vivo and in normal and type 2 diabetic rats.

    Science.gov (United States)

    Chukwuma, Chika Ifeanyi; Islam, Md Shahidul

    2017-04-01

    Previous studies have suggested that sorbitol, a known polyol sweetener, possesses glycemic control potentials. However, the effect of sorbitol on intestinal glucose absorption and muscle glucose uptake still remains elusive. The present study investigated the effects of sorbitol on intestinal glucose absorption and muscle glucose uptake as possible anti-hyperglycemic or glycemic control potentials using ex vivo and in vivo experimental models. Sorbitol (2.5% to 20%) inhibited glucose absorption in isolated rat jejuna (IC50 = 14.6% ± 4.6%) and increased glucose uptake in isolated rat psoas muscle with (GU50 = 3.5% ± 1.6%) or without insulin (GU50 = 7.0% ± 0.5%) in a concentration-dependent manner. Furthermore, sorbitol significantly delayed gastric emptying, accelerated digesta transit, inhibited intestinal glucose absorption, and reduced blood glucose increase in both normoglycemic and type 2 diabetic rats after 1 h of coingestion with glucose. Data of this study suggest that sorbitol exhibited anti-hyperglycemic potentials, possibly via increasing muscle glucose uptake ex vivo and reducing intestinal glucose absorption in normal and type 2 diabetic rats. Hence, sorbitol may be further investigated as a possible anti-hyperglycemic sweetener.

  20. 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... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell culture...

  1. 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.)

  2. Endoscopic submucosal dissection training with ex vivo human gastric remnants.

    Science.gov (United States)

    Pham, David V; Shah, Anand; Borao, Frank J; Gorcey, Steven

    2014-01-01

    Endoscopic submucosal dissection (ESD) offers en bloc resection of early cancer or precancerous lesions, potentially saving patients from major organ resection, such as gastrectomy, colectomy, and esophagectomy. Japan now leads the world in ESD due to its high rate of gastric cancer. Western countries, with their lower gastric cancer rates, do not get as much experience with the technique. Training in ESD utilizing both in vivo and ex vivo porcine stomach has been shown to decrease rates of perforation and operative time. Both models can be prohibitively expensive or not generally available to the majority of endoscopists on a regular basis. This study describes the framework for using human gastric remnants from sleeve gastrectomy patients for ESD training. Patients undergoing sleeve gastrectomy for morbid obesity were consented for use of their gastric specimen before surgery. The specimen was weighed and measured by the pathologist and then used for ESD training. The specimen was mounted to a 15-mm laparoscopic port and secured using a pursestring suture. ESD was then performed through this port. We were able to successfully use this model to resect multiple marked out lesions in an en bloc fashion. Training using this model has improved our dissection times from approximately 2 h to 30 min for a 2-cm simulated lesion. ESD requires the endoscopist to perform a surgical dissection. Until now, development of these skills required intensive training on porcine models that are not widely available. We were able to create a method using the excised portion from sleeve gastrectomy patients, providing a more accessible and cost-effective model for ESD training and potentially other endoscopic therapeutic modalities.

  3. Ex vivo

    Science.gov (United States)

    Matsuda, Kant M; Lopes-Calcas, Ana; Honke, Michael L; O'Brien-Moran, Zoe; Buist, Richard; West, Michael; Martin, Melanie

    2017-07-01

    To advance magnetic resonance imaging (MRI) technologies further for in vivo tissue characterization with histopathologic validation, we investigated the feasibility of ex vivo tissue imaging of a surgically removed human brain tumor as a comprehensive approach for radiology-pathology correlation in histoanatomically identical fashion in a rare case of pigmented ganglioglioma with complex paramagnetic properties. Pieces of surgically removed ganglioglioma, containing melanin and hemosiderin pigments, were imaged with a small bore 7-T MRI scanner to obtain T1-, T2-, and T2*-weighted image and diffusion tensor imaging (DTI). Corresponding histopathological slides were prepared for routine hematoxylin and eosin stain and special stains for melanin and iron/hemosiderin to correlate with MRI signal characteristics. Furthermore, mean diffusivity (MD) maps were generated from DTI data and correlated with cellularity using image analysis. While the presence of melanin was difficult to interpret in in vivo MRI with certainty due to concomitant hemosiderin pigments and calcium depositions, ex vivo tissue imaging clearly demonstrated pieces of tissue exhibiting the characteristic MR signal pattern for melanin with pathologic confirmation in a histoanatomically identical location. There was also concordant correlation between MD and cellularity. Although it is still in an initial phase of development, ex vivo tissue imaging is a promising approach, which offers radiology-pathology correlation in a straightforward and comprehensive manner.

  4. Functional and Molecular Characterization of Ex Vivo Cultured Epiretinal Membrane Cells from Human Proliferative Diabetic Retinopathy

    Directory of Open Access Journals (Sweden)

    Zoltán Veréb

    2013-01-01

    Full Text Available Characterization of the cell surface marker phenotype of ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs from proliferative diabetic retinopathy (PDR can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherently ex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNFα activation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when cultured ex vivo, which were negative for endothelial cell markers (CD31, VEGFR2, partially positive for hematopoietic- (CD34, CD47 and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFRβ, and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1 were detected upon TNFα activation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDR ex vivo.

  5. Ex Vivo Expanded Human NK Cells Survive and Proliferate in Humanized Mice with Autologous Human Immune Cells.

    Science.gov (United States)

    Vahedi, Fatemeh; Nham, Tina; Poznanski, Sophie M; Chew, Marianne V; Shenouda, Mira M; Lee, Dean; Ashkar, Ali A

    2017-09-21

    Adoptive immune cell therapy is emerging as a promising immunotherapy for cancer. Particularly, the adoptive transfer of NK cells has garnered attention due to their natural cytotoxicity against tumor cells and safety upon adoptive transfer to patients. Although strategies exist to efficiently generate large quantities of expanded NK cells ex vivo, it remains unknown whether these expanded NK cells can persist and/or proliferate in vivo in the absence of exogenous human cytokines. Here, we have examined the adoptive transfer of ex vivo expanded human cord blood-derived NK cells into humanized mice reconstituted with autologous human cord blood immune cells. We report that ex vivo expanded NK cells are able to survive and possibly proliferate in vivo in humanized mice without exogenous cytokine administration, but not in control mice that lack human immune cells. These findings demonstrate that the presence of autologous human immune cells supports the in vivo survival of ex vivo expanded human NK cells. These results support the application of ex vivo expanded NK cells in cancer immunotherapy and provide a translational humanized mouse model to test the lifespan, safety, and functionality of adoptively transferred cells in the presence of autologous human immune cells prior to clinical use.

  6. EX VIVO MODEL OF RABBIT INTESTINAL EPITHELIUM APPLIED TO THE STUDY OF COLONIZATION BY ENTEROAGGREGATIVE ESCHERICHIA COLI

    Directory of Open Access Journals (Sweden)

    Ricardo Luís Lopes BRAGA

    2017-03-01

    Full Text Available ABSTRACT BACKGROUND The diarrheal syndrome is considered a serious public health problem all over the world and is considered a major cause of morbidity and mortality in developing countries. The high incidence of enteroaggregative Escherichia coli in diarrheal syndromes classified as an emerging pathogen of gastrointestinal infections. After decades of study, your pathogenesis remains uncertain and has been investigated mainly using in vitro models of adhesion in cellular lines. OBJECTIVE The present study investigated the interaction of enteroaggregative Escherichia coli strains isolated from childhood diarrhea with rabbit ileal and colonic mucosa ex vivo, using the in vitro organ culture model. METHODS The in vitro adhesion assays using cultured tissue were performed with the strains co-incubated with intestinal fragments of ileum and colon over a period of 6 hours. Each strain was tested with three intestinal fragments for each region. The fragments were analysed by scanning electron microscopy. RESULTS Through scanning electron microscopy we observed that all strains adhered to rabbit ileal and colonic mucosa, with the typical aggregative adherence pattern of “stacked bricks” on the epithelium. However, the highest degree of adherence was observed on colonic mucosa. Threadlike structures were found in greater numbers in the ileum compared to the colon. CONCLUSION These data showed that enteroaggregative Escherichia coli may have a high tropism for the human colon, which was ratified by the higher degree of adherence on the rabbit colonic mucosa. Finally, data indicated that in vitro organ culture of intestinal mucosa from rabbit may be used to elucidate the enteroaggregative Escherichia coli pathogenesis.

  7. 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...... responses were observed but data could be reproduced within individual donors. Monocyte chemoattractant protein-1, a potent basophil secretagogue, did not induce histamine release in lung tissue which indicated mast cells to be the histamine source. Substance P did not release histamine in the lung tissue....... CONCLUSIONS: The microdialysis technique allowed measurements of histamine release from mast cells in intact lung ex vivo. The method may prove useful since a number of experiments can be performed in a few hours in intact lung tissue without any dispersion or enzymatic treatment....

  8. Ex vivo culture of human fetal gonads

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...... fetal testis development after treatment with RA indicates that abnormal meiosis regulation can potentially cause gonadal dysgenesis. Further studies will elucidate the exact mechanisms and timing of observed effects. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by an ESPE Research...

  9. Transfer of meropenem in the ex vivo human placenta perfusion model.

    OpenAIRE

    Roger E. Bawdon; Michael Hnat

    2005-01-01

    OBJECTIVES: To determine maternal-fetal transplacental passage of meropenem in the ex vivo human perfusion model. STUDY DESIGN: Term placentae (n = 6) were collected immediately after delivery. A single cotyledon was localized, perfused and stabilized with physiologic Eagles minimal essential medium containing 3% bovine albumin and heparin as described by Chalier (Chalier JC. Criteria for evaluating perfusion experiments and presentation results. Contrib Gynecol Obstet 1985; 13:32 - 39). Mero...

  10. Transfer of Meropenem in the ex Vivo Human Placenta perfusion Model

    OpenAIRE

    Michael Hnat; Roger E. Bawdon

    2005-01-01

    Objectives. To determine maternal-fetal transplacental passage of meropenem in the ex vivo human perfusion model.Study design. Term placentae (n = 6) were collected immediately after delivery. A single cotyledon was localized, perfused and stabilized with physiologic Eagles minimal essential medium containing 3% bovine albumin and heparin as described by Chalier (Chalier JC. Criteria for evaluating perfusion experiments and presentation results. Contrib Gynecol Obstet 1985; 13:32–39). Meropen...

  11. Ex Vivo Behaviour of Human Bone Tumor Endothelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Infante, Teresa [SDN-Foundation, Institute of Diagnostic and Nuclear Development, IRCCS, 80143 Naples (Italy); Cesario, Elena [Department of Biochemistry and Biophysics, Second University of Naples, 80138 Naples (Italy); Gallo, Michele; Fazioli, Flavio [Division of Skeletal Muscles Oncology Surgery, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); De Chiara, Annarosaria [Anatomic Pathology Unit, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); Tutucci, Cristina; Apice, Gaetano [Medical Oncology of Bone and Soft Sarcoma tissues Unit, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); Nigris, Filomena de, E-mail: filomena.denigris@unina2.it [Department of Biochemistry and Biophysics, Second University of Naples, 80138 Naples (Italy)

    2013-04-11

    Cooperation between endothelial cells and bone in bone remodelling is well established. In contrast, bone microvasculature supporting the growth of primary tumors and metastasis is poorly understood. Several antiangiogenic agents have recently been undergoing trials, although an extensive body of clinical data and experimental research have proved that angiogenic pathways differ in each tumor type and stage. Here, for the first time, we characterize at the molecular and functional level tumor endothelial cells from human bone sarcomas at different stages of disease and with different histotypes. We selected a CD31{sup +} subpopulation from biopsies that displayed the capability to grow as adherent cell lines without vascular endothelial growth factor (VEGF). Our findings show the existence in human primary bone sarcomas of highly proliferative endothelial cells expressing CD31, CD44, CD105, CD146 and CD90 markers. These cells are committed to develop capillary-like structures and colony formation units, and to produce nitric oxide. We believe that a better understanding of tumor vasculature could be a valid tool for the design of an efficacious antiangiogenic therapy as adjuvant treatment of sarcomas.

  12. 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.

  13. Kinetic analysis of ex vivo human blood infection by Leishmania.

    Directory of Open Access Journals (Sweden)

    Inmaculada Moreno

    invasion of human leukocytes is an extremely rapid and efficient reaction, and suggest that the IA reaction constitutes a central strategy for this parasite in subverting host innate immune defenses.

  14. Vaginal Lactobacillus Inhibits HIV-1 Replication in Human Tissues Ex Vivo

    Science.gov (United States)

    Ñahui Palomino, Rogers A.; Zicari, Sonia; Vanpouille, Christophe; Vitali, Beatrice; Margolis, Leonid

    2017-01-01

    Lactobacillus species, which dominate vaginal microbiota of healthy reproductive-age women, lower the risks of sexually transmitted infections, including the risk of human immunodeficiency virus (HIV) acquisition. The exact mechanisms of this protection remain to be understood. Here, we investigated these mechanisms in the context of human cervico-vaginal and lymphoid tissues ex vivo. We found that all six Lactobacillus strains tested in these systems significantly suppressed HIV type-1 (HIV-1) infection. We identified at least three factors that mediated this suppression: (i) Acidification of the medium. The pH of the undiluted medium conditioned by lactobacilli was between 3.8 and 4.6. Acidification of the culture medium with hydrochloric acid (HCl) to this pH in control experiments was sufficient to abrogate HIV-1 replication. However, the pH of the Lactobacillus-conditioned medium (CM) diluted fivefold, which reached ∼6.9, was also suppressive for HIV-1 infection, while in control experiments HIV-1 infection was not abrogated when the pH of the medium was brought to 6.9 through the use of HCl. This suggested the existence of other factors responsible for HIV-1 inhibition by lactobacilli. (ii) Lactic acid. There was a correlation between the concentration of lactic acid in the Lactobacillus-CM and its ability to suppress HIV-1 infection in human tissues ex vivo. Addition of lactic acid isomers D and L to tissue culture medium at the concentration that corresponded to their amount released by lactobacilli resulted in HIV-1 inhibition. Isomer L was produced in higher quantities than isomer D and was mostly responsible for HIV-1 inhibition. These results indicate that lactic acid, in particular its L-isomer, inhibits HIV-1 independently of lowering of the pH. (iii) Virucidal effect. Incubation of HIV-1 in Lactobacillus-CM significantly suppressed viral infectivity for human tissues ex vivo. Finally, lactobacilli adsorb HIV-1, serving as a sink decreasing the

  15. Validation and Optimization of an Ex Vivo Assay of Intestinal Mucosal Biopsies in Crohn's Disease: Reflects Inflammation and Drug Effects.

    Science.gov (United States)

    Vadstrup, Kasper; Galsgaard, Elisabeth Douglas; Gerwien, Jens; Vester-Andersen, Marianne Kajbæk; Pedersen, Julie Steen; Rasmussen, Julie; Neermark, Søren; Kiszka-Kanowitz, Marianne; Jensen, Teis; Bendtsen, Flemming

    2016-01-01

    Crohn's disease (CD) is a chronic illness demanding better therapeutics. The marketed biologics only benefit some patients or elicit diminishing effect over time. To complement the known methods in drug development and to obtain patient specific drug responses, we optimized and validated a known human explant method to test drug candidates and pathophysiological conditions in CD intestinal biopsies. Mucosal biopsies from 27 CD patients and 6 healthy individuals were collected to validate an explant assay test where the polarized tissue was cultured on a novel metal mesh disk, slightly immersed in medium imitating an air-liquid interphase. After culture in high oxygen for 24 hours with or without biological treatment in the medium, biopsy integrity and penetration of antibodies was measured by immunohistochemistry (IHC). Nine cytokines were quantified in the conditioned medium as a read-out for degree of inflammation in individual biopsies and used to evaluate treatment efficacy. The biopsies were well-preserved, showing few structural changes. IHC revealed tissue penetration of antibodies demonstrating ability to test therapeutic antibodies. The cytokine release to the medium showed that the assay can distinguish between inflammation states and then validate the known effect of two treatment biologics confirmed by a detection panel of five specific cytokines. Our data also suggest that the assay would be able to indicate which patients are responders to anti-TNF-α therapeutics, and which are non-responders. This study demonstrates this version of an ex vivo culture as a valid and robust assay to assess inflammation in mucosal biopsies and test of the efficacy of novel drug candidates and current treatments on individual patients-potentially for a personalized medicine approach.

  16. Fatty acids penetration into human skin ex vivo: A TOF-SIMS analysis approach.

    Science.gov (United States)

    Čižinauskas, Vytis; Elie, Nicolas; Brunelle, Alain; Briedis, Vitalis

    2017-03-02

    Linoleic, oleic, palmitoleic, palmitic, and stearic fatty acids (FAs) are commonly used in dermatological formulations. They differ by their structure, presence in the skin, and mode of application in pharmaceuticals and cosmetics compounding. These FAs are also known as chemical penetration enhancers, but their mechanisms of penetration enhancement and effect on barrier characteristics of the skin require additional study. In this study, the authors conducted an ex vivo analysis of the distribution of lipid components in the epidermis and dermis of human skin after applying individual FAs. The goal was to elucidate possible mechanisms of penetration enhancement and FA effects on barrier characteristics of the skin. FA penetration studies were conducted ex vivo on human skin and time-of-flight secondary ion mass spectrometry (TOF-SIMS) bioimaging analysis was performed to visualize and analyze distribution of FAs in skin sections. The current study demonstrated that TOF-SIMS imaging was effective in visualizing the distribution of linoleic, oleic, palmitoleic, palmitic, and stearic acid in the human skin ex vivo after the skin penetration experiment of individual FAs. The integration of the obtained TOF-SIMS images allowed a semiquantitative comparison of the effects induced by individual FA applications on the human skin ex vivo. FAs showed varying abilities to penetrate the skin and disorder the FAs within the skin, based on their structures and physicochemical properties. Linoleic acid penetrated the skin and changed the distribution of all the analyzed FAs. Skin treatment with palmitoleic or oleic acid increased the amounts of singular FAs in the skin. Penetration of saturated FAs was low, but it increased the detected amounts of linoleic acid in both skin layers. The results indicate that application of FAs on the skin surface induce redistribution of native FAs not only in the stratum corneum layer of epidermis but also in the lipid content of full epidermis

  17. Ex Vivo Model of Human Penile Transplantation and Rejection: Implications for Erectile Tissue Physiology.

    Science.gov (United States)

    Sopko, Nikolai A; Matsui, Hotaka; Lough, Denver M; Miller, Devin; Harris, Kelly; Kates, Max; Liu, Xiaopu; Billups, Kevin; Redett, Richard; Burnett, Arthur L; Brandacher, Gerald; Bivalacqua, Trinity J

    2017-04-01

    Penile transplantation is a potential treatment option for severe penile tissue loss. Models of human penile rejection are lacking. Evaluate effects of rejection and immunosuppression on cavernous tissue using a novel ex vivo mixed lymphocyte reaction (MLR) model. Cavernous tissue and peripheral blood mononuclear cells (PBMCs) from 10 patients undergoing penile prosthesis operations and PBMCs from a healthy volunteer were obtained. Ex vivo MLRs were prepared by culturing cavernous tissue for 48h in media alone, in media with autologous PBMCs, or in media with allogenic PBMCs to simulate control, autotransplant, and allogenic transplant conditions with or without 1μM cyclosporine A (CsA) or 20nM tacrolimus (FK506) treatment. Rejection was characterized by PBMC flow cytometry and gene expression transplant array. Cavernous tissues were evaluated by histomorphology and myography to assess contraction and relaxation. Data were analyzed using two-way analysis of variance and unpaired Student t test. Flow cytometry and tissue array demonstrated allogenic PBMC activation consistent with rejection. Rejection impaired cavernous tissue physiology and was associated with cellular infiltration and apoptosis. CsA prevented rejection but did not improve tissue relaxation. CsA treatment impaired relaxation in tissues cultured without PBMCs compared with media and FK506. Study limitations included the use of penile tissue with erectile dysfunction and lack of cross-matching data. This model could be used to investigate the effects of penile rejection and immunosuppression. Additional studies are needed to optimize immunosuppression to prevent rejection and maximize corporal tissue physiology. This report describes a novel ex vivo model of human penile transplantation rejection. Tissue rejection impaired erectile tissue physiology. This report suggests that cyclosporin A might hinder corporal physiology and that other immunosuppressant agents, such as FK506, might be better suited

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

    Directory of Open Access Journals (Sweden)

    Dénes Ádám

    2010-05-01

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

  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. In vivo Prevention of Transplant Arteriosclerosis by ex vivo Expanded Human Regulatory T Cells

    Science.gov (United States)

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

    2010-01-01

    Transplant arteriosclerosis (TA) is the hallmark of chronic allograft dysfunction (CAD) affecting transplanted organs in the long term [1,2]. These fibroproliferative lesions lead to neointimal thickening of arteries in all transplanted allografts [2]. Luminal narrowing then leads to graft ischemia and organ demise. To date, there are no known tolerance induction strategies that prevent TA [3,4]. Therefore, this study was designed to test the hypothesis that human regulatory T cells (Treg cells) expanded ex vivo could prevent TA. Here we show the comparative capacity of Treg cells, sorted via two separate strategies, to prevent TA in a clinically relevant chimeric humanized mouse system. We found that the in vivo development of TA in human arteries was prevented with the treatment of ex vivo expanded human Treg cells. Additionally, we show that Treg cells sorted based on the low expression of CD127 (IL-7Rα) provide a more potent therapy to conventional Treg cells. Our results demonstrate, for the first time, that human Treg cells can inhibit TA by impairing effector function and graft infiltration. We anticipate our findings to serve as a foundation for the clinical development of therapeutics targeting TA in both allograft transplantation and other immune-mediated causes of vasculopathy [5]. PMID:20473306

  1. 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...... studied was either plasmid DNA, naked plant DNA or plant DNA embedded in maize flour. Ex vivo experiments performed by incubating plant DNA in intestinal samples, showed that DNA is rapidly degraded in the upper part of the GI tract whereas degradation is less severe in the lower part. In contrast...

  2. An immunohistological study of anhydrous topical ascorbic acid compositions on ex vivo human skin.

    Science.gov (United States)

    Heber, Geoffrey K; Markovic, Boban; Hayes, Amanda

    2006-06-01

    Ascorbic acid has numerous essential and beneficial functions in normal and photoaged skin. Ionisation of ascorbic acid in aqueous topical formulations leads to oxidative degradation. Ascorbic acid in an anhydrous vehicle would inherently have greater stability. The objective of this study was to observe the effects of two anhydrous formulations containing microfine particles of ascorbic acid on neocollagenesis and cytokeratin production in ex vivo human skin. Vitamin C preparations were applied topically onto the surface of freshly excised human abdominal skin. Following an exposure time of 48 h with appropriate controls, skin discs were cut into sections, placed on slides and assessed using immunohistochemical (antibodies: collagen type I, III, cytokeratin) staining. Analysis was performed using microscopy and descriptive rating. Both formulations resulted in increased production of collagen types I and III and cytokeratin. The application of anhydrous formulations containing microfine particles of ascorbic acid to ex vivo human skin in this study resulted in neocollagenesis and increased production of cytokeratin. This approach appears to enable biological effects of ascorbic acid in the skin using a vehicle which would provide it greater stability than an aqueous vehicle.

  3. MALDI-MS imaging of lipids in ex vivo human skin.

    Science.gov (United States)

    Hart, Philippa J; Francese, Simona; Claude, Emmanuelle; Woodroofe, M Nicola; Clench, Malcolm R

    2011-07-01

    Lipidomics is a rapidly expanding area of scientific research and there are a number of analytical techniques that are employed to facilitate investigations. One such technique is matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Previous MALDI-MS studies involving lipidomic investigation have included the analysis of a number of different ex vivo tissues, most of which were obtained from animal models, with only a few being of human origin. In this study, we describe the use of MALDI-MS, MS/MS and MS imaging methods for analysing lipids within cross-sections of ex vivo human skin. It has been possible to tentatively identify lipid species via accurate mass measurement MALDI-MS and also to confirm the identity of a number of these species via MALDI-MS/MS, in experiments carried out directly on tissue. The main lipid species detected include glycerophospholipids and sphingolipids. MALDI images have been generated at a spatial resolution of 150 and 30 μm, using a MALDI quadrupole time-of-flight Q-Star Pulsar-i (TM) (Applied Biosystems/MDS Sciex, Concord, ON, Canada) and a MALDI high-definition MS (HDMS) SYNAPT G2-HDMS(TM) system (Waters, Manchester, UK), respectively. These images show the normal distribution of lipids within human skin, which will provide the basis for assessing alterations in lipid profiles linked to specific skin conditions e.g. sensitisation, in future investigations.

  4. Ex Vivo Correlation of the Permeability of Metoprolol Across Human and Porcine Buccal Mucosa

    DEFF Research Database (Denmark)

    Meng-Lund, Emil; Marxen, Eva; Pedersen, Anne Marie Lynge

    2014-01-01

    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...

  5. Cryosectioning the intestinal crypt-villus axis: An ex vivo method to study the dynamics of epigenetic modifications from stem cells to differentiated cells

    OpenAIRE

    Vincent, Audrey; Kazmierczak, Catherine; Duchêne, Belinda; Jonckheere, Nicolas; Leteurtre, Emmanuelle; Seuningen, Isabelle Van

    2015-01-01

    The intestinal epithelium is a particularly attractive biological adult model to study epigenetic mechanisms driving adult stem cell renewal and cell differentiation. Since epigenetic modifications are dynamic, we have developed an original ex vivo approach to study the expression and epigenetic profiles of key genes associated with either intestinal cell pluripotency or differentiation by isolating cryosections of the intestinal crypt-villus axis. Gene expression, DNA methylation and histone...

  6. Human ex-vivo action potential model for pro-arrhythmia risk assessment.

    Science.gov (United States)

    Page, Guy; Ratchada, Phachareeya; Miron, Yannick; Steiner, Guido; Ghetti, Andre; Miller, Paul E; Reynolds, Jack A; Wang, Ken; Greiter-Wilke, Andrea; Polonchuk, Liudmila; Traebert, Martin; Gintant, Gary A; Abi-Gerges, Najah

    2016-01-01

    While current S7B/E14 guidelines have succeeded in protecting patients from QT-prolonging drugs, the absence of a predictive paradigm identifying pro-arrhythmic risks has limited the development of valuable drug programs. We investigated if a human ex-vivo action potential (AP)-based model could provide a more predictive approach for assessing pro-arrhythmic risk in man. Human ventricular trabeculae from ethically consented organ donors were used to evaluate the effects of dofetilide, d,l-sotalol, quinidine, paracetamol and verapamil on AP duration (APD) and recognized pro-arrhythmia predictors (short-term variability of APD at 90% repolarization (STV(APD90)), triangulation (ADP90-APD30) and incidence of early afterdepolarizations at 1 and 2Hz to quantitatively identify the pro-arrhythmic risk. Each drug was blinded and tested separately with 3 concentrations in triplicate trabeculae from 5 hearts, with one vehicle time control per heart. Electrophysiological stability of the model was not affected by sequential applications of vehicle (0.1% dimethyl sulfoxide). Paracetamol and verapamil did not significantly alter anyone of the AP parameters and were classified as devoid of pro-arrhythmic risk. Dofetilide, d,l-sotalol and quinidine exhibited an increase in the manifestation of pro-arrhythmia markers. The model provided quantitative and actionable activity flags and the relatively low total variability in tissue response allowed for the identification of pro-arrhythmic signals. Power analysis indicated that a total of 6 trabeculae derived from 2 hearts are sufficient to identify drug-induced pro-arrhythmia. Thus, the human ex-vivo AP-based model provides an integrative translational assay assisting in shaping clinical development plans that could be used in conjunction with the new CiPA-proposed approach. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Ex vivo photometric and polarimetric multilayer characterization of human healthy colon by multispectral Mueller imaging

    Science.gov (United States)

    Pierangelo, Angelo; Manhas, Sandeep; Benali, Abdelali; Fallet, Clément; Antonelli, Maria-Rosaria; Novikova, Tatiana; Gayet, Brice; Validire, Pierre; De Martino, Antonello

    2012-06-01

    Healthy human colon samples were analyzed ex vivo with a multispectral imaging Mueller polarimeter operating from 500 to 700 nm in a backscattering configuration with diffuse light illumination impinging on the innermost tissue layer, the mucosa. The intensity and polarimetric responses were taken on whole tissues first and after progressive exfoliation of the outer layers afterwards. Moreover, these measurements were carried out with two different substrates (one bright and the other dark) successively placed beneath each sample, allowing a reasonably accurate evaluation of the contributions to the overall backscattered light by the various layers. For the shorter investigated wavelengths (500 to 550 nm) the major contribution comes from mucosa and submucosa, while for the longer wavelengths (650 to 700 nm) muscular tissue and fat also contribute significantly. The depolarization has also been studied and is found to be stronger in the red part of the spectrum, mainly due to the highly depolarizing power of the muscular and fat layers.

  8. 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.

  9. Viability of human chondrocytes in an ex vivo model in relation to temperature and cartilage depth.

    Science.gov (United States)

    Drobnic, M; Mars, T; Alibegović, A; Bole, V; Balazic, J; Grubic, Z; Brecelj, J

    2005-01-01

    Chondrocytes in human articular cartilage remain viable post-mortem. It has however not been established yet how the storage temperature affects their survival, which is essential information when post-mortem cartilage is used for toxicologic studies. Our aim was to construct a simple model of explanted knee cartilage and to test the influences of time and temperature on the viability of chondrocytes in the ex vivo conditions. Osteochondral cylinders were procured from the cadaveric femoral condyles. The cylinders were embedded in water-tight rubber tubes, which formed separate chondral and osteal compartments. Tubes were filled with normal saline, without additives, to keep chondrocytes under close-to-normal conditions. The samples were divided into two groups stored at 4 degrees C and 35 degrees C, respectively. Three samples of each of these two groups were analysed at the time of removal, and then three and nine days later. Images of Live-Dead staining were scanned by a confocal laser microscope. Count of viable chondrocytes in four regions, from surface to bone, was obtained using image analysis software. The regression model revealed that the number of viable chondrocytes decreased every day by 19% and that an increase in temperature by 1 degree C decreased their viability by 5.8%. The temperature effect fell by 0.2 percentage points for every 100 microm from the surface to the bone. Herein we demonstrate that chondrocytes remain viable in the ex vivo model of human knee cartilage long enough to be able to serve as a model for toxicologic studies. Their viability is, however, significantly influenced by time and temperature.

  10. Transfer of meropenem in the ex vivo human placenta perfusion model.

    Science.gov (United States)

    Hnat, Michael; Bawdon, Roger E

    2005-12-01

    To determine maternal-fetal transplacental passage of meropenem in the ex vivo human perfusion model. Term placentae (n = 6) were collected immediately after delivery. A single cotyledon was localized, perfused and stabilized with physiologic Eagles minimal essential medium containing 3% bovine albumin and heparin as described by Chalier (Chalier JC. Criteria for evaluating perfusion experiments and presentation results. Contrib Gynecol Obstet 1985; 13:32 - 39). Meropenem was added to the maternal medium in concentrations similar to maternal serum peak and trough levels, then perfused through the maternal circulation of the cotyledon. To assess transfer and accumulation, fluid aliquots from both the maternal and fetal compartments were collected over an hour at defined intervals in an open and closed system. Antipyrine 14C was added to the medium in order to calculate the transport fraction and clearance indexes. Meropenem and antipyrine 14C concentrations were determined by High-pressure Liquid Chromatography and liquid scintillation, respectively. Mean antipyrine transport fraction was 2.33 + 0.25. Maternal and fetal mean meropenem peak concentrations were 54.3 + 3.3 microg/ml and 2.2 + 0.18 microg/ml, respectively. Whereas, maternal and fetal mean trough concentrations were 12.7 + 1.3 microg/ml and 0.41 + 0.10 microg/ml, respectively. Mean peak clearance index was 0.077 + 0.007 and the mean trough was 0.052 + 0.015. Mean accumulation for the peak and trough concentrations of meropenem were 0.9 and 2.95 microg/ml, respectively. Transplacental passage of meropenem was incomplete in the ex vivo human placental perfusion model. Accumulation was also noted in the fetal compartment.

  11. Transplacental passage of vancomycin in the ex vivo human perfusion model.

    Science.gov (United States)

    Hnat, Michael D; Gainer, Julie; Bawdon, Roger E; Wendel, George D

    2004-06-01

    To determine maternal-fetal transplacental passage of vancomycin in the ex vivo human placental perfusion model. Six term placentas were collected immediately after delivery and perfused with physiologic medium using the single cotyledon perfusion system. Vancomycin was added to the maternal medium and perfused through the maternal circulation of the cotyledon. Over a 1-h period in an open system, samples of the perfusate were collected at defined intervals from the fetal venous catheter and from the maternal effluence to assess vancomycin transfer. Thereafter, the system was closed for 1-5 h to establish accumulation. Transport fraction and clearance indexes were calculated by perfusing antipyrine 14C (positive control). Vancomycin was estimated by high pressure liquid chromatography and antipyrine 14C concentration was determined by liquid scintillation. Mean vancomycin maternal peak and trough concentrations ranged from 30.0 to 51.5 microg/ml and 7.7 to 16.4 microg/ml, respectively. Clearance indexes were minimal with a mean peak range of 0.000-0.080 and a mean trough range of 0.00-0.17. For each placenta, transport fraction for antipyrine 14C was > 1.85 with a single pass of > 40%. No accumulation of vancomycin was noted when the system was closed for 1-5 h. The mean peak clearance index was zero after perfusing the placenta for up to 5 h with 35.8 microg/ml of vancomycin. Transplacental passage of vancomycin was minimal in the ex vivo human placental perfusion model, with no detectable accumulation.

  12. Aberrant response to commensal Bacteroides thetaiotaomicron in Crohn's disease: an ex vivo human organ culture study.

    Science.gov (United States)

    Edwards, L A; Lucas, M; Edwards, E A; Torrente, F; Heuschkel, R B; Klein, N J; Murch, S H; Bajaj-Elliott, M; Phillips, A D

    2011-05-01

    Human ex vivo evidence indicating that an inappropriate immune response(s) to nonpathogenic bacteria contributes to disease pathogenesis in pediatric Crohn's disease (CD) is limited. The aim of the present study was to compare and contrast the early innate immune response of pediatric "healthy" versus CD mucosa to pathogenic, probiotic, and commensal bacteria. "Healthy control" and CD pediatric mucosal biopsies (terminal ileum and transverse colon) were cocultured for 8 hours with E. coli O42, Lactobacillus GG (LGG), Bacteroidesthetaiotaomicron (B. theta), or stimulated with interleukin (IL)-1β (positive control). Matched nonstimulated biopsies served as experimental controls. IL-8 was the immune marker of choice. IL-8 mRNA and protein levels were quantified by quantitative polymerase chain reaction and sandwich enzyme-linked immunosorbent assay, respectively. IL-8 secretion was observed when control, ileal biopsies were exposed to pathogenic O42 and probiotic LGG, with no response noted to commensal B. theta. In comparison, Crohn's ileal biopsies showed impaired ability to induce IL-8 in response to O42 and LGG. Control colonic tissue showed a limited response to O42 or B. theta and LGG significantly reduced IL-8 secretion. Unlike control tissue, however, Crohn's ileal and colonic tissue did respond to B. theta, with more enhanced expression in the colon. We provide the first ex vivo data to support the notion that aberrant mucosal recognition of commensal bacteria may contribute to pediatric CD. While IL-8 responses to O42 and LGG varied with disease status and anatomical location, B. theta consistently induced significant IL-8 both in ileal and colonic CD tissue, which was not seen in control, healthy tissue. Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.

  13. 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 (ppolarimetry together with PLS statistics hold promise for automated pathology classification. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Correlations between in vivo (1)H MRS and ex vivo (1)H HRMAS metabolite measurements in adult human gliomas.

    NARCIS (Netherlands)

    Opstad, K.S.; Wright, A.J.; Bell, B.A.; Griffiths, J.R.; Howe, F.A.

    2010-01-01

    PURPOSE: To assess how accurately ex vivo high-resolution magic angle spinning (HRMAS) proton magnetic resonance spectroscopy ((1)H MRS) from small biopsy tissues relate to in vivo (1)H MRS (from larger tumor volumes) in human astrocytomas. MATERIALS AND METHODS: In vivo (PRESS, TE = 30 msec) and ex

  15. Quantitative assessment of human T lymphocytes in RAG2(-/-)gammac(-/-) mice: the impact of ex vivo manipulation on in vivo functionality

    NARCIS (Netherlands)

    van Rijn, Rozemarijn S.; Simonetti, Elles R.; Hagenbeek, Anton; Bonyhadi, Mark; Storm, Gert; Martens, Anton C. M.; Ebeling, Saskia B.

    2007-01-01

    OBJECTIVE: Recent clinical trials of adoptive immunotherapy showed diminished reactivity of human T cells upon ex vivo manipulation. For a safe and effective clinical application of human T cells, it is necessary to improve ex vivo manipulation procedures and evaluate their impact on in vivo

  16. Human fetal liver cells for regulated ex vivo erythropoietin gene therapy

    Directory of Open Access Journals (Sweden)

    Ebtisam El Filali

    2014-01-01

    Full Text Available Possible risks and lack of donor livers limit application of liver transplantation. Liver cell transplantation is, at this moment, not a feasible alternative because engraftment in the liver is poor. Furthermore, there is also shortage of cells suitable for transplantation. Fetal liver cells are able to proliferate in cell culture and could therefore present an alternative source of cells for transplantation. In this study, we investigated the utility of human fetal liver cells for therapeutic protein delivery. We transplanted human fetal liver cells in immunodeficient mice but were not able to detect engraftment of human hepatocytes. In contrast, transplantation of human adult hepatocytes led to detectable engraftment of hepatocytes in murine liver. Transplantation of fetal liver cells did lead to abundant reconstitution of murine liver with human endothelium, indicating that endothelial cells are the most promising cell type for ex vivo liver cell gene therapy. Human liver endothelial cells were subsequently transduced with a lentiviral autoregulatory erythropoietin expression vector. After transplantation in immunodeficient mice, these cells mediated long-term regulation of murine hematocrits. Our study shows the potential of human liver endothelial cells for long-term regulated gene therapy.

  17. In vivo and ex vivo inflammatory markers of common metabolic phenotypes in humans

    DEFF Research Database (Denmark)

    Mærkedahl, Rasmus Baadsgaard; Frøkiær, Hanne; Stenbæk, Marie Grøntved

    2018-01-01

    BACKGROUND: Low-grade systemic inflammation (LGSI) is often characterized by elevated levels of interleukin (IL)6, tumor necrosis factor (TNF)α, and C-reactive protein (CRP). Other serum proteins, ex vivo-stimulated cytokine production, and leukocyte count have, however, also been suggested LGSI-markers...... also correlated with leukocyte count. Ex vivo-produced cytokines were intercorrelated and correlated with leukocyte count, but did not correlate with the serum immune markers. MS score, body mass index, and glycated hemoglobin (HbA1c) were associated with 8%-16% higher inflammatory score per standard...... fasting serum markers of LGSI and leukocyte counts associated best with measures of MS-associated LGSI, whereas ex vivo cytokine production was only associated with prevailing glycemia and dyslipidemia. Taken together, this indicates that the relationship between in vivo and ex vivo inflammatory markers...

  18. Flagellin Induces β-Defensin 2 in Human Colonic Ex vivo Infection with Enterohemorrhagic Escherichia coli.

    Science.gov (United States)

    Lewis, Steven B; Prior, Alison; Ellis, Samuel J; Cook, Vivienne; Chan, Simon S M; Gelson, William; Schüller, Stephanie

    2016-01-01

    Enterohemorrhagic E.coli (EHEC) is an important foodborne pathogen in the developed world and can cause life-threatening disease particularly in children. EHEC persists in the human gut by adhering intimately to colonic epithelium and forming characteristic attaching/effacing lesions. In this study, we investigated the innate immune response to EHEC infection with particular focus on antimicrobial peptide and protein expression by colonic epithelium. Using a novel human colonic biopsy model and polarized T84 colon carcinoma cells, we found that EHEC infection induced expression of human β-defensin 2 (hBD2), whereas hBD1, hBD3, LL-37, and lysozyme remained unchanged. Infection with specific EHEC deletion mutants demonstrated that this was dependent on flagellin, and apical exposure to purified flagellin was sufficient to stimulate hBD2 and also interleukin (IL)-8 expression ex vivo and in vitro. Flagellin-mediated hBD2 induction was significantly reduced by inhibitors of NF-κB, MAP kinase p38 and JNK but not ERK1/2. Interestingly, IL-8 secretion by polarized T84 cells was vectorial depending on the side of stimulation, and apical exposure to EHEC or flagellin resulted in apical IL-8 release. Our results demonstrate that EHEC only induces a modest immune response in human colonic epithelium characterized by flagellin-dependent induction of hBD2 and low levels of IL-8.

  19. Innovations in preclinical biology: ex vivo engineering of a human kidney tissue microperfusion system.

    Science.gov (United States)

    Kelly, Edward J; Wang, Zhican; Voellinger, Jenna L; Yeung, Cathy K; Shen, Danny D; Thummel, Kenneth E; Zheng, Ying; Ligresti, Giovanni; Eaton, David L; Muczynski, Kimberly A; Duffield, Jeremy S; Neumann, Thomas; Tourovskaia, Anna; Fauver, Mark; Kramer, Greg; Asp, Elizabeth; Himmelfarb, Jonathan

    2013-01-01

    Kidney disease is a public health problem that affects more than 20 million people in the US adult population, yet little is understood about the impact of kidney disease on drug disposition. Consequently there is a critical need to be able to model the human kidney and other organ systems, to improve our understanding of drug efficacy, safety, and toxicity, especially during drug development. The kidneys in general, and the proximal tubule specifically, play a central role in the elimination of xenobiotics. With recent advances in molecular investigation, considerable information has been gathered regarding the substrate profiles of the individual transporters expressed in the proximal tubule. However, we have little knowledge of how these transporters coupled with intracellular enzymes and influenced by metabolic pathways form an efficient secretory and reabsorptive mechanism in the renal tubule. Proximal tubular secretion and reabsorption of xenobiotics is critically dependent on interactions with peritubular capillaries and the interstitium. We plan to robustly model the human kidney tubule interstitium, utilizing an ex vivo three-dimensional modular microphysiological system with human kidney-derived cells. The microphysiological system should accurately reflect human physiology, be usable to predict renal handling of xenobiotics, and should assess mechanisms of kidney injury, and the biological response to injury, from endogenous and exogenous intoxicants.

  20. Ex-vivo UV autofluorescence imaging and fluorescence spectroscopy of atherosclerotic pathology in human aorta

    Science.gov (United States)

    Lewis, William; Williams, Maura; Franco, Walfre

    2017-02-01

    The aim of our study was to identify fluorescence excitation-emission pairs correlated with atherosclerotic pathology in ex-vivo human aorta. Wide-field images of atherosclerotic human aorta were captured using UV and visible excitation and emission wavelength pairs of several known fluorophores to investigate correspondence with gross pathologic features. Fluorescence spectroscopy and histology were performed on 21 aortic samples. A matrix of Pearson correlation coefficients were determined for the relationship between relevant histologic features and the intensity of emission for 427 wavelength pairs. A multiple linear regression analysis indicated that elastin (370/460 nm) and tryptophan (290/340 nm) fluorescence predicted 58% of the variance in intima thickness (R-squared = 0.588, F(2,18) = 12.8, p=.0003), and 48% of the variance in media thickness (R-squared = 0.483, F(2,18) = 8.42, p=.002), suggesting that endogenous fluorescence intensity at these wavelengths can be utilized for improved pathologic characterization of atherosclerotic plaques.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Effective thermal penetration depth in photo-irradiated ex vivo human tissues.

    Science.gov (United States)

    Stolik, Suren; Delgado, José Alberto; Anasagasti, Lorenzo; Pérez, Arllene Mariana

    2011-10-01

    In this work, a model of bioheat distribution is discussed for ex vivo human tissue samples, and the thermal penetration depth measurements performed on several tissues are presented. Optical radiation is widely applied in the treatment and diagnosis of different pathologies. A power density of incident light at 100 mW/cm(2) is sufficiently high enough to induce a temperature increase of >5°C in irradiated human tissue. In this case, knowledge of the thermal properties of the tissue is needed to achieve a better understanding of the therapeutic effects. The application of the diffusion approximation of the radiative transfer equation for the distribution of optical radiation, the experimental setup, and the results thereof are presented and discussed. The effective thermal penetration depth in the studied tissues has been determined to be in the range of 4.3-7.0 mm. The effective thermal penetration depth has been defined, and this could be useful for developing models to describe the thermal effects with a separate analysis of the tissue itself and the blood that irrigates it.

  3. Human whole-blood culture system for ex vivo characterization of designer-cell function.

    Science.gov (United States)

    Schukur, Lina; Geering, Barbara; Fussenegger, Martin

    2016-03-01

    Encapsulated designer cells implanted into mice are currently used to validate the efficacy of therapeutic gene networks for the diagnosis and treatment of various human diseases in preclinical research. Because many human conditions cannot be adequately replicated by animal models, complementary and alternative procedures to test future treatment strategies are required. Here we describe a novel approach utilizing an ex vivo human whole-blood culture system to validate synthetic biology-inspired designer cell-based treatment strategies. The viability and functionality of transgenic mammalian designer cells co-cultured with primary human immune cells were characterized. We demonstrated that transgenic mammalian designer cells required adequate insulation from the human blood microenvironment to maintain viability and functionality. The biomaterial alginate-(poly-l-lysine)-alginate used to encapsulate the transgenic designer cells did neither affect the viability of primary granulocytes and lymphocytes nor the functionality of lymphocytes. Additionally, alginate-encapsulated transgenic designer cells remained responsive to the release of the pro-inflammatory cytokine tumor necrosis factor (TNF) from the whole-blood culture upon exposure to bacterial lipopolysaccharide (LPS). TNF diffused into the alginate capsules, bound to the specific TNF receptors on the transgenic designer cells' surface and triggered the expression of the reporter gene SEAP (human placental secreted alkaline phosphatase) that was rewired to the TNF-specific signaling cascade. Human whole-blood culture systems can therefore be considered as valuable complementary assays to animal models for the validation of synthetic circuits in genetically modified mammalian cells and may speed up preclinical research in a world of personalized medicine. © 2015 Wiley Periodicals, Inc.

  4. Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis.

    Science.gov (United States)

    Pinnock, Abigail; Shivshetty, Nagaveni; Roy, Sanhita; Rimmer, Stephen; Douglas, Ian; MacNeil, Sheila; Garg, Prashant

    2017-02-01

    In the study of microbial keratitis, in vivo animal models often require a large number of animals, and in vitro monolayer cell culture does not maintain the three-dimensional structure of the tissues or cell-to-cell communication of in vivo models. Here, we propose reproducible ex vivo models of single- and dual-infection keratitis as an alternative to in vivo and in vitro models. Excised rabbit and human corneoscleral rims maintained in organ culture were infected using 108 cells of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans or Fusarium solani. The infection was introduced by wounding with a scalpel and exposing corneas to the microbial suspension or by intrastromal injection. Post-inoculation, corneas were maintained for 24 and 48 h at 37 °C. After incubation, corneas were either homogenised to determine colony-forming units (CFU)/cornea or processed for histological examination using routine staining methods. Single- and mixed-species infections were compared. We observed a significant increase in CFU after 48 h compared to 24 h with S. aureus and P. aeruginosa. However, no such increase was observed in corneas infected with C. albicans or F. solani. The injection method yielded an approximately two- to 100-fold increase (p keratitis, particularly when this might be due to two infective organisms.

  5. Effectiveness of hand washing on the removal of iron oxide nanoparticles from human skin ex vivo.

    Science.gov (United States)

    Lewinski, Nastassja A; Berthet, Aurélie; Maurizi, Lionel; Eisenbeis, Antoine; Hopf, Nancy B

    2017-08-01

    In this study, the effectiveness of washing with soap and water in removing nanoparticles from exposed skin was investigated. Dry, nanoscale hematite (α-Fe2O3) or maghemite (γ-Fe2O3) powder, with primary particle diameters between 20-30 nm, were applied to two samples each of fresh and frozen ex vivo human skin in two independent experiments. The permeation of nanoparticles through skin, and the removal of nanoparticles after washing with soap and water were investigated. Bare iron oxide nanoparticles remained primarily on the surface of the skin, without penetrating beyond the stratum corneum. Skin exposed to iron oxide nanoparticles for 1 and 20 hr resulted in removal of 85% and 90%, respectively, of the original dose after washing. In the event of dermal exposure to chemicals, removal is essential to avoid potential local irritation or permeation across skin. Although manufactured at an industrial scale and used extensively in laboratory experiments, limited data are available on the removal of engineered nanoparticles after skin contact. Our finding raises questions about the potential consequences of nanoparticles remaining on the skin and whether alternative washing methods should be proposed. Further studies on skin decontamination beyond use of soap and water are needed to improve the understanding of the potential health consequences of dermal exposure to nanoparticles.

  6. Recombinant Lucilia sericata chymotrypsin in a topical hydrogel formulation degrades human wound eschar ex vivo.

    Science.gov (United States)

    Britland, Stephen; Smith, Annie; Finter, Wayne; Eagland, Donald; Vowden, Kath; Vowden, Peter; Telford, Gary; Brown, Alan; Pritchard, David

    2011-01-01

    Larval biotherapy is a debridement tool used in wound management. The mechanism of action involves degradation of eschar by serine proteases including chymotrypsin within the alimentary fluids of first instar Lucilia sericata. With the rationale of obviating some limitations of biotherapy, including cost, complexity of use, and patient reticence, the present study describes a mobile hydrogel formulation containing freeze-dried recombinant L. sericata chymotrypsin designed for topical application. Neither freeze-drying nor formulation into the hydrogel significantly attenuated the measured activity of released enzyme compared to fresh-frozen enzyme in aqueous solution. Gel electrophoresis confirmed qualitatively that the chymotrypsin/hydrogel formulation both with and without supplementary urea at 10% (w) /(v) degraded human chronic wound eschar ex vivo. Mindful that the hallmark of intractability of chronic wounds is aberrant biochemistry, the pH activity profile for the enzyme/hydrogel formulation was compared with exudate pH in chronic wounds of mixed aetiology in a cohort of 48 hospital in-patients. Five patients' wounds were acidic, however, the remainder were predominantly alkaline and coincided with the pH optimum for the insect enzyme. Thus, a recombinant L. sericata chymotrypsin and hydrogel formulation could represent a pragmatic alternative to larval therapy for the management of chronic wounds. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

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

    Science.gov (United States)

    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

    2015-01-01

    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. PMID:26712205

  8. Evaluation of superporous hydrogel (SPH) and SPH composite in porcine intestine ex-vivo: assessment of drug transport, morphology effect, and mechanical fixation to intestinal wall.

    Science.gov (United States)

    Dorkoosh, Farid A; Borchard, Gerrit; Rafiee-Tehrani, Morteza; Verhoef, J Coos; Junginger, Hans E

    2002-03-01

    The objective of this study was to investigate the potential of superporous hydrogel (SPH) and SPH composite (SPHC) polymers to enhance the transport of N-alpha-benzoyl-L-arginine ethylester (BAEE) and fluorescein isothiocyanate-dextran 4400 (FD4) across porcine intestinal epithelium ex-vivo, and to study any possible morphological damage to the epithelium by applying these polymers. In addition, the ability of these polymers to attach to the gut wall by mechanical pressure was examined by using a specifically designed centrifuge model. The transport of BAEE and FD4 across the intestinal mucosa was enhanced 2- to 3-fold by applying SPHC polymer in comparison to negative control. No significant morphological damage was observed by applying these polymers inside the intestinal lumen. Moreover, the SPH and SPHC polymers were able to attach mechanically to the intestinal wall by swelling and did not move in the intestinal lumen even when a horizontal force of 13 gms(-2) was applied. In conclusion, these polymers are appropriate vehicles for enhancing the intestinal absorption of peptide and protein drugs.

  9. Ex vivo

    Science.gov (United States)

    Liang, Tao; Dolai, Subhankar; Xie, Li; Winter, Erin; Orabi, Abrahim I; Karimian, Negar; Cosen-Binker, Laura I; Huang, Ya-Chi; Thorn, Peter; Cattral, Mark S; Gaisano, Herbert Y

    2017-04-07

    A genuine understanding of human exocrine pancreas biology and pathobiology has been hampered by a lack of suitable preparations and reliance on rodent models employing dispersed acini preparations. We have developed an organotypic slice preparation of the normal portions of human pancreas obtained from cancer resections. The preparation was assessed for physiologic and pathologic responses to the cholinergic agonist carbachol (Cch) and cholecystokinin (CCK-8), including 1) amylase secretion, 2) exocytosis, 3) intracellular Ca 2+ responses, 4) cytoplasmic autophagic vacuole formation, and 5) protease activation. Cch and CCK-8 both dose-dependently stimulated secretory responses from human pancreas slices similar to those previously observed in dispersed rodent acini. Confocal microscopy imaging showed that these responses were accounted for by efficient apical exocytosis at physiologic doses of both agonists and by apical blockade and redirection of exocytosis to the basolateral plasma membrane at supramaximal doses. The secretory responses and exocytotic events evoked by CCK-8 were mediated by CCK-A and not CCK-B receptors. Physiologic agonist doses evoked oscillatory Ca 2+ increases across the acini. Supraphysiologic doses induced formation of cytoplasmic autophagic vacuoles and activation of proteases (trypsin, chymotrypsin). Maximal atropine pretreatment that completely blocked all the Cch-evoked responses did not affect any of the CCK-8-evoked responses, indicating that rather than acting on the nerves within the pancreas slice, CCK cellular actions directly affected human acinar cells. Human pancreas slices represent excellent preparations to examine pancreatic cell biology and pathobiology and could help screen for potential treatments for human pancreatitis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  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

    . 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......The placental passage of three compounds with different physicochemical properties was recently investigated in ex vivo human placental perfusion experiments (caffeine, benzoic acid, and glyphosate) [Mose, T., Kjaerstad, M.B., Mathiesen, L., Nielsen, J.B., Edelfors, S., Knudsen, L.E., 2008...... 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. Metabolic profiling during ex vivo machine perfusion of the human liver

    NARCIS (Netherlands)

    Bruinsma, Bote G.; Sridharan, Gautham V.; Weeder, Pepijn D.; Avruch, James H.; Saeidi, Nima; Oezer, Sinan; Geerts, Sharon; Porte, Robert J.; Heger, Michal; van Gulik, Thomas M.; Martins, Paulo N.; Markmann, James F.; Yeh, Heidi; Uygun, Korkut

    2016-01-01

    As donor organ shortages persist, functional machine perfusion is under investigation to improve preservation of the donor liver. The transplantation of donation after circulatory death (DCD) livers is limited by poor outcomes, but its application may be expanded by ex vivo repair and assessment of

  12. Visualization of ex vivo human ciliated epithelium and induced flow using optical coherence tomography (Conference Presentation)

    Science.gov (United States)

    Ling, Yuye; Gamm, Uta A.; Yao, Xinwen; Arteaga-Solis, Emilio; Emala, Charles W.; Choma, Michael A.; Hendon, Christine P.

    2017-04-01

    The ciliated epithelium is important to the human respiratory system because it clears mucus that contains harmful microorganisms and particulate matter. We report the ex vivo visualization of human trachea/bronchi ciliated epithelium and induced flow characterized by using spectral-domain optical coherence tomography (SD-OCT). A total number of 17 samples from 7 patients were imaged. Samples were obtained from Columbia University Department of Anesthesiology's tissue bank. After excision, the samples were placed in Gibco Medium 199 solution with oxygen at 4°C until imaging. The samples were maintained at 36.7°C throughout the experiment. The imaging protocol included obtaining 3D volumes and 200 consecutive B-scans parallel to the head-to-feet direction (superior-inferior axis) of the airway, using Thorlabs Telesto system at 1300 nm at 28 kHz A-line rate and a custom built high resolution SDOCT system at 800nm at 32 kHz A-line rate. After imaging, samples were processed with H and E histology. Speckle variance of the time resolved datasets demonstrate significant contrast at the ciliated epithelium sites. Flow images were also obtained after injecting 10μm polyester beads into the solution, which shows beads traveling trajectories near the ciliated epithelium areas. In contrary, flow images taken in the orthogonal plane show no beads traveling trajectories. This observation is in line with our expectation that cilia drive flow predominantly along the superior-inferior axis. We also observed the protective function of the mucus, shielding the epithelium from the invasion of foreign objects such as microspheres. Further studies will be focused on the cilia's physiological response to environmental changes such as drug administration and physical injury.

  13. Irrigation of human prepared root canal – ex vivo based computational fluid dynamics analysis

    Science.gov (United States)

    Šnjarić, Damir; Čarija, Zoran; Braut, Alen; Halaji, Adelaida; Kovačević, Maja; Kuiš, Davor

    2012-01-01

    Aim To analyze the influence of the needle type, insertion depth, and irrigant flow rate on irrigant flow pattern, flow velocity, and apical pressure by ex-vivo based endodontic irrigation computational fluid dynamics (CFD) analysis. Methods Human upper canine root canal was prepared using rotary files. Contrast fluid was introduced in the root canal and scanned by computed tomography (CT) providing a three-dimensional object that was exported to the computer-assisted design (CAD) software. Two probe points were established in the apical portion of the root canal model for flow velocity and pressure measurement. Three different CAD models of 27G irrigation needles (closed-end side-vented, notched open-end, and bevel open-end) were created and placed at 25, 50, 75, and 95% of the working length (WL). Flow rates of 0.05, 0.1, 0.2, 0.3, and 0.4 mL/s were simulated. A total of 60 irrigation simulations were performed by CFD fluid flow solver. Results Closed-end side-vented needle required insertion depth closer to WL, regarding efficient irrigant replacement, compared to open-end irrigation needle types, which besides increased velocity produced increased irrigant apical pressure. For all irrigation needle types and needle insertion depths, the increase of flow rate was followed by an increased irrigant apical pressure. Conclusions The human root canal shape obtained by CT is applicable in the CFD analysis of endodontic irrigation. All the analyzed values –irrigant flow pattern, velocity, and pressure – were influenced by irrigation needle type, as well as needle insertion depth and irrigant flow rate. PMID:23100209

  14. PAR4 (Protease-Activated Receptor 4) Antagonism With BMS-986120 Inhibits Human Ex Vivo Thrombus Formation.

    Science.gov (United States)

    Wilson, Simon J; Ismat, Fraz A; Wang, Zhaoqing; Cerra, Michael; Narayan, Hafid; Raftis, Jennifer; Gray, Timothy J; Connell, Shea; Garonzik, Samira; Ma, Xuewen; Yang, Jing; Newby, David E

    2018-02-01

    BMS-986120 is a novel first-in-class oral PAR4 (protease-activated receptor 4) antagonist with potent and selective antiplatelet effects. We sought to determine for the first time, the effect of BMS-986120 on human ex vivo thrombus formation. Forty healthy volunteers completed a phase 1 parallel-group PROBE trial (Prospective Randomized Open-Label Blinded End Point). Ex vivo platelet activation, platelet aggregation, and thrombus formation were measured at 0, 2, and 24 hours after (1) oral BMS-986120 (60 mg) or (2) oral aspirin (600 mg) followed at 18 hours with oral aspirin (600 mg) and oral clopidogrel (600 mg). BMS-986120 demonstrated highly selective and reversible inhibition of PAR4 agonist peptide (100 μM)-stimulated P-selectin expression, platelet-monocyte aggregates, and platelet aggregation (PURL: http://www.clinicaltrials.gov. Unique identifier: NCT02439190. © 2017 The Authors.

  15. Ex vivo intestinal adhesion of Escherichia coli LF82 in Crohn’s disease

    DEFF Research Database (Denmark)

    Jensen, Stina Rikke; Fink, Lisbeth Nielsen; Nielsen, Ole Haagen

    2011-01-01

    Adherent-invasive Escherichia coli (AIEC) are reported to inhabit the gut mucosa in Crohn’s disease (CD), however, little is known about the importance of host factors for the interplay between AIEC and the human gut.To examine if differences in bacterial adhesion patterns are disease associated......, the AIEC-prototype strain LF82 was evaluated for its ability to adhere to ileal and colonic biopsies from CD and healthy controls (HC). Moreover, the efficacy of the non-pathogenic E. coli Nissle 1917 (ECN) in averting LF82 adhesion to ileal mucosa was assessed.Similar numbers of LF82 adhered to biopsies...... from CD and HC. A significantly greater LF82 attachment to ileal versus colonic mucosa was found in HC (P adhesion of LF82 to ileal specimens in CD or HC.These results show that enhanced bacterial adhesion ability is unlikely to play any significant...

  16. An automated discontinuous venous blood sampling system for ex vivo glucose determination in humans.

    Science.gov (United States)

    Schaller, Roland; Feichtner, Franz; Köhler, Hans; Bodenlenz, Manfred; Plank, Johannes; Wutte, Andrea; Mader, Julia K; Ellmerer, Martin; Hainisch, Reinhard; Pieber, Thomas R; Schaupp, Lukas

    2009-01-01

    Intensive insulin therapy reduces mortality and morbidity in critically ill patients but places great demands on medical staff who must take frequent blood samples for the determination of glucose levels. A cost-effective solution to this resourcing problem could be provided by an effective and reliable automated blood sampling (ABS) system suitable for ex vivo glucose determination. The primary study aim was to compare the performance of a prototype ABS system with a manual reference system over a 30 h sampling period under controlled conditions in humans. Two venous cannulae were inserted to connect the ABS system and the reference system. Blood samples were taken with both systems at 15, 30, and 60 min intervals and analyzed using a Beckman glucose analyzer. During the study, blood glucose levels were altered through four meal ingestions. The median Pearson coefficient of correlation between manually and automatically withdrawn blood samples was 0.976 (0.953-0.996). The system error was -3.327 ± 5.546% (-6.03-0.49). Through Clark error grid analysis, 420 data pairs were analyzed, showing that 98.6% of the data were in zone A and 1.4% were in zone B. Insulin titration error grid analysis revealed an acceptable treatment in 100% of cases. A 17.5-fold reduction in the occurrence of blood-withdrawal failures through occluded catheters was moreover achieved by the added implementation in the ABS system of a "keep vein open" saline infusion. Our study showed that the ABS system described provides a user-friendly, reliable automated means for reproducible and accurate blood sampling from a peripheral vein for blood glucose determination and thus represents a promising alternative to frequent manual blood sampling. © Diabetes Technology Society

  17. Antiandrogenic actions of medroxyprogesterone acetate on epithelial cells within normal human breast tissues cultured ex vivo.

    Science.gov (United States)

    Ochnik, Aleksandra M; Moore, Nicole L; Jankovic-Karasoulos, Tanja; Bianco-Miotto, Tina; Ryan, Natalie K; Thomas, Mervyn R; Birrell, Stephen N; Butler, Lisa M; Tilley, Wayne D; Hickey, Theresa E

    2014-01-01

    Medroxyprogesterone acetate (MPA), a component of combined estrogen-progestin therapy (EPT), has been associated with increased breast cancer risk in EPT users. MPA can bind to the androgen receptor (AR), and AR signaling inhibits cell growth in breast tissues. Therefore, the aim of this study was to investigate the potential of MPA to disrupt AR signaling in an ex vivo culture model of normal human breast tissue. Histologically normal breast tissues from women undergoing breast surgical operation were cultured in the presence or in the absence of the native AR ligand 5α-dihydrotestosterone (DHT), MPA, or the AR antagonist bicalutamide. Ki67, bromodeoxyuridine, B-cell CLL/lymphoma 2 (BCL2), AR, estrogen receptor α, and progesterone receptor were detected by immunohistochemistry. DHT inhibited the proliferation of breast epithelial cells in an AR-dependent manner within tissues from postmenopausal women, and MPA significantly antagonized this androgenic effect. These hormonal responses were not commonly observed in cultured tissues from premenopausal women. In tissues from postmenopausal women, DHT either induced or repressed BCL2 expression, and the antiandrogenic effect of MPA on BCL2 was variable. MPA significantly opposed the positive effect of DHT on AR stabilization, but these hormones had no significant effect on estrogen receptor α or progesterone receptor levels. In a subset of postmenopausal women, MPA exerts an antiandrogenic effect on breast epithelial cells that is associated with increased proliferation and destabilization of AR protein. This activity may contribute mechanistically to the increased risk of breast cancer in women taking MPA-containing EPT.

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

    Directory of Open Access Journals (Sweden)

    Lang Dagmar S

    2007-06-01

    Full Text Available Abstract Background Non-small cell lung cancer (NSCLC causes most of cancer related deaths in humans and is characterized by poor prognosis regarding efficiency of chemotherapeutical treatment and long-term survival of the patients. The purpose of the present study was the development of a human ex vivo tissue culture model and the analysis of the effects of conventional chemotherapy, which then can serve as a tool to test new chemotherapeutical regimens in NSCLC. Methods In a short-term tissue culture model designated STST (Short-Term Stimulation of Tissues in combination with the novel *HOPE-fixation and paraffin embedding method we examined the responsiveness of 41 human NSCLC tissue specimens to the individual cytotoxic drugs carboplatin, vinorelbine or gemcitabine. Viability was analyzed by LIFE/DEAD assay, TUNEL-staining and colorimetric MTT assay. Expression of Ki-67 protein and of BrdU (bromodeoxyuridine uptake as markers for proliferation and of cleaved (activated effector caspase-3 as indicator of late phase apoptosis were assessed by immunohistochemistry. Transcription of caspase-3 was analyzed by RT-PCR. Flow cytometry was utilized to determine caspase-3 in human cancer cell lines. Results Viability, proliferation and apoptosis of the tissues were moderately affected by cultivation. In human breast cancer, small-cell lung cancer (SCLC and human cell lines (CPC-N, HEK proliferative capacity was clearly reduced by all 3 chemotherapeutic agents in a very similar manner. Cleavage of caspase-3 was induced in the chemo-sensitive types of cancer (breast cancer, SCLC. Drug-induced effects in human NSCLC tissues were less evident than in the chemo-sensitive tumors with more pronounced effects in adenocarcinomas as compared to squamous cell carcinomas. Conclusion Although there was high heterogeneity among the individual tumor tissue responses as expected, we clearly demonstrate specific multiple drug-induced effects simultaneously. Thus, STST

  19. 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.

  20. Human Lung Cancer Cells Grown in an Ex Vivo 3D Lung Model Produce Matrix Metalloproteinases Not Produced in 2D Culture

    Science.gov (United States)

    Mishra, Dhruva K.; Sakamoto, Jason H.; Thrall, Michael J.; Baird, Brandi N.; Blackmon, Shanda H.; Ferrari, Mauro; Kurie, Jonathan M.; Kim, Min P.

    2012-01-01

    We compared the growth of human lung cancer cells in an ex vivo three-dimensional (3D) lung model and 2D culture to determine which better mimics lung cancer growth in patients. A549 cells were grown in an ex vivo 3D lung model and in 2D culture for 15 days. We measured the size and formation of tumor nodules and counted the cells after 15 days. We also stained the tissue/cells for Ki-67, and Caspase-3. We measured matrix metalloproteinase (MMP) levels in the conditioned media and in blood plasma from patients with adenocarcinoma of the lung. Organized tumor nodules with intact vascular space formed in the ex vivo 3D lung model but not in 2D culture. Proliferation and apoptosis were greater in the ex vivo 3D lung model compared to the 2D culture. After 15 days, there were significantly more cells in the 2D culture than the 3D model. MMP-1, MMP-9, and MMP-10 production were significantly greater in the ex vivo 3D lung model. There was no production of MMP-9 in the 2D culture. The patient samples contained MMP-1, MMP-2, MMP-9, and MMP-10. The human lung cancer cells grown on ex vivo 3D model form perfusable nodules that grow over time. It also produced MMPs that were not produced in 2D culture but seen in human lung cancer patients. The ex vivo 3D lung model may more closely mimic the biology of human lung cancer development than the 2D culture. PMID:23028922

  1. Retinoic acid and rapamycin differentially affect and synergistically promote the ex vivo expansion of natural human T regulatory cells.

    Directory of Open Access Journals (Sweden)

    Tatiana N Golovina

    Full Text Available Natural T regulatory cells (Tregs are challenging to expand ex vivo, and this has severely hindered in vivo evaluation of their therapeutic potential. All trans retinoic acid (ATRA plays an important role in mediating immune homeostasis in vivo, and we investigated whether ATRA could be used to promote the ex vivo expansion of Tregs purified from adult human peripheral blood. We found that ATRA helped maintain FOXP3 expression during the expansion process, but this effect was transient and serum-dependent. Furthermore, natural Tregs treated with rapamycin, but not with ATRA, suppressed cytokine production in co-cultured effector T cells. This suppressive activity correlated with the ability of expanded Tregs to induce FOXP3 expression in non-Treg cell populations. Examination of CD45RA+ and CD45RA- Treg subsets revealed that ATRA failed to maintain suppressive activity in either population, but interestingly, Tregs expanded in the presence of both rapamycin and ATRA displayed more suppressive activity and had a more favorable epigenetic status of the FOXP3 gene than Tregs expanded in the presence of rapamycin only. We conclude that while the use of ATRA as a single agent to expand Tregs for human therapy is not warranted, its use in combination with rapamycin may have benefit.

  2. The effect of standard and transepithelial ultraviolet collagen cross-linking on human corneal nerves: an ex vivo study.

    Science.gov (United States)

    Al-Aqaba, Mouhamed; Calienno, Roberta; Fares, Usama; Otri, Ahmad Muneer; Mastropasqua, Leonardo; Nubile, Mario; Dua, Harminder S

    2012-02-01

    To evaluate the early effect of standard and transepithelial collagen cross-linking on human corneal nerves in donor eyes by ex vivo confocal microscopy and acetylcholinesterase staining. Experimental laboratory investigation. Eight human eye bank corneal buttons (mean age, 73.6 years) were included. Ultraviolet A collagen cross-linking was performed postmortem on 3 corneas with the standard protocol involving epithelial debridement and 4 corneas by the transepithelial approach. One cornea served as a control. Corneal nerves were evaluated using confocal microscopy and acetylcholinesterase histology. Confocal microscopy demonstrated the absence of subbasal nerves in corneas treated by the standard technique. These nerves were preserved in corneas treated by the transepithelial approach. Stromal nerves were visible in both groups. Histology of corneas treated by the standard technique revealed localized swellings of the stromal nerves with disruption of axonal membrane and loss of axonal continuity within the treatment zone. These changes were absent in corneas treated by the transepithelial approach. This study highlights the immediate effects of collagen cross-linking on the corneal nerves in an ex vivo model. The absence of subbasal nerves in the early phase of treatment appears to be attributable mainly to mechanical removal of epithelium, rather than ultraviolet light-induced damage. Localized swelling of the stromal nerves was the main difference between the 2 treatment protocols. Further research on laboratory animals would be necessary to verify these changes over a specified time course without the super-addition of postmortem changes. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. In vitro efficacy of tavaborole topical solution, 5% after penetration through nail polish on ex vivo human fingernails.

    Science.gov (United States)

    Gupta, Aditya K; Vlahovic, Tracey C; Foley, Kelly A; Lowe, Nicole Gellings; Turner, Rob; Brown, Marc; Hall, Steve

    2018-01-10

    Topical antifungal treatments for onychomycosis are applied to clean, unpolished nails for 48 weeks or longer. Patients often wish to mask their infection with nail polish yet there is no evidence to suggest antifungal efficacy in the presence of nail polish. To determine if tavaborole retains the ability to penetrate the nail plate and inhibit fungal growth in the presence of nail polish. Tavaborole was applied to human fingernails painted with 2 or 4 coats of nail polish, and unpainted nails in an ex vivo model. Nails were mounted on TurChub ® chambers seeded with Trichophyton rubrum and allowed to incubate for 7 days. Antifungal activity was assessed by measuring zones of inhibition. Tavaborole exhibited antifungal activity in all experimental groups. The zones of inhibition of T. rubrum for all experimental groups (2 or 4 coats of polish, unpolished) were greater than infected controls (polished and unpolished), p s  polished nails and kills T. rubrum in this ex vivo model.

  4. Toxic and therapeutic effects of Nifurtimox and Benznidazol on Trypanosoma cruzi ex vivo infection of human placental chorionic villi explants.

    Science.gov (United States)

    Rojo, Gemma; Castillo, Christian; Duaso, Juan; Liempi, Ana; Droguett, Daniel; Galanti, Norbel; Maya, Juan Diego; López-Muñoz, Rodrigo; Kemmerling, Ulrike

    2014-04-01

    Nifurtimox (Nfx) and Benznidazole (Bnz) are the only available drugs in use for the treatment of Chagas disease. These drugs are recommended but not fully validated in evidence-based medicine and reports about the differential toxicity of both drugs are controversial. Here, we evaluated the toxic and therapeutic effects of Nfx and Bnz on human placental chorionic villi explants (HPCVE) during ex vivo infection of Trypanosoma cruzi, performing histopathological, histochemical, immunohistochemical as well as immunofluorescence analysis of the tissue. Additionally, we determined the effect of both drugs on parasite load by real time PCR. Bnz prevents the parasite induced tissue damage in ex vivo infected HPCVE compared to Nfx, which is toxic per se. The presence of T. cruzi antigens and DNA in infected explants suggests that these drugs do not impair parasite invasion into the HPCVE. Additionally, our results confirm reports suggesting that Bnz is less toxic than Nfx and support the need for the development of more effective and better-tolerated drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. 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.

  6. Cryosectioning the intestinal crypt-villus axis: an ex vivo method to study the dynamics of epigenetic modifications from stem cells to differentiated cells.

    Science.gov (United States)

    Vincent, Audrey; Kazmierczak, Catherine; Duchêne, Belinda; Jonckheere, Nicolas; Leteurtre, Emmanuelle; Van Seuningen, Isabelle

    2015-01-01

    The intestinal epithelium is a particularly attractive biological adult model to study epigenetic mechanisms driving adult stem cell renewal and cell differentiation. Since epigenetic modifications are dynamic, we have developed an original ex vivo approach to study the expression and epigenetic profiles of key genes associated with either intestinal cell pluripotency or differentiation by isolating cryosections of the intestinal crypt-villus axis. Gene expression, DNA methylation and histone modifications were studied by qRT-PCR, methylation-specific PCR and micro-chromatin immunoprecipitation, respectively. Using this approach, it was possible to identify segment-specific methylation and chromatin profiles. We show that (i) expression of intestinal stem cell markers (Lgr5, Ascl2) exclusively in the crypt is associated with active histone marks, (ii) promoters of all pluripotency genes studied and transcription factors involved in intestinal cell fate (Cdx2) harbour a bivalent chromatin pattern in the crypts and (iii) expression of differentiation markers (Muc2, Sox9) along the crypt-villus axis is associated with DNA methylation. Hence, using an original model of cryosectioning along the crypt-villus axis that allows in situ detection of dynamic epigenetic modifications, we demonstrate that regulation of pluripotency and differentiation markers in healthy intestinal mucosa involves different and specific epigenetic mechanisms. Copyright © 2015. Published by Elsevier B.V.

  7. Cryosectioning the intestinal crypt-villus axis: An ex vivo method to study the dynamics of epigenetic modifications from stem cells to differentiated cells

    Directory of Open Access Journals (Sweden)

    Audrey Vincent

    2015-01-01

    Full Text Available The intestinal epithelium is a particularly attractive biological adult model to study epigenetic mechanisms driving adult stem cell renewal and cell differentiation. Since epigenetic modifications are dynamic, we have developed an original ex vivo approach to study the expression and epigenetic profiles of key genes associated with either intestinal cell pluripotency or differentiation by isolating cryosections of the intestinal crypt-villus axis. Gene expression, DNA methylation and histone modifications were studied by qRT-PCR, methylation-specific PCR and micro-chromatin immunoprecipitation, respectively. Using this approach, it was possible to identify segment-specific methylation and chromatin profiles. We show that (i expression of intestinal stem cell markers (Lgr5, Ascl2 exclusively in the crypt is associated with active histone marks, (ii promoters of all pluripotency genes studied and transcription factors involved in intestinal cell fate (Cdx2 harbour a bivalent chromatin pattern in the crypts and (iii expression of differentiation markers (Muc2, Sox9 along the crypt-villus axis is associated with DNA methylation. Hence, using an original model of cryosectioning along the crypt-villus axis that allows in situ detection of dynamic epigenetic modifications, we demonstrate that regulation of pluripotency and differentiation markers in healthy intestinal mucosa involves different and specific epigenetic mechanisms.

  8. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Gastrointestinal absorption and metabolism of apple polyphenols ex vivo by the pig intestinal mucosa in the Ussing chamber.

    Science.gov (United States)

    Deusser, Hannah; Rogoll, Dorothee; Scheppach, Wolfgang; Volk, Antje; Melcher, Ralph; Richling, Elke

    2013-03-01

    Polyphenols contained in food have various positive effects on human health. The absorption and metabolism of polyphenols in the intestinal tract needs to be studied to estimate these effects. The Ussing chamber technique was used to investigate the transport behavior of apple polyphenols through pig small intestinal mucosa, which served as a model for human gastrointestinal mucosa. The identities and concentrations of polyphenols and their metabolites in the half-chambers (luminal and basolateral) within an incubation period of 4 h were determined by HPLC-MS/MS and HPLC-DAD (DAD = diode-array detection). Flux values were also measured. It was found that 5-caffeoylquinic acid and caffeic acid were absorbed and translocated to the basolateral side (1.9 and 3.7%, respectively), but other compounds, including glycosides of phloretin and quercetin, were observed without translocation. A Ussing chamber utilizing pig small intestinal mucosa is a suitable model for assessing the effect of apple polyphenols on mucosal integrity and nutrition absorption across porcine mucosa. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. An ex vivo study of nitric oxide efflux from human erythrocytes in both genders.

    Science.gov (United States)

    Duarte, Catarina; Napoleão, Patrícia; Freitas, Teresa; Saldanha, Carlota

    2016-01-01

    Acetylcholinesterase (AChE) is located on outer surface of erythrocyte membrane. Gender-related differences in erythrocyte AChE enzyme activity had been verified in young adults. It is also known that binding of acetylcholine (ACh) with AChE on erythrocyte membrane initiates a signal transduction mechanism that stimulates nitric oxide (NO) efflux. This ex vivo study was done to compare the amount of NO efflux obtained from erythrocytes of healthy donors in males and females. We included 66 gender age-matched healthy donors (40-60 years old). We performed quantification of erythrocyte NO efflux from erythrocytes and of the membrane AChE enzyme activity. There are no significant differences in NO efflux from erythrocytes between men and women. Regarding AChE enzyme activity values, in this range of age, no differences between genders were obtained. However, the values of AChE enzyme activity in the third quartile of NO efflux values were significantly higher (p gender. For the same range of values of NO efflux from erythrocytes, in both gender, it was verified higher values of AChE enzyme activity in women.

  11. Influence of a preadsorbed terpolymer on human platelet accumulation, fibrinogen adsorption, and ex vivo blood activation in hemodialysis hollow fibers.

    Science.gov (United States)

    Yan, F; Déjardin, P; Mulvihill, J N; Cazenave, J P; Crost, T; Thomas, M; Pusineri, C

    1992-01-01

    Results are presented on kinetics of platelet accumulation in charged polyacrylonitrile (AN69) hollow fibers by continuous data recording under flow conditions (wall shear rate 108-1050 s-1), using suspensions of washed 111In-labeled human platelets in Tyrode's-albumin buffer, containing washed red blood cells (0-40%). Preadsorption of a terpolymer of acrylonitrile, poly(ethyleneoxide) methacrylate and trimethylaminoethyl chloride methacrylate leads to very efficient passivation with respect to platelet accumulation and fibrinogen adsorption. In human ex vivo tests, evaluation of complement peptide C3a, platelet beta-thromboglobulin, leucocyte-polymorphonuclear neutrophile elastase and fibrinopeptide A shows no detectable activation. Furthermore, preadsorption appears to result in simultaneous improvement in hemocompatibility of the blood lines leading to and from the dialysis module. This single pretreatment of dialysis membranes should allow injection of lower doses of anticoagulant to patients submitted to hemodialysis.

  12. Human memory FOXP3+ Tregs secrete IL-17 ex vivo and constitutively express the T(H)17 lineage-specific transcription factor RORgamma t.

    Science.gov (United States)

    Ayyoub, Maha; Deknuydt, Florence; Raimbaud, Isabelle; Dousset, Christelle; Leveque, Lucie; Bioley, Gilles; Valmori, Danila

    2009-05-26

    Recent studies have suggested a close relationship between CD4(+)FOXP3(+) regulatory T cells (Tregs) and proinflammatory IL-17-producing T helper cells (T(H)17) expressing the lineage-specific transcription factor RORgamma t. We report here the unexpected finding that human memory Tregs secrete IL-17 ex vivo and constitutively express RORgamma t. IL-17-secreting Tregs share some phenotypic and functional features with conventional T(H)17 cells, expressing high levels of CCR4 and CCR6 and low levels of CXCR3. However, unlike conventional T(H)17 cells, they express low levels of CD161 and mostly fail to cosecrete IL-22 and TNF-alpha ex vivo. Ex vivo secretion of IL-17 and constitutive expression of RORgamma t by human memory Tregs suggest that, in addition to their well-known suppressive functions, these cells likely play additional, as yet undescribed, proinflammatory functions.

  13. Human memory FOXP3+ Tregs secrete IL-17 ex vivo and constitutively express the TH17 lineage-specific transcription factor RORγt

    Science.gov (United States)

    Ayyoub, Maha; Deknuydt, Florence; Raimbaud, Isabelle; Dousset, Christelle; Leveque, Lucie; Bioley, Gilles; Valmori, Danila

    2009-01-01

    Recent studies have suggested a close relationship between CD4+FOXP3+ regulatory T cells (Tregs) and proinflammatory IL-17-producing T helper cells (TH17) expressing the lineage-specific transcription factor RORγt. We report here the unexpected finding that human memory Tregs secrete IL-17 ex vivo and constitutively express RORγt. IL-17-secreting Tregs share some phenotypic and functional features with conventional TH17 cells, expressing high levels of CCR4 and CCR6 and low levels of CXCR3. However, unlike conventional TH17 cells, they express low levels of CD161 and mostly fail to cosecrete IL-22 and TNF-α ex vivo. Ex vivo secretion of IL-17 and constitutive expression of RORγt by human memory Tregs suggest that, in addition to their well-known suppressive functions, these cells likely play additional, as yet undescribed, proinflammatory functions. PMID:19439651

  14. Evaluation of UVA Cytotoxicity for Human Endothelium in an Ex Vivo Corneal Cross-linking Experimental Setting.

    Science.gov (United States)

    Mooren, Pepijn; Gobin, Laure; Bostan, Nezahat; Wouters, Kristien; Zakaria, Nadia; Mathysen, Danny G P; Koppen, Carina

    2016-01-01

    To evaluate endothelial cytotoxicity after exposure of human corneas to ultraviolet-A (UVA) (λ = 365 nm; 5.4 J/cm(2)) in an experimental ex vivo corneal cross-linking setting. Sixteen pairs of human donor corneas were cut into two pieces. One piece of each cornea was treated with 0.025% riboflavin solution prior to UVA irradiation (5 minutes; 18 mW/cm(2)), whereas the other piece was not irradiated but treated with riboflavin (right eye) or preservation medium (left eye). By irradiating from the endothelial side, the UVA dosage applied to endothelial cells exceeded at least eight times the cytotoxic threshold established in animal models (0.65 J/cm(2)). Endothelial cell counts were performed by two independent investigators after storage (4 to 5 days at 31 °C) and staining (trypan blue, alizarin red). Normality (Q-Q plot; Shapiro-Wilk test) and equivalence (mixed-effects modeling with a 10% equivalence threshold) of the endothelial cell counts of the different groups were evaluated. Equivalence of mean endothelial cell density between both groups was observed: 2,237 ± 208 cells/mm(2) in UVA-irradiated pieces and 2,290 ± 281 cells/mm(2) in control pieces (mean difference of 53 ± 240 cells/mm(2) between both groups). Despite direct irradiation of human donor endothelium using the clinical dosage for cross-linking, equivalence in endothelial cell counts was observed between irradiated tissues and controls. Ex vivo human corneal endothelial cells seem to be far more resistant to riboflavin-enhanced UVA irradiation than previously estimated by animal experiments. Copyright 2016, SLACK Incorporated.

  15. 1,8-Cineol Reduces Mucus-Production in a Novel Human Ex Vivo Model of Late Rhinosinusitis.

    Science.gov (United States)

    Sudhoff, Holger; Klenke, Christin; Greiner, Johannes F W; Müller, Janine; Brotzmann, Viktoria; Ebmeyer, Jörg; Kaltschmidt, Barbara; Kaltschmidt, Christian

    2015-01-01

    Inflammatory diseases of the respiratory system such as rhinosinusitis, chronic obstructive pulmonary disease, or bronchial asthma are strongly associated with overproduction and hypersecretion of mucus lining the epithelial airway surface. 1,8-cineol, the active ingredient of the pharmaceutical drug Soledum, is commonly applied for treating such inflammatory airway diseases. However, its potential effects on mucus overproduction still remain unclear.In the present study, we successfully established ex vivo cultures of human nasal turbinate slices to investigate the effects of 1,8-cineol on mucus hypersecretion in experimentally induced rhinosinusitis. The presence of acetyl-α-tubulin-positive cilia confirmed the integrity of the ex vivo cultured epithelium. Mucin-filled goblet cells were also detectable in nasal slice cultures, as revealed by Alcian Blue and Periodic acid-Schiff stainings. Treatment of nasal slice cultures with lipopolysaccharides mimicking bacterial infection as observed during late rhinosinusitis led to a significantly increased number of mucin-filled goblet cells. Notably, the number of mucin-filled goblet cells was found to be significantly decreased after co-treatment with 1,8-cineol. On a molecular level, real time PCR-analysis further showed 1,8-cineol to significantly reduce the expression levels of the mucin genes MUC2 and MUC19 in close association with significantly attenuated NF-κB-activity. In conclusion, we demonstrate for the first time a 1,8-cineol-dependent reduction of mucin-filled goblet cells and MUC2-gene expression associated with an attenuated NF-κB-activity in human nasal slice cultures. Our findings suggest that these effects partially account for the clinical benefits of 1,8-cineol-based therapy during rhinosinusitis. Therefore, topical application of 1,8-cineol may offer a novel therapeutic approach to reduce bacteria-induced mucus hypersecretion.

  16. 1,8-Cineol Reduces Mucus-Production in a Novel Human Ex Vivo Model of Late Rhinosinusitis.

    Directory of Open Access Journals (Sweden)

    Holger Sudhoff

    Full Text Available Inflammatory diseases of the respiratory system such as rhinosinusitis, chronic obstructive pulmonary disease, or bronchial asthma are strongly associated with overproduction and hypersecretion of mucus lining the epithelial airway surface. 1,8-cineol, the active ingredient of the pharmaceutical drug Soledum, is commonly applied for treating such inflammatory airway diseases. However, its potential effects on mucus overproduction still remain unclear.In the present study, we successfully established ex vivo cultures of human nasal turbinate slices to investigate the effects of 1,8-cineol on mucus hypersecretion in experimentally induced rhinosinusitis. The presence of acetyl-α-tubulin-positive cilia confirmed the integrity of the ex vivo cultured epithelium. Mucin-filled goblet cells were also detectable in nasal slice cultures, as revealed by Alcian Blue and Periodic acid-Schiff stainings. Treatment of nasal slice cultures with lipopolysaccharides mimicking bacterial infection as observed during late rhinosinusitis led to a significantly increased number of mucin-filled goblet cells. Notably, the number of mucin-filled goblet cells was found to be significantly decreased after co-treatment with 1,8-cineol. On a molecular level, real time PCR-analysis further showed 1,8-cineol to significantly reduce the expression levels of the mucin genes MUC2 and MUC19 in close association with significantly attenuated NF-κB-activity. In conclusion, we demonstrate for the first time a 1,8-cineol-dependent reduction of mucin-filled goblet cells and MUC2-gene expression associated with an attenuated NF-κB-activity in human nasal slice cultures. Our findings suggest that these effects partially account for the clinical benefits of 1,8-cineol-based therapy during rhinosinusitis. Therefore, topical application of 1,8-cineol may offer a novel therapeutic approach to reduce bacteria-induced mucus hypersecretion.

  17. Ex Vivo Transepithelial Collagen Cross-linking in Porcine and Human Corneas Using Human Decorin Core Protein.

    Science.gov (United States)

    Metzler, Kimberly M; Roberts, Cynthia J; Mahmoud, Ashraf M; Agarwal, Gunjan; Liu, Jun

    2016-06-01

    To investigate changes in corneal biomechanics after cross-linking with human decorin core protein (decoron), which is a small, naturally occurring proteoglycan that bridges collagen fibrils, organizing and stabilizing lamellar collagen architecture. Five human donor pairs (10 eyes) and 5 porcine pairs (10 eyes) had one random eye treated transepithelially with decoron, with the untreated fellow eye serving as control. Pretreatment (45 to 60 seconds) and penetration enhancer (45 to 60 seconds) preceded instillation of decoron (45 to 60 seconds). Total treatment time was less than 4 minutes per eye. Human donor eyes were evaluated using the CorVis ST (Oculus Optikgeräte GmbH, Wetzlar, Germany) at 15, 20, 30, 40, and 50 mm Hg of intraocular pressure. Elastic modulus was calculated for human corneas, using parameters derived from Scheimpflug images. Analysis of variance was performed. Porcine corneas underwent uniaxial tensile testing with a Rheometrics Systems Analyzer (RSA III; TA Instruments, New Castle, DE). Secant modulus was calculated and paired t tests were performed between treated and control groups. One human eye pair was excluded based on initial corneal thickness greater than 850 µm. Analysis of variance of the included four pairs demonstrated a significant treatment effect (P cross-linking. Elastic modulus demonstrated a significant treatment effect with a higher elastic modulus in the treatment group. In porcine eye pairs, the secant modulus was significantly higher in the treated than the untreated corneas at 4%, 5%, and 6% strain (P biomechanical behavior and higher elastic modulus in both human and porcine corneas in this preliminary ex vivo study. Further studies are needed to evaluate clinical safety, efficacy, and long-term stability. [J Refract Surg. 2016;32(6):410-417.]. Copyright 2016, SLACK Incorporated.

  18. Degranulation of mast cells due to compound 48/80 induces concentration-dependent intestinal contraction in rainbow trout (Oncorhynchus mykiss Walbaum) ex vivo.

    Science.gov (United States)

    Manera, M; Giammarino, A; Borreca, C; Giari, L; Dezfuli, B S

    2011-10-01

    Rainbow trout (Oncorhynchus mykiss) intestinal strips (n = 10) were mounted in an isolated organ bath and the effect of incremental doses of compound 48/80 was recorded. Compound 48/80 induced concentration-related contraction in all the examined strips following a sigmoidal dose-response curve fit. Values for maximal contraction (E(max) , g cm(-2)), negative logarithm of the EC(50) (pD(2)), and hill slope were, respectively (mean±standard error), 12.88 ± 0.51, 1.88 ± 0.05, 1.49 ± 0.27. The histological modification induced on mast cells (MCs) due to compound 48/80 was characterized by mean of gray-levels and texture analysis. Significant differences were observed between gray-levels values (Linear mixed model, Pcompound 48/80-treated strips compared with MCs from untreated strips. Moreover, maximal intestinal contraction (due to compound 48/80) correlates positively and significantly (Pearson and Spearman correlations, Pcompound 48/80 induces the degranulation of trout intestinal MCs ex vivo, and that the aforementioned degranulation promotes a concentration-dependent intestinal contraction. Copyright © 2011 Wiley-Liss, Inc., A Wiley Company.

  19. In vitro and ex vivo intestinal tissue models to measure mucoadhesion of poly (methacrylate) and N-trimethylated chitosan polymers.

    Science.gov (United States)

    Keely, Simon; Rullay, Atvinder; Wilson, Carolyn; Carmichael, Adrian; Carrington, Steve; Corfield, Anthony; Haddleton, David M; Brayden, David J

    2005-01-01

    The adhesion of a range of polymers based on poly(2-(dimethylamino-ethyl) methacrylate (pDMAEMA) was assessed using human mucus-secreting and non mucus-secreting intestinal cell monolayers, HT29-MTX-E12 (E12) and HT29 monolayers, as well as excised non-everted intestinal sacs from rats. Differentiation of mucoadhesion from bioadhesion was achieved by pre-treatment with the mucolytic agent, N-acetyl cysteine (NAC). Adherence of pDMAEMA polymers was compared to that obtained with the mucoadhesive, N-trimethylated chitosan (TMC). The quantity of adherent coumarin 343-conjugated polymers to HT29, E12, and intestinal sacs was measured by fluorescence. Confocal laser scanning microscopy (CLSM), light microscopy, and fluorescent microscopy were used to provide direct evidence. Measurements of transepithelial electrical resistance (TEER), permeability to FITC-dextran 4000 (FD-4), and the release of lactate dehydrogenase (LDH) were used to assess potential cytotoxicity of polymers. Adherence of unquaternized and of 10%, 24%, and 32% methyl iodide-quaternized pDMAEMA polymers was measured in E12, HT29, and sacs. All pDMAEMA polymers showed significantly higher levels of adhesion to mucus (mucoadhesion) than to epithelium (bioadhesion). Colocalization of pDMAEMA with mucus was confirmed in E12 by microscopy. TMC showed equally high levels of mucoadhesion as unquaternized and 24% quaternized pDMAEMA, but displayed higher levels of bioadhesion. pDMAEMA-based polymers demonstrated lower levels of adherence to E12 and rat sacs in the presence of NAC, whereas adherence of TMC was unchanged. pDMAEMA significantly decreased the permeability of FD-4 across E12 monolayers and sacs and was less cytotoxic in E12 than in HT29. In contrast, TMC increased the permeability of FD-4 across E12 and sacs and was less cytotoxic in E12 than in HT29. Human mucus-producing E12 monolayers can be used to assess polymer mucoadhesion and give similar data to isolated rat intestinal sacs. p

  20. A new approach to predict human intestinal absorption using porcine intestinal tissue and biorelevant matrices

    NARCIS (Netherlands)

    Westerhout, J.; Steeg, E. van de; Grossouw, D.; Zeijdner, E.E.; Krul, C.A.M.; Verwei, M.; Wortelboer, H.M.

    2014-01-01

    A reliable prediction of the oral bioavailability in humans is crucial and of high interest for pharmaceutical and food industry. The predictive value of currently used in silico methods, in vitro cell lines, ex vivo intestinal tissue and/or in vivo animal studies for human intestinal absorption,

  1. Ex vivo culture of human fetal gonads: manipulation of meiosis signalling by retinoic acid treatment disrupts testis development.

    Science.gov (United States)

    Jørgensen, A; Nielsen, J E; Perlman, S; Lundvall, L; Mitchell, R T; Juul, A; Rajpert-De Meyts, E

    2015-10-01

    What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads? RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal phenotype in fetal testis cultures. 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. 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 outcomes included tissue integrity and morphology, cell proliferation and survival and the expression of markers of meiosis and sex differentiation. Tissue from 24 first trimester human fetuses was included in this study, all from elective terminations at gestational week (GW) 7-12. Gonads were cultured for 2 weeks with and without addition of 1 µM RA. Samples were subsequently formalin-fixed and investigated by immunohistochemistry and cell counting. Proteins investigated and quantified included; octamer-binding transcription factor 4 (OCT4), transcription factor AP-2 gamma (AP2γ) (embryonic germ cell markers), SRY (sex determining region Y)-box 9 (SOX9), anti-Müllerian hormone (AMH) (immature Sertoli cell markers), COUP transcription factor 2 (COUP-TFII) (marker of interstitial cells), forkhead box L2 (FOXL2) (granulosa cell marker), H2A histone family, member X (γH2AX) (meiosis marker), doublesex and mab-3 related transcription factor 1 (DMRT1) (meiosis regulator), cleaved poly ADP ribose polymerase (PARP), cleaved Caspase 3 (apoptosis markers) and Ki-67 antigen (Ki-67) (proliferation marker). Also, proliferation was determined using a 5'-bromo-2

  2. Synthetic Secoisolariciresinol Diglucoside (LGM2605 Protects Human Lung in an Ex Vivo Model of Proton Radiation Damage

    Directory of Open Access Journals (Sweden)

    Anastasia Velalopoulou

    2017-11-01

    Full Text Available Radiation therapy for the treatment of thoracic malignancies has improved significantly by directing of the proton beam in higher doses on the targeted tumor while normal tissues around the tumor receive much lower doses. Nevertheless, exposure of normal tissues to protons is known to pose a substantial risk in long-term survivors, as confirmed by our work in space-relevant exposures of murine lungs to proton radiation. Thus, radioprotective strategies are being sought. We established that LGM2605 is a potent protector from radiation-induced lung toxicity and aimed in the current study to extend the initial findings of space-relevant, proton radiation-associated late lung damage in mice by looking at acute changes in human lung. We used an ex vivo model of organ culture where tissue slices of donor living human lung were kept in culture and exposed to proton radiation. We exposed donor human lung precision-cut lung sections (huPCLS, pretreated with LGM2605, to 4 Gy proton radiation and evaluated them 30 min and 24 h later for gene expression changes relevant to inflammation, oxidative stress, and cell cycle arrest, and determined radiation-induced senescence, inflammation, and oxidative tissue damage. We identified an LGM2605-mediated reduction of proton radiation-induced cellular senescence and associated cell cycle changes, an associated proinflammatory phenotype, and associated oxidative tissue damage. This is a first report on the effects of proton radiation and of the radioprotective properties of LGM2605 on human lung.

  3. Ex vivo visualization of human ciliated epithelium and quantitative analysis of induced flow dynamics by using optical coherence tomography.

    Science.gov (United States)

    Ling, Yuye; Yao, Xinwen; Gamm, Ute A; Arteaga-Solis, Emilio; Emala, Charles W; Choma, Michael A; Hendon, Christine P

    2017-03-01

    Cilia-driven mucociliary clearance is an important self-defense mechanism of great clinical importance in pulmonary research. Conventional light microscopy possesses the capability to visualize individual cilia and its beating pattern but lacks the throughput to assess the global ciliary activities and flow dynamics. Optical coherence tomography (OCT), which provides depth-resolved cross-sectional images, was recently introduced to this area. Fourteen de-identified human tracheobronchial tissues are directly imaged by two OCT systems: one system centered at 1,300 nm with 6.5 μm axial resolution and 15 μm lateral resolution, and the other centered at 800 nm with 2.72 μm axial resolution and 5.52 μm lateral resolution. Speckle variance images are obtained in both cross-sectional and volumetric modes. After imaging, sample blocks are sliced along the registered OCT imaging plane and processed with hematoxylin and eosin (H&E) stain for comparison. Quantitative flow analysis is performed by tracking the path-lines of microspheres in a fixed cross-section. Both the flow rate and flow direction are characterized. The speckle variance images successfully segment the ciliated epithelial tissue from its cilia-denuded counterpart, and the results are validated by corresponding H&E stained sections. A further temporal frequency analysis is performed to extract the ciliary beat frequency (CBF) at cilia cites. By adding polyester microspheres as contrast agents, we demonstrate ex vivo imaging of the flow induced by cilia activities of human tracheobronchial samples. This manuscript presents an ex vivo study on human tracheobronchial ciliated epithelium and its induced mucous flow by using OCT. Within OCT images, intact ciliated epithelium is effectively distinguished from cilia-denuded counterpart, which serves as a negative control, by examining the speckle variance images. The cilia beat frequency is extracted by temporal frequency analysis. The flow rate, flow

  4. Ex vivo-growth response of porcine small intestinal bacterial communities to pharmacological doses of dietary zinc oxide.

    Directory of Open Access Journals (Sweden)

    Ingo C Starke

    Full Text Available Piglets were fed diets containing 57 (low or 2425 (high mg zinc from analytical grade zinc oxide (ZnO ·kg(-1 feed. Digesta samples from the stomach and jejuna of 32, 39, 46 and 53 d old animals (n = 6 per group were incubated in media containing 80, 40, 20 and 0 µg·mL(-1 soluble zinc from ZnO. Turbidity was recorded for 16 h and growth parameters were calculated. Additionally, DNA extracts of selected samples were analyzed via qPCR for different bacterial groups. Samples from animals fed the low dietary zinc concentration always showed highest rate of growth and lowest lag times in media without added zinc. However, media supplemented with zinc displayed highest growth rates and lowest lag time in the high dietary zinc group. Specific growth rates and lag time showed significant differences on day 32 and 39 of age, but rarely on days 46 and 53 of age. Bacterial growth in digesta samples from the high dietary zinc group was less influenced by zinc and recovered growth more rapidly than in the low dietary zinc group. Specific growth rates and bacterial cell numbers from qPCR results showed that lactobacilli were most susceptible to zinc, while bifidobacteria, enterobacteria and enterococci exhibited increased growth rates in samples of animals from the high dietary zinc treatment. No treatment related differences were observed for clostridial cluster IV and the Bacteroides-Prevotella-Porphyromonas cluster. The diversity of enterobacteria after incubation was always higher in the high dietary zinc treatment or in medium supplemented with 80 µg·mL(-1 soluble ZnO. This study has shown that a pharmacological dosage of ZnO leads to a reduced ex vivo-bacterial growth rate of bacteria from the stomach and jejunum of weaned piglets. In view of the rapid bacterial adaptation to dietary zinc, the administration of ZnO in feeds for weaned piglets might only be beneficial in a short period after weaning.

  5. Laminin-511 and -521-based matrices for efficient ex vivo-expansion of human limbal epithelial progenitor cells.

    Science.gov (United States)

    Polisetti, Naresh; Sorokin, Lydia; Okumura, Naoki; Koizumi, Noriko; Kinoshita, Shigeru; Kruse, Friedrich E; Schlötzer-Schrehardt, Ursula

    2017-07-11

    Optimization of culture conditions for human limbal epithelial stem/progenitor cells (LEPC) that incorporate the in vivo cell-matrix interactions are essential to enhance LEPC ex vivo-expansion and transplantation efficiency. Here, we investigate the efficacy of laminin (LN) isoforms preferentially expressed in the limbal niche as culture matrices for epithelial tissue engineering. Analyses of expression patterns of LN chains in the human limbal niche provided evidence for enrichment of LN-α2, -α3, -α5, -β1, -β2, -β3, -γ1, -γ2 and -γ3 chains in the limbal basement membrane, with LN-α5 representing a signature component specifically produced by epithelial progenitor cells. Recombinant human LN-521 and LN-511 significantly enhanced in vitro LEPC adhesion, migration and proliferation compared to other isoforms, and maintained phenotype stability. The bioactive LN-511-E8 fragment carrying only C-terminal domains showed similar efficacy as full-length LN-511. Functional blocking of α3β1 and α6β1 integrins suppressed adhesion of LEPC to LN-511/521-coated surfaces. Cultivation of LEPC on fibrin-based hydrogels incorporating LN-511-E8 resulted in firm integrin-mediated adhesion to the scaffold and well-stratified epithelial constructs, with maintenance of a progenitor cell phenotype in their (supra)basal layers. Thus, the incorporation of chemically defined LN-511-E8 into biosynthetic scaffolds represents a promising approach for xeno-free corneal epithelial tissue engineering for ocular surface reconstruction.

  6. Ex vivo analysis of human memory B lymphocytes specific for A and B influenza hemagglutinin by polychromatic flow-cytometry.

    Science.gov (United States)

    Bardelli, Monia; Alleri, Liliana; Angiolini, Francesca; Buricchi, Francesca; Tavarini, Simona; Sammicheli, Chiara; Nuti, Sandra; Degl'Innocenti, Elena; Isnardi, Isabelle; Fragapane, Elena; Del Giudice, Giuseppe; Castellino, Flora; Galli, Grazia

    2013-01-01

    Understanding the impact that human memory B-cells (MBC), primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA) is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA⁺ B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses.

  7. Ex vivo analysis of human memory B lymphocytes specific for A and B influenza hemagglutinin by polychromatic flow-cytometry.

    Directory of Open Access Journals (Sweden)

    Monia Bardelli

    Full Text Available Understanding the impact that human memory B-cells (MBC, primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA⁺ B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses.

  8. 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.

  9. 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)

  10. 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.

  11. Analysis of Human and Porcine Skin in vivo/ex vivo for Penetration of Selected Oils by Confocal Raman Microscopy.

    Science.gov (United States)

    Choe, ChunSik; Lademann, Jürgen; Darvin, Maxim E

    2015-01-01

    The subject of oil penetration into the skin is controversially discussed in the scientific literature. Confocal Raman microscopy was used for analyzing oil penetration into the skin. The following methods were applied in the study: methods based on tracking specific peaks (method 1), the nonrestricted multiple least square fit (method 2), analyzing the lipid-to-keratin peak ratio using the perpendicular drop-down cutoff procedure (method 3), and the Gaussian function-based deconvolution procedure (method 4). The results obtained using methods 1, 2 and 4 show that the investigated oils do not penetrate deeper than 11 µm into human and porcine skin. Petrolatum has a prominent swelling effect on the stratum corneum (32% in vivo, 28% ex vivo), while the other oils exhibit no significant swelling effect. By using method 3, the penetration profile of oils, and especially of petrolatum, into the skin was interpreted incorrectly for various reasons that are addressed herein below. Predominantly remaining in the uppermost corneocyte layers of the stratum corneum, topically applied oils do not reach the viable cells of the stratum spinosum. To exclude any possible mistakes when using the lipid-keratin Raman peak (2,820-3,030 cm-1), the penetration analysis should be performed using the Gaussian function-based deconvolution procedure. © 2015 S. Karger AG, Basel.

  12. FEM simulation of the human lens compared to ex vivo porcine lens cutting pattern: a possible treatment of presbyopia

    Science.gov (United States)

    Ripken, T.; Breitenfeld, P.; Fromm, M.; Oberheide, U.; Gerten, G.; Lubatschowski, H.

    2006-02-01

    The most probable reason for presbyopia is an age related loss of elasticity of the lens. It progresses typically during the whole life and at the age of about 45 it leads to a considerable loss of the ability to accommodate within the next decade. However, both, the ciliary muscle and the lens capsule stay active and elastic, respectively. With respect to this, one concept is to regain the deformability of the lens without changing the capsule or zonular apparatus. Since the investigations of Ripken et al. proofed that the flexibility of the presbyopic lens tissue can be increased through the creation of fs-laser induced microcuts inside the lens, this is one possible approach to treat presbyopia. On this account a finite-element-method model with ANSYS of the human lens during accommodation will be presented. The analysis premises all lens materials to be linear elastic and allow large displacements. A first analysis of this method for the treatment of presbyopia is accomplished. Therefore the mechanical analysis of untreated and treated lens are compared. In addition ex-vivo elasticity measurements of untreated and treated lenses will be presented. As a result an improvement of the flexibility of the lens tissue is found and as its consequence a change of the lens radii of curvature is established. After suitable processing of the output data the change in optical power between untreated and treated lenses are calculated. The finite element simulation shows similar behaviour compared to the treated porcine lenses.

  13. Comparison of In Vivo and Ex Vivo MRI of the Human Hippocampal Formation in the Same Subjects.

    Science.gov (United States)

    Wisse, L E M; Adler, D H; Ittyerah, R; Pluta, J B; Robinson, J L; Schuck, T; Trojanowski, J Q; Grossman, M; Detre, J A; Elliott, M A; Toledo, J B; Liu, W; Pickup, S; Das, S R; Wolk, D A; Yushkevich, P A

    2017-11-01

    Multiple techniques for quantification of hippocampal subfields from in vivo MRI have been proposed. Linking in vivo MRI to the underlying histology can help validate and improve these techniques. High-resolution ex vivo MRI can provide an intermediate modality to map information between these very different imaging modalities. This article evaluates the ability to match information between in vivo and ex vivo MRI in the same subjects. We perform rigid and deformable registration on 10 pairs of in vivo (3 T, 0.4 × 0.4 × 2.6 mm3) and ex vivo (9.4 T, 0.2 × 0.2 × 0.2 mm3) scans, and describe differences in MRI appearance between these modalities qualitatively and quantitatively. The feasibility of using this dataset to validate in vivo segmentation is evaluated by applying an automatic hippocampal subfield segmentation technique (ASHS) to in vivo scans and comparing SRLM (stratum/radiatum/lacunosum/moleculare) surface to manual tracing on corresponding ex vivo scans (and in 2 cases, histology). Regional increases in thickness are detected in ex vivo scans adjacent to the ventricles and were not related to scanner, resolution differences, or susceptibility artefacts. Satisfactory in vivo/ex vivo registration and subvoxel accuracy of ASHS segmentation of hippocampal SRLM demonstrate the feasibility of using this dataset for validation, and potentially, improvement of in vivo segmentation methods. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. A novel monoclonal antibody of human stem cell factor inhibits umbilical cord blood stem cell ex vivo expansion

    Directory of Open Access Journals (Sweden)

    Fan Jie

    2012-12-01

    Full Text Available Abstract Stem cell factor (SCF activates hematopoietic stem cell (HSC self-renewal and is being used to stimulate the ex vivo expansion of HSCs. The mechanism by which SCF supports expansion of HSCs remains poorly understood. In cord blood ex vivo expansion assays, a newly produced anti-SCF monoclonal antibody (clone 23C8 was found to significantly inhibit the expansion of CD34+ cells. This antibody appears to bind directly to a part of SCF that is critical for biological activity toward expansion of CD34+ cells, which is located in the first 104 amino acids from the NH2-terminus.

  15. Ex Vivo Expansion of Human Limbal Epithelial Cells Using Human Placenta-Derived and Umbilical Cord-Derived Mesenchymal Stem Cells

    OpenAIRE

    Sang Min Nam; Yong-Sun Maeng; Eung Kweon Kim; Kyoung Yul Seo; Helen Lew

    2017-01-01

    Ex vivo culture of human limbal epithelial cells (LECs) is used to treat limbal stem cell (LSC) deficiency, a vision loss condition, and suitable culture systems using feeder cells or serum without animal elements have been developed. This study evaluated the use of human umbilical cord or placenta mesenchymal stem cells (C-MSCs or P-MSCs, resp.) as feeder cells in an animal/serum-free coculture system with human LECs. C-/P-MSCs stimulated LEC colony formation of the stem cell markers (p63, A...

  16. Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

    Science.gov (United States)

    Zhu, Yaqi; Yang, Yan; Guo, Juanjuan; Dai, Ying; Ye, Lina; Qiu, Jianbin; Zeng, Zhihong; Wu, Xiaoting; Xing, Yanmei; Long, Xiang; Wu, Xufeng; Ye, Lin; Wang, Shubin; Li, Hui

    2017-02-28

    Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

  17. Expansion of human and murine hematopoietic stem and progenitor cells ex vivo without genetic modification using MYC and Bcl-2 fusion proteins.

    Directory of Open Access Journals (Sweden)

    Gregory A Bird

    Full Text Available The long-term repopulating hematopoietic stem cell (HSC population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs. We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use.

  18. Ghrelin-mediated inhibition of the TSH-stimulated function of differentiated human thyrocytes ex vivo.

    Science.gov (United States)

    Barington, Maria; Brorson, Marianne Møller; Hofman-Bang, Jacob; Rasmussen, Åse Krogh; Holst, Birgitte; Feldt-Rasmussen, Ulla

    2017-01-01

    Ghrelin is a peptide hormone produced mainly in the gastrointestinal tract known to regulate several physiological functions including gut motility, adipose tissue accumulation and hunger sensation leading to increased bodyweight. Studies have found a correlation between the plasma levels of thyroid hormones and ghrelin, but an effect of ghrelin on the human thyroid has never been investigated even though ghrelin receptors are present in the thyroid. The present study shows a ghrelin-induced decrease in the thyroid-stimulating hormone (TSH)-induced production of thyroglobulin and mRNA expression of thyroperoxidase in a primary culture of human thyroid cells obtained from paranodular tissue. Accordingly, a trend was noted for an inhibition of TSH-stimulated expression of the sodium-iodine symporter and the TSH-receptor. Thus, this study suggests an effect of ghrelin on human thyrocytes and thereby emphasizes the relevance of examining whether ghrelin also influences the metabolic homeostasis through altered thyroid hormone production.

  19. Replication of H9 influenza viruses in the human ex vivo respiratory tract, and the influence of neuraminidase on virus release.

    Science.gov (United States)

    Chan, Renee W Y; Chan, Louisa L Y; Mok, Chris K P; Lai, Jimmy; Tao, Kin P; Obadan, Adebimpe; Chan, Michael C W; Perez, Daniel R; Peiris, J S Malik; Nicholls, John M

    2017-07-24

    H9N2 viruses are the most widespread influenza viruses in poultry in Asia. We evaluated the infection and tropism of human and avian H9 influenza virus in the human respiratory tract using ex vivo respiratory organ culture. H9 viruses infected the upper and lower respiratory tract and the majority of H9 viruses had a decreased ability to release virus from the bronchus rather than the lung. This may be attributed to a weak neuraminidase (NA) cleavage of carbon-6-linked sialic acid (Sia) rather than carbon-3-linked Sia. The modified cleavage of N-acetlylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) by NA in H9 virus replication was observed by reverse genetics, and recombinant H9N2 viruses with amino acids (38KQ) deleted in the NA stalk, and changing the amino acid at position 431 from Proline-to-Lysine. Using recombinant H9 viruses previously evaluated in the ferret, we found that viruses which replicated well in the ferret did not replicate to the same extent in the human ex vivo cultures. The existing risk assessment models for H9N2 viruses in ferrets may not always have a strong correlation with the replication in the human upper respiratory tract. The inclusion of the human ex vivo cultures would further strengthen the future risk-assessment strategies.

  20. Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas.

    Directory of Open Access Journals (Sweden)

    David Kryza

    Full Text Available The human Matrix MetalloProtease-9 (hMMP-9 is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B, fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of 99mTc-MAG-aptamer and 111In-DOTA-F3B. 99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of 111In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05 higher for 111In-DOTA-F3B with 2.0%ID/g than for the 111In-DOTA-control oligonucleotide (0.7%ID/g with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor.

  1. Transplantation of human neonatal foreskin stromal cells in ex vivo organotypic cultures of embryonic chick femurs

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Vishnubalaji, Radhakrishnan

    2017-01-01

    We have previously reported that human neonatal foreskin stromal cells (hNSSCs) promote angiogenesis in vitro and in chick embryo chorioallantoic membrane (CAM) assay in vivo. To examine the in vivo relevance of this observation, we examined in the present study the differentiation potential of h...

  2. Precision-cut rat, mouse, and human intestinal slices as novel models for the early-onset of intestinal fibrosis

    NARCIS (Netherlands)

    Pham, Bao Tung; van Haaften, Wouter Tobias; Oosterhuis, Dorenda; Nieken, Judith; de Graaf, Inge Anne Maria; Olinga, Peter

    Intestinal fibrosis (IF) is a major complication of inflammatory bowel disease. IF research is limited by the lack of relevant in vitro and in vivo models. We evaluated precision-cut intestinal slices (PCIS) prepared from human, rat, and mouse intestine as ex vivo models mimicking the early-onset of

  3. Advancing the Capabilities of an Authentic Ex Vivo Model of Primary Human Prostate Cancer

    Science.gov (United States)

    2014-10-01

    The lactate dehydrogenase (LDH) assay is one of the most widely used assays for quantifying cytotoxicity in organotypic cultures. We performed LDH...R. Keshari et al., Metabolic reprogramming and validation of hyperpolarized 13C lactate as a prostate cancer biomarker using a human prostate tissue...inherent drawbacks hinder their clinical relevance.1,2 Cell lines, often artificially immortalized, acquire mutations in culture and lack the

  4. CMKLR1 activation ex vivo does not increase proportionally to serum total chemerin in obese humans

    Directory of Open Access Journals (Sweden)

    Jay Toulany

    2016-11-01

    Full Text Available Prochemerin is the inactive precursor of the adipokine chemerin. Proteolytic processing is obligatory for the conversion of prochemerin into active chemerin and subsequent regulation of cellular processes via the chemokine-like receptor 1 (CMKLR1. Elevated plasma or serum chemerin concentrations and differential processing of prochemerin have been reported in obese humans. The impact of these changes on CMKLR1 signalling in humans is unknown. The objective of this pilot study was to develop a cellular bioassay to measure CMKLR1 activation by chemerin present in human serum and to characterise how obesity modifies serum activation of CMKLR1 under fasted and fed conditions. Blood samples were collected from control (N = 4, BMI 20–25 and obese (N = 4, BMI >30 female subjects after an overnight fast (n = 2 and at regular intervals (n = 7 following consumption of breakfast over a period of 6 h. A cellular CMKLR1-luminescent reporter assay and a pan-chemerin ELISA were used to determine CMKLR1 activation and total chemerin concentrations, respectively. Serum total chemerin concentration (averaged across all samples was higher in obese vs control subjects (17.9 ± 1.8 vs 10.9 ± 0.5 nM, P < 0.05, but serum activation of CMKLR1 was similar in both groups. The CMKLR1 activation/total chemerin ratio was lower in obese vs control subjects (0.33 ± 0.04 vs 0.58 ± 0.05, P < 0.05. After breakfast, serum total chemerin or CMKLR1 activation did not differ from baseline values. In conclusion, the unexpected observation that obese serum activation of CMKLR1 did not match increased total chemerin concentrations suggests impaired processing to and/or enhanced degradation of active chemerin in serum of obese humans.

  5. The effects of pravastatin on the normal human placenta: Lessons from ex-vivo models.

    Directory of Open Access Journals (Sweden)

    Adelina Balan

    Full Text Available Research in animal models and preliminary clinical studies in humans support the use of pravastatin for the prevention of preeclampsia. However, its use during pregnancy is still controversial due to limited data about its effect on the human placenta and fetus.In the present study, human placental cotyledons were perfused in the absence or presence of pravastatin in the maternal reservoir (PraM. In addition, placental explants were treated with pravastatin for 5, 24 and 72 h under normoxia and hypoxia. We monitored the secretion of placental growth factor (PlGF, soluble fms-like tyrosine kinase-1 (sFlt-1, soluble endoglin (sEng, endothelial nitric oxide synthase (eNOS expression and activation and the fetal vasoconstriction response to angiotensin-II.The concentrations of PlGF, sFlt-1 and sEng were not significantly altered by pravastatin in PraM cotyledons and in placental explants compared to control. Under hypoxic conditions, pravastatin decreased sFlt-1 concentrations. eNOS expression was significantly increased in PraM cotyledons but not in pravastatin-treated placental explants cultured under normoxia or hypoxia. eNOS phosphorylation was not significantly affected by pravastatin. The feto-placental vascular tone and the fetal vasoconstriction response to angiotensin-II, did not change following exposure of the maternal circulation to pravastatin.We found that pravastatin does not alter the essential physiological functions of the placenta investigated in the study. The relevance of the study lays in the fact that it expands the current knowledge obtained thus far regarding the effect of the drug on the normal human placenta. This data is reassuring and important for clinicians that consider the treatment of high-risk patients with pravastatin, a treatment that exposes some normal pregnancies to the drug.

  6. Cytotoxicity evaluation of three light-cured dentin adhesive materials on human gingival fibroblasts, ex vivo.

    Science.gov (United States)

    Kierklo, A; Pawińska, M; Tokajuk, G; Popławska, B; Bielawska, A

    2012-01-01

    To evaluate the cytotoxic effects of three current light-cured dentin adhesives, in both uncured and post-cured conditions, on human gingival fibroblasts. The materials tested were Heliobond, Adper Single Bond 2 and Xeno V, which are characterized by various compositions and application procedures. Each agent, in volumes of 5 and 10 μL, was tested after polymerization, and those unpolymerized were diluted in DMEM to 10-3 and 10-5. The cytotoxicity of the adhesives was assessed on the basis of a test of cell viability in a culture of human gingival fibroblasts, with the use of tetrazolic salt (MTT assay). The results showed that, among the adhesive/bonding systems tested, Xeno V was the least cytotoxic. There were statistically significant differences in cell survival between polymerized Xeno V, Adper Single Bond 2 and Heliobond in the amount of 5 μL as well as between the Xeno V and Adper Single Bond 2 in 10-5 dilutions. The tested adhesives were more toxic in the polymerized form than in the dilutions. Samples of 10 μL resulted in a lower survival percentage of fibroblasts compared to 5 μL. All the tested adhesives demonstrated cytopathic effects towards human gingival fibroblasts, but varied in their cytotoxicity. This has clinical implications. Dentists should follow the rules of adhesive application, precisely dose them and not allow direct contact with the gums as, even after polymerization, adhesive agents exhibit potential cytotoxic activity.

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

    NARCIS (Netherlands)

    Wilcks, A.; Hoek, van A.H.A.M.; Joosten, R.G.; Jacobsen, B.B.L.; Aarts, H.J.M.

    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

  8. Lack of effect of selected sunscreens applied on ex vivo human skin for 5-methyl-aminolevulinic acid penetration and protoporphyrin IX photoactivation.

    Science.gov (United States)

    Osman-Ponchet, Hanan; Sevin, Karine; Gaborit, Alexandre; Kouidhi, Magali; Hanaizi, Johanna; Comby, Pierre; Ruty, Bernard; Bouvier, Guy

    2017-03-01

    Photodynamic therapy (PDT) is a successful treatment for non-melanoma skin cancers. Methyl-aminolevulinate (MAL) is metabolized to protoporphyrin IX (PpIX) which accumulates in the skin lesion and which generates a painful photochemical toxic reaction upon red light exposure. PDT using daylight (DL) exposure is now used to reduce pain and subjects are advised to protect the areas with sunscreen. This work investigated the effect of sunscreen on MAL penetration and PpIX photoactivation in ex vivo human skin. To measure skin penetration of MAL, particle-free sunscreens were applied on ex vivo human skin samples mounted on diffusion cells before application of Metvix cream containing [14C]-MAL for 2.5h. To circumvent the absence of skin penetration of PpIX, skin samples were first treated with microneedles and mounted on diffusion cells before the application of PpIX solution for 1h followed by sunscreens. Skin samples were then exposed to solar simulator for 1h. Concentrations of [14C]-MAL or PpIX were measured in both total skin and receptor liquid. The results showed that the in vitro skin penetration of MAL and the PpIX photoactivation on ex vivo human skin samples are not modified by pretreatments of ex vivo human skin with sunscreens. This study demonstrates that neither in vitro skin penetration of MAL nor PpIX photoactivation were modified by pretreatments with Cetaphil SPF 30 Dermacontrol and Actinica® Lotion SPF 50+. This supports the efficacy and safety of MAL DL-PDT in the clinical situation. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Using infrared and Raman microspectroscopies to compare ex vivo involved psoriatic skin with normal human skin

    Science.gov (United States)

    Leroy, Marie; Lefèvre, Thierry; Pouliot, Roxane; Auger, Michèle; Laroche, Gaétan

    2015-06-01

    Psoriasis is a chronic dermatosis that affects around 3% of the world's population. The etiology of this autoimmune pathology is not completely understood. The barrier function of psoriatic skin is known to be strongly altered, but the structural modifications at the origin of this dysfunction are not clear. To develop strategies to reduce symptoms of psoriasis or adequate substitutes for modeling, a deep understanding of the organization of psoriatic skin at a molecular level is required. Infrared and Raman microspectroscopies have been used to obtain direct molecular-level information on psoriatic and healthy human skin biopsies. From the intensities and positions of specific vibrational bands, the lipid and protein distribution and the lipid order have been mapped in the different layers of the skin. Results showed a similar distribution of lipids and collagen for normal and psoriatic human skin. However, psoriatic skin is characterized by heterogeneity in lipid/protein composition at the micrometer scale, a reduction in the definition of skin layer boundaries and a decrease in lipid chain order in the stratum corneum as compared to normal skin. A global decrease of the structural organization is exhibited in psoriatic skin that is compatible with an alteration of its barrier properties.

  10. [Ultrasonic lipoemulsification: a working definition and ex-vivo study on human adipose tissue].

    Science.gov (United States)

    Palmieri, B; Criscuolo, M; Gozzi, G

    1994-01-01

    On the basis of previous reports by other authors which have become increasingly numerous over the past years, we have focused our attention on the use of ultrasonic energy in the medical field to resolve medical and cosmetic problems, such as lipodystrophy and diffuse subcutaneous adiposity. In order to standardise the dissolution times of human subcutaneous adipose tissue we used a ultrasonic generator operating at a constant frequency of 19800 Hz, but which was able to emit a range of power from 0 to 100 Watt. The ultrasounds were applied (according to a scale of power) using a titanium probe to fat samples with a volume of 1 cm until each sample had fully dissolved. This allowed the levels of greatest working efficiency to be established for the most commonly used probes.

  11. Atmospheric-Pressure Cold Plasma Induces Transcriptional Changes in Ex Vivo Human Corneas.

    Directory of Open Access Journals (Sweden)

    Umberto Rosani

    Full Text Available Atmospheric pressure cold plasma (APCP might be considered a novel tool for tissue disinfection in medicine since the active chemical species produced by low plasma doses, generated by ionizing helium gas in air, induces reactive oxygen species (ROS that kill microorganisms without substantially affecting human cells.In this study, we evaluated morphological and functional changes in human corneas exposed for 2 minutes (min to APCP and tested if the antioxidant n-acetyl l-cysteine (NAC was able to inhibit or prevent damage and cell death.Immunohistochemistry and western blotting analyses of corneal tissues collected at 6 hours (h post-APCP treatment demonstrated no morphological tissue changes, but a transient increased expression of OGG1 glycosylase that returned to control levels in 24 h. Transcriptome sequencing and quantitative real time PCR performed on different corneas revealed in the treated corneas many differentially expressed genes: namely, 256 and 304 genes showing expression changes greater than ± 2 folds in the absence and presence of NAC, respectively. At 6 h post-treatment, the most over-expressed gene categories suggested an active or enhanced cell functioning, with only a minority of genes specifically concerning oxidative DNA damage and repair showing slight over-expression values (<2 folds. Moreover, time-related expression analysis of eight genes up-regulated in the APCP-treated corneas overall demonstrated the return to control expression levels after 24 h.These findings of transient oxidative stress accompanied by wide-range transcriptome adjustments support the further development of APCP as an ocular disinfectant.

  12. Cyclin D1 Gene Silencing by siRNA in Ex Vivo Human Tissue Cultures.

    Science.gov (United States)

    Piazza, Ornella; Russo, Ilaria; Bochicchio, Sabrina; Barba, Anna Angela; Lamberti, Gaetano; Zeppa, Pio; Crescenzo, Vincenzo Di; Carrizzo, Albino; Vecchione, Carmine; Ciacci, Carolina

    2017-01-01

    Short interfering RNAs (siRNAs) are double-stranded RNA molecules able to specifically targeting genes products responsible for human diseases. Cyclin D1 (CyD1) is a cell cycleregulatory molecule, up-regulated at sites of inflammation in several tissues. CyD1 is a very interesting potential target in lung and colon inflammatory diseases. The aim of this paper was testing CyD1 expression in human lung and colon tissues after the application of an inflammatory stimulus, and verifying its gene silencing by using siRNA for CyD1 (siCyD1). Colon and pulmonary biopsies were treated with siCyD1 by using two different transfection carriers: a) invivofectamine and b) ad hoc produced nanoliposomes. After 24 hours of incubation with nanoliposomes encapsulating siRNA or invivofectamine-CyD1siRNA, in presence or absence of ECLPS, we analysed the protein expression of CyD1 through Western-Blotting. After EC-LPS treatment, in both colon and pulmonary biopsies, an overexpression of CyD1was found (about 64% and 40% respectively). Invivofectamine-CyD1 siRNA reduced the expression of CyD1 approximately by 46% compared to the basal condition, and by around 65% compared to EC-LPS treated colon samples. In lung, following in vivo fectamine siRNA silencing in the presence of EC-LPS, no reduction was observed. Ad hoc nanoliposomes were able to enter colon and lung tissues, but CyD1 silencing was reported in 2 colon samples out of 4 and no efficacy was demonstrated in the only lung sample we studied. The silencing of Cyclin D1 expression in vitro "organ culture" model is possible. Our preliminary results encourage further investigations, using different siRNA concentrations delivered by nanoliposomes. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. Effect of pheromone induction on transfer of the Enterococcus faecalis plasmid pCF10 in intestinal mucus ex vivo

    DEFF Research Database (Denmark)

    Licht, Tine Rask; Hammerum, Anette Marie; Jensen, Lars Bogø

    2001-01-01

    The effect of synthetic sex pheromone on pheromone-inducible conjugation between the isogenic Enterococcus faecalis strains OG1RF and OG1SS was investigated in (i) Todd-Hewitt broth medium and (ii) intestinal mucus isolated from germ-free rats. In broth, the presence of synthetic pheromone cCF10...... had no detectable effect on the transfer kinetics observed for the tetracycline resistance encoding plasmid pCF10. In MUCUS, presence of the same pheromone significantly increased the transfer efficiency observed during the first 2 h of conjugation, while the effect was less pronounced later...... in the experiment. We suggest that due to differences in diffusion rates and medium-binding of the pheromones, the effect of the synthetic cCF10 was immediately dominated by the effect of pheromones produced by the recipient E. faecalis strain in broth, while this happened later in mucus....

  14. A novel automated discontinuous venous blood monitoring system for ex vivo glucose determination in humans.

    Science.gov (United States)

    Schaller, R; Feichtner, F; Köhler, H; Bodenlenz, M; Plank, J; Wutte, A; Mader, J K; Ellmerer, M; Hellmich, R; Wedig, H; Hainisch, R; Pieber, T R; Schaupp, L

    2009-03-15

    Intensive insulin therapy reduces mortality and morbidity in critically ill patients but imposes great demands on medical staff who must take frequent blood samples for the determination of glucose levels. A solution to this resourcing problem would be provided by an automated blood monitoring system. The aim of the present clinical study was to evaluate such a system comprising an automatic blood sampling unit linked to a glucose biosensor. Our approach was to determine the correlation and system error of the sampling unit alone and of the combined system with respect to reference levels over 12h in humans. Two venous cannulae were inserted to connect the automatic and reference systems to the subjects. Blood samples were taken at 15 and 30 min intervals. The median Pearson coefficient of correlation between manually and automatically withdrawn blood samples was 0.982 for the sampling unit alone and 0.950 for the complete system. The biosensor had a linear range up to 20 mmoll(-1) and a 95% response time of blood sampling system reduced blood withdrawal failures through occluded catheters fourfold. In summary, automated blood sampling from a peripheral vein coupled with automatic glucose determination is a promising alternative to frequent manual blood sampling.

  15. Intratubular Antibacterial Effect of Polyethyleneimine Nanoparticles: An Ex Vivo Study in Human Teeth

    Directory of Open Access Journals (Sweden)

    Itzhak Abramovitz

    2015-01-01

    Full Text Available Enterococcus faecalis is a facultative gram positive bacterium which can remain in the teeth root canals and cause refractory or persistent periapical diseases. E. faecalis bacteria that penetrate the dentinal tubules can be the source of intracanal infection and endodontic disease. Quaternary ammonium polyethyleneimine (QPEI nanopolymers were shown to have long lasting antibacterial activity against gram positive and gram negative bacteria. The present study evaluated the intratubular antibacterial effect of an epoxy resin sealer incorporating 1% QPEI against E. faecalis in a human dentin model. Root canals of extracted teeth were inoculated with E. faecalis for 7 days prior to standard endodontic treatment. The antibacterial effect of an epoxy-amine resin endodontic sealer was tested at concentration of 0% or 1% (wt/wt added QPEI nanoparticles. Reduction in bacterial viability p<0.01 was depicted in the dentinal tubules of the root canals obturated with the sealer incorporating QPEI nanoparticles. In conclusion, QPEI nanoparticles when incorporated in a small percentage into epoxy-resin based sealer may target E. faecalis in the dentinal tubules, producing a potent antibacterial effect that reduces significantly bacterial viability.

  16. Preliminary interlaboratory comparison of the ex vivo dual human placental perfusion system

    DEFF Research Database (Denmark)

    Myllynen, Päivi; Mathiesen, Line; Weimer, Marc

    2010-01-01

    As a part of EU-project ReProTect, a comparison of the dual re-circulating human placental perfusion system was carried out, by two independent research groups. The detailed placental transfer data of model compounds [antipyrine, benzo(a)pyrene, PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b......)pyridine) and IQ (2-amino-3-methylimidazo(4,5-f)quinoline] has been/will be published separately. For this project, a comparative re-analysis was done, by curve fitting the data and calculating two endpoints: AUC(120), defined as the area under the curve between time 0 and time 120min and as t(0.5), defined...... as the time when the fetal to maternal concentration ratio is expected to be 0.5. The transport of the compounds from maternal to fetal circulation across the perfused placenta could be ranked in the order of antipyrine>IQ>PhIP in terms of both t(0.5) and AUC(120) by both partners. For benzo...

  17. A novel approach to quantify different iron forms in ex-vivo human brain tissue

    Science.gov (United States)

    Kumar, Pravin; Bulk, Marjolein; Webb, Andrew; van der Weerd, Louise; Oosterkamp, Tjerk H.; Huber, Martina; Bossoni, Lucia

    2016-12-01

    We propose a novel combination of methods to study the physical properties of ferric ions and iron-oxide nanoparticles in post-mortem human brain, based on the combination of Electron Paramagnetic Resonance (EPR) and SQUID magnetometry. By means of EPR, we derive the concentration of the low molecular weight iron pool, as well as the product of its electron spin relaxation times. Additionally, by SQUID magnetometry we identify iron mineralization products ascribable to a magnetite/maghemite phase and a ferrihydrite (ferritin) phase. We further derive the concentration of magnetite/maghemite and of ferritin nanoparticles. To test out the new combined methodology, we studied brain tissue of an Alzheimer’s patient and a healthy control. Finally, we estimate that the size of the magnetite/maghemite nanoparticles, whose magnetic moments are blocked at room temperature, exceeds 40-50 nm, which is not compatible with the ferritin protein, the core of which is typically 6-8 nm. We believe that this methodology could be beneficial in the study of neurodegenerative diseases such as Alzheimer’s Disease which are characterized by abnormal iron accumulation in the brain.

  18. 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.

  19. ex vivo DNA assembly

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    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.

  20. The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia.

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    Blanco, Teresa Mortera; Mantalaris, Athanasios; Bismarck, Alexander; Panoskaltsis, Nicki

    2010-03-01

    Acute myeloid leukaemia (AML) is a cancer of haematopoietic cells that develops in three-dimensional (3-D) bone marrow niches in vivo. The study of AML has been hampered by lack of appropriate ex vivo models that mimic this microenvironment. We hypothesised that fabrication and optimisation of suitable biomimetic scaffolds for culturing leukaemic cells ex vivo might facilitate the study of AML in its native 3-D niche. We evaluated the growth of three leukaemia subtype-specific cell lines, K-562, HL60 and Kasumi-6, on highly porous scaffolds fabricated from biodegradable and non-biodegradable polymeric materials, such as poly (L-lactic-co-glycolic acid) (PLGA), polyurethane (PU), poly (methyl-methacrylate), poly (D, L-lactade), poly (caprolactone), and polystyrene. Our results show that PLGA and PU supported the best seeding efficiency and leukaemic growth. Furthermore, the PLGA and PU scaffolds were coated with extracellular matrix (ECM) proteins, collagen type I (62.5 or 125 microg/ml) and fibronectin (25 or 50 microg/ml) to provide biorecognition signals. The 3 leukaemia subtype-specific lines grew best on PU scaffolds coated with 62.5 microg/ml collagen type I over 6 weeks in the absence of exogenous growth factors. In conclusion, PU-collagen scaffolds may provide a practical model to study the biology and treatment of primary AML in an ex vivo mimicry. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  1. In Vitro Nail Penetration of Tavaborole Topical Solution, 5%, Through Nail Polish on Ex Vivo Human Fingernails.

    Science.gov (United States)

    Vlahovic, Tracey; MPharm, Tejal Merchant; Chanda, Sanjay; Zane, Lee T; Coronado, Dina

    2015-07-01

    Onychomycosis is a common infection of the toenails that causes nail thickening and discoloration. The physical appearance of the infected nail can diminish self-image and negatively impact quality of life. Patients may use nail polish to mask the appearance of infected nails. To evaluate the in vitro nail penetration properties of tavaborole topical solution, 5%, through nail polish using ex vivo, non-diseased human fingernails. In study 1, tavaborole penetration was evaluated over 20 days of dosing using the Franz finite dose technique and modified Franz diffusion cells. Nails received either 1 coat of over-the-counter (OTC) typical polish or were left unpolished (controls). In study 2, tavaborole penetration was measured over 14 days of dosing using the finite dose technique and vertical diffusion cells. Nails were polished with either 4 coats or 1 coat of salon typical polish or with 2 coats or 1 coat of OTC typical polish, or they were left unpolished. In study 1, the mean ± standard deviation (SD) cumulative tavaborole penetration at day 21 was numerically higher, though not statistically significant, through polished nails (3,526 ± 1,433 μg/cm(2))vs unpolished nails (2,661 ± 1,319 μg/cm(2)).In study 2, the mean cumulative tavaborole penetration was also numerically higher (statistical significance not assessed) through all nails that received polish vs unpolished nails. At day 15, mean ± SD cumulative tavaborole nail penetration was 1,179 ± 554 μg/cm(2) through 4 coats of salon typical polish, 1,227 ± 974 μg/cm(2) through 1 coat of salon typical polish, 1,493 ± 1,322 μg/cm(2) through 2 coats of OTC typical polish, 1,428 ± 841 μg/cm(2) through 1 coat of OTC typical polish, and 566 ± 318 μg/cm(2) through unpolished nails. Results from these in vitro studies demonstrated that tavaborole penetrated through human nails with up to 4 layers of nail polish.

  2. Ex vivo differentiation of human bone marrow-derived stem cells into neuronal cell-like lineages

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    Al-Zoubi A

    2016-06-01

    Full Text Available Adeeb Al-Zoubi,1,2 Feras Altwal,3 Farah Khalifeh,2 Jamil Hermas,4 Ziad Al-Zoubi,5 Emad Jafar,5 Mohammed El-Khateeb,6,7 1Department of Surgery, University of Illinois College of Medicine at Peoria, Peoria, IL, USA; 2Stem Cells of Arabia, Amman, Jordan; 3Department of Neuroscience, School of Graduate and Postdoctoral Studies, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA; 4Stem Cell Division, Al-Yamama Company, 5Jordan Orthopedic and Spinal Center, 6National Center for Diabetes, Endocrinology and Genetics, 7Department of Pathology, Faculty of Medicine, University of Jordan, Amman, Jordan Background: Methods to obtain safe and practical populations of stem cells (SCs at a clinical grade that are able to differentiate into neuronal cell lineages are yet to be developed. In a previous study, we showed that mouse bone marrow-derived SCs (BM-SCs differentiated into neuronal cell-like lineages when put in a neuronal-like environment, which is a special media supplemented with the necessary growth factors needed for the differentiation of SCs into neuronal cell-like lineages. Aim: In this study, we aim to assess the potentials of adult human CD34+ and CD133+ SCs to differentiate into neuronal cell-like lineages ex vivo when placed in a neuronal-like microenvironment. Methods: The neuronal-like microenvironment was created by culturing cells in nonhematopoietic expansion media (NHEM supplemented with growth factors that favor differentiation into neuronal cell lineages (low-affinity nerve growth factor [LNGF], mouse spinal cord extract [mSpE], or both. Cultured cells were assessed for neuronal differentiation by cell morphologies and by expression of GFAP. Results: Our results show that culturing unpurified human BM-derived mononuclear cells (hBM-MNCs in NHEM+LNGF+mSpE did not lead to neuronal differentiation. In contrast, culturing of purified CD34+ hBM-SCs in NHEM+LNGF+mSpE favored their differentiation into astrocyte

  3. Objective human tooth colour measurements as a means of determining chronologic age in vivo and ex vivo.

    Science.gov (United States)

    Devos, N; Willems, G; Wood, R

    2009-12-01

    Colour is a subjective sensation and as such is difficult to use in a quantitative study. However a number of clinical studies on extracted teeth have shown a good correlation between tooth colour and age. The purpose of this study was to examine the usefulness of a specific spectrophotometer in determining tooth colour on extracted and non- extracted teeth and to look for a possible age relationship. There were two parts in this study. An ex vivo study concentrated on collected tooth material. Single rooted teeth were selected out of each of the 5- year-age groups (ages ranged from 15-89 years). Colour measurements were performed on the mesial and vestibular aspects of the roots as well on the mid-vestibular aspects of the enamel crown. An in vivo study concentrated on the use of this specific shade taking system in living patients (n=70). Statistical analysis of the results revealed regression formulas for both ex vivo and in vivo situations displaying adjusted R-squares between 0.48 and 0.56. It may be concluded that age related trends were found. Having its shortcomings, the shade taking system was found to perform well as a convenient adjunct to dental age estimation in both the living and the deceased.

  4. Induction of reactive oxygen intermediates-dependent programmed cell death in human malignant ex vivo glioma cells and inhibition of the vascular endothelial growth factor production by taurolidine.

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    Rodak, Roksana; Kubota, Hisashi; Ishihara, Hideyuki; Eugster, Hans-Pietro; Könü, Dilek; Möhler, Hanns; Yonekawa, Yasuhiro; Frei, Karl

    2005-06-01

    Taurolidine, a derivative of the amino acid taurin, was recently found to display a potent antineoplastic effect both in vitro and in vivo. The authors therefore initiated studies to assess the potential antineoplastic activity of taurolidine in human glioma cell lines and in ex vivo malignant cell cultures. They also studied the mechanisms that induce cell death and the impact of taurolidine on tumor-derived vascular endothelial growth factor (VEGF) production. Cytotoxicity and clonogenic assays were performed using crystal violet staining. In the cytotoxicity assay 100% of glioma cell lines (eight of eight) and 74% of ex vivo glioma cultures (14 of 19) demonstrated sensitivity to taurolidine, with a mean median effective concentration (EC50) of 51 +/- 28 microg/ml and 56 +/- 23 microg/ml, respectively. Colony formation was inhibited by taurolidine, with a mean EC50 of 7 +/- 3 microg/ml for the cell lines and a mean EC50 of 3.5 +/- 1.7 microg/ml for the ex vivo glioma cultures. On observing this high activity of taurolidine in both assays, the authors decided to evaluate its cell death mechanisms. Fragmentation of DNA, externalization of phosphatidylserine, activation of poly(adenosine diphosphate-ribose) polymerase, loss of the mitochondrial membrane potential followed by a release of apoptosis-inducing factor, and typical apoptotic features were found after taurolidine treatment. Cell death was preceded by the generation of reactive O2 intermediates, which was abrogated by N-acetylcysteine but not by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Moreover, taurolidine also induced suppression of VEGF production on the protein and messenger RNA level, as shown by an enzyme-linked immunosorbent assay and by reverse transcription-polymerase chain reaction. Given all these findings, taurolidine may be a promising new agent in the treatment of malignant gliomas; it displays a combination of antineoplastic and antiangiogenic activities, inducing tumor cell

  5. Administration of the cyclic peptide COR-1 in humans (phase I study): ex vivo measurements of anti-β1-adrenergic receptor antibody neutralization and of immune parameters.

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    Münch, Götz; Boivin-Jahns, Valerie; Holthoff, Hans-Peter; Adler, Kristin; Lappo, Mariola; Truöl, Stephan; Degen, Heidrun; Steiger, Nina; Lohse, Martin J; Jahns, Roland; Ungerer, Martin

    2012-11-01

    A novel concept for the treatment of heart failure is the neutralization of antibodies against the β(1)-adrenergic receptor (anti-β(1)AR-ab). In a rat model of autoimmune cardiomyopathy, the cyclic peptide COR-1 (given i.v. once monthly) neutralized anti-β(1)AR-abs and prevented anti-β(1)AR-ab-induced myocardial damage, and completely reverted cardiac dysfunction over 3-6 months. A clinical phase I trial was designed as a single-blinded, placebo-controlled study. Fifty human volunteers received COR-1 or matching placebo as a single i.v. administration with ascending doses (10-240 mg). Primary endpoints were safety and tolerability, while the pharmacokinetic profile of COR-1 was assessed as a secondary endpoint. All five investigated dose groups were well tolerated; no drug-related side effects occurred. Pharmacokinetics revealed a favourable profile with an almost complete plasma clearance within 60 min after administration. Pharmacodynamic investigation showed dose-dependent efficacy with almost complete scavenging of pathological anti-β(1)AR-abs ex vivo at the two highest doses. No anti-COR-1 autoantibodies occurred. No other effects on the immune system (such as an increase of crucial cytokines) were observed up to 43 days after drug administration, nor upon incubation of anti-β(1)AR-ab-positive patient blood samples with COR-1 ex vivo. COR-1 was shown to be safe after i.v. administration in vivo; no relevant side effects occurred. Efficacy was estimated from ex vivo investigation of the potency to neutralize specific anti-β(1)-AR-abs. NCT 01043146, Eudra CT 2008-007745-31.

  6. The hollow fiber bioreactor as a stroma-supported, serum-free ex vivo expansion platform for human umbilical cord blood cells.

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    Xue, Cao; Kwek, Kenneth Y C; Chan, Jerry K Y; Chen, Qingfeng; Lim, Mayasari

    2014-07-01

    The bone marrow microenvironment plays an integral role in the regulation of hematopoiesis. Residing stromal cells and the extracellular matrix in the bone marrow microenvironment provide biological signals that control hematopoietic stem cell (HSC) function. In this study, we developed a bio-mimetic co-culture platform using the hollow fiber bioreactor (HFBR) for ex vivo expansion of HSCs. We evaluated the efficacy of such a platform in comparison to standard cultures performed on tissue culture polystyrene (TCP), using a human stromal cell line (HS-5) as stromal support, co-cultured with lineage-depleted human cord blood cells in serum-free medium supplemented with a cytokine cocktail. Our results showed that the performance of the HFBR in supporting total cell and CD34(+) progenitor cell expansion was comparable to that of cultures on TCP. Cells harvested from the HFBR had a higher clonogenic ability. The performance of ex vivo-expanded cells from the HFBR in hematopoietic reconstitution in humanized mice was comparable to that of the TCP control. Scanning electron microscopy revealed that stroma cell growth inside the HFBR created a three-dimensional cell matrix architecture. These findings demonstrate the feasibility of utilizing the HFBR for creating a complex cell matrix architecture, which may provide good in vitro mimicry of the bone marrow, supporting large-scale expansion of HSCs. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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    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.

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Ex vivo generation of human alloantigen-specific regulatory T cells from CD4(posCD25(high T cells for immunotherapy.

    Directory of Open Access Journals (Sweden)

    Jorieke H Peters

    Full Text Available BACKGROUND: Regulatory T cell (Treg based immunotherapy is a potential treatment for several immune disorders. By now, this approach proved successful in preclinical animal transplantation and auto-immunity models. In these models the success of Treg based immunotherapy crucially depends on the antigen-specificity of the infused Treg population. For the human setting, information is lacking on how to generate Treg with direct antigen-specificity ex vivo to be used for immunotherapy. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that in as little as two stimulation cycles with HLA mismatched allogeneic stimulator cells and T cell growth factors a very high degree of alloantigen-specificity was reached in magnetic bead isolated human CD4(posCD25(high Treg. Efficient increases in cell numbers were obtained. Primary allogeneic stimulation appeared a prerequisite in the generation of alloantigen-specific Treg, while secondary allogeneic or polyclonal stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies enriched alloantigen-specificity and cell yield to a similar extent. CONCLUSIONS/SIGNIFICANCE: The ex vivo expansion protocol that we describe will very likely increase the success of clinical Treg-based immunotherapy, and will help to induce tolerance to selected antigens, while minimizing general immune suppression. This approach is of particular interest for recipients of HLA mismatched transplants.

  10. Micro-endoscopy of the human vas deferens: a feasibility study of a novel device in several ex vivo models.

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    Trottmann, M; Sroka, R; Braun, C; Liedl, B; Schaaf, H; Graw, M; Becker, A J; Stief, C G; Khoder, W Y

    2017-01-01

    The aim of this study was to show limitation as well as potential of micro-endoscopy techniques as an innovative diagnostic and therapeutic approach in andrology. Two kinds of custom-made micro-endoscopes (ME) were tested in ex vivo vas deferens specimen and in post-mortem whole body. The semi-rigid ME included a micro-optic (0.9 mm outer diameter [OD], 10.000 pixels, 120° vision angle [VE], 3-20 mm field depth [FD]) and an integrated fibre-optic light source. The flexible ME was composed of a micro-optic (OD = 0.6 mm, 6.000 pixels, 120° VE, 3-20 mm FD). The ex vivo study included retrograde investigation of the vas deferens (surgical specimen n = 9, radical prostatectomy n = 3). The post-mortem investigation (n = 4) included the inspection of the vas deferens via both approaches. The results showed that antegrade and retrograde rigid endoscopy of the vas deferens were achieved as a diagnostic tool. The working channel enabled therapeutic use including biopsies or baskets. Using the flexible ME, the orifices of the ejaculatory ducts were identified. In vivo cadaveric retrograde cannulation of the orifices was successful. Post-mortem changes of verumontanum hindered the examinations beyond. Orifices were identified shaded behind a thin transparent membrane. Antegrade vasoscopy using flexible ME was possible up to the internal inguinal ring. Further advancement was impossible because of anatomical angle and lack adequate vision guidance. The vas deferens interior was clearly visible and was documented by pictures and movies. Altogether, the described ME techniques were feasible and effective, offering the potential of innovative diagnostic and therapeutic approaches for use in the genital tract. Several innovative indications could be expected. © 2016 American Society of Andrology and European Academy of Andrology.

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

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    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.

  12. Performance assessment of Pulse Wave Imaging using conventional ultrasound in canine aortas ex vivo and normal human arteries in vivo.

    Science.gov (United States)

    Li, Ronny X; Qaqish, William; Konofagou, Elisa E

    2015-09-01

    The propagation behavior of the arterial pulse wave may provide valuable diagnostic information for cardiovascular pathology. Pulse Wave Imaging (PWI) is a noninvasive, ultrasound imaging-based technique capable of mapping multiple wall motion waveforms along a short arterial segment over a single cardiac cycle, allowing for the regional pulse wave velocity (PWV) and propagation uniformity to be evaluated. The purpose of this study was to improve the clinical utility of PWI using a conventional ultrasound system. The tradeoff between PWI spatial and temporal resolution was evaluated using an ex vivo canine aorta (n = 2) setup to assess the effects of varying image acquisition and signal processing parameters on the measurement of the PWV and the pulse wave propagation uniformity r2. PWI was also performed on the carotid arteries and abdominal aortas of 10 healthy volunteers (24.8 ± 3.3 y.o.) to determine the waveform tracking feature that would yield the most precise PWV measurements and highest r2 values in vivo. The ex vivo results indicated that the highest precision for measuring PWVs ~ 2.5 - 3.5 m/s was achieved using 24-48 scan lines within a 38 mm image plane width (i.e. 0.63 - 1.26 lines/mm). The in vivo results indicated that tracking the 50% upstroke of the waveform would consistently yield the most precise PWV measurements and minimize the error in the propagation uniformity measurement. Such findings may help establish the optimal image acquisition and signal processing parameters that may improve the reliability of PWI as a clinical measurement tool.

  13. Performance assessment and optimization of Pulse Wave Imaging (PWI) in ex vivo canine aortas and in vivo normal human arteries.

    Science.gov (United States)

    Li, Ronny X; Qaqish, William W; Shahmirzadi, Danial; Konofagou, Elisa E

    2012-01-01

    The amplitude, velocity, and morphology of the arterial pulse wave may all provide valuable diagnostic information for cardiovascular pathology. Pulse Wave Imaging (PWI) is an ultrasound-based method developed by our group to noninvasively visualize and map the spatio-temporal variations of the pulse wave-induced vessel wall motion. Because PWI is capable of acquiring multiple wall motion waveforms successively along an imaged arterial segment over a single cardiac cycle in vivo, the regional morphological changes, amplitudes, and velocity (i.e. pulse wave velocity, or PWV) of the pulse wave can all be evaluated. In this study, an ex vivo setup was used to assess the effects of varying PWI image acquisition variables (beam density/frame rate and scanning orientation) and signal processing methods (beam sweep compensation scheme and waveform feature tracking) on the PWV estimation in order to validate the optimal parameters. PWI was also performed on the carotid arteries and abdominal aortas of six healthy volunteers for identification of several salient features of the waveforms over the entire cardiac cycle that may aid in assessing the morphological changes of the pulse wave. The ex vivo results suggest that the PWI temporal resolution is more important for PWV estimation than the PWI spatial resolution, and also that the reverse scanning orientation (i.e. beam sweeping direction opposite the direction of fluid flow) is advantageous due to higher precision and less dependence on the frame rate. In the in vivo waveforms, the highest precision PWV measurements were obtained by tracking the 50% upstroke of the waveforms. Finally, the dicrotic notch, reflected wave, and several inflection points were qualitatively identified in the carotid and aortic anterior wall motion waveforms and shown in one representative subject.

  14. Selective venous vasodilator properties of the analgesic metamizole (dipyrone) in a human ex vivo model-implications for postoperative pain management.

    Science.gov (United States)

    Hoenicka, Markus; Gorki, Hagen; Traeger, Karl; Liebold, Andreas

    2017-05-01

    Metamizole (dipyrone) is a first-line, non-opioid analgesic used for postoperative pain management. Clinical data and animal experiments indicate a possible vasodilator action of this drug. We investigated the effects of metamizole on human artery and vein tone in an ex vivo model to assess potential contributions to venous pooling. Excess segments of bypass grafts were harvested during coronary artery bypass grafting procedures. Tensions were measured in an organ bath for 120 min after adding metamizole to the preconstricted vessels. Contribution of endothelium was assessed in endothelium-denuded vessels, and indometacin was used to identify cyclooxygenase-mediated effects. Internal mammary arteries (n = 6) constricted after addition of 1, 3, and 10 μM metamizole and remained constricted at the lower doses. Transient constrictions also occurred in saphenous veins (n = 20), but veins relaxed below solvent controls after 20 min at all concentrations. Endothelium removal (n = 12) and cyclooxygenase inhibition (n = 12) suppressed the vasoconstrictor effect but not the vasodilator effect. Metamizole and its metabolites display counteracting effects on blood vessel tone ex vivo. The vasoconstrictor effect is mediated by cyclooxygenase-derived products. The net effect is site-specific, resulting in a selective venous vasodilator action. This may exacerbate unwanted venous pooling during postoperative pain therapy.

  15. A Simple Method for Establishing Adherent Ex Vivo Explant Cultures from Human Eye Pathologies for Use in Subsequent Calcium Imaging and Inflammatory Studies

    Science.gov (United States)

    Veréb, Zoltán; Facskó, Andrea; Hawlina, Marko

    2014-01-01

    A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes (fvERMs) from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh) stimulation and indicate presence of ACh receptors in these cells. Furthermore, an ex vivo study model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment. PMID:25276840

  16. A Simple Method for Establishing Adherent Ex Vivo Explant Cultures from Human Eye Pathologies for Use in Subsequent Calcium Imaging and Inflammatory Studies

    Directory of Open Access Journals (Sweden)

    Sofija Andjelic

    2014-01-01

    Full Text Available A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs from cataract surgery and proliferative diabetic retinopathy (PDR fibrovascular epiretinal membranes (fvERMs from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh stimulation and indicate presence of ACh receptors in these cells. Furthermore, an ex vivo study model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment.

  17. Evaluation of Corneal Cross-Linking for Treatment of Fungal Keratitis: Using Confocal Laser Scanning Microscopy on an Ex Vivo Human Corneal Model.

    Science.gov (United States)

    Alshehri, Jawaher M; Caballero-Lima, David; Hillarby, M Chantal; Shawcross, Susan G; Brahma, Arun; Carley, Fiona; Read, Nick D; Radhakrishnan, Hema

    2016-11-01

    Some previous reports have established the use of photoactivated chromophore-induced corneal cross-linking (PACK-CXL) in treating fungal keratitis. The results of these case reports have often been conflicting. To systematically study the effect of PACK-CXL in the management of Fusarium keratitis, we have developed an ex vivo model of human corneal infection using eye-banked human corneas. Sixteen healthy ex vivo human corneas were divided into four study groups: (1) untreated control, (2) cross-linked, (3) infected with fungal spores, and (4) infected with fungal spores and then cross-linked. All infected corneas were inoculated with Fusarium oxysporum spores. The PACK-CXL procedure was performed 24 hours post inoculation for group 4. For PACK-CXL treatment, the corneas were debrided of epithelium; then 1% (wt/vol) isotonic riboflavin was applied dropwise at 5-minute intervals for 30 minutes and during the course of UV-A cross-linking for another 30 minutes. The corneas were imaged using a confocal microscope at 48 hours post inoculation, and the Fusarium hyphal volume and spore concentration were calculated. The infected and then cross-linked group had a significantly lower volume of Fusarium hyphae, compared to the infected (P = 0.001) group. In the infected and then cross-linked group there was significant inhibition of Fusarium sporulation compared with the infected (P = 0.007) group. A model of human corneal infection was successfully developed for investigation of the effects of PACK-CXL on fungal keratitis. A treatment regimen of combined UV-A/riboflavin-induced corneal cross-linking appears to be a valuable approach to inhibit the growth and sporulation of Fusarium and suppress the progression of fungal keratitis.

  18. Ex vivo expansion of Primate CD34+ Cells isolated from Bone Marrow and Human Bone Marrow Mononuclear Cells using a Novel Scaffold

    Directory of Open Access Journals (Sweden)

    Devaprasad D

    2009-01-01

    Full Text Available Bone marrow derived CD34+ cells have been in clinical application in patients with haematological malignancies. One of the major problems with this treatment is the non-availability of matched donors or the necessity of multiple transfusions depending upon the pathology. Recently evidences have been accumulating to prove the safety and efficacy of autologous CD34+ cells in diseases such as myocardial dysfunction, peripheral vascular diseases and neurological certain conditions. However there are only a few reports in the literature on ex vivo expansion of the bone marrow derived CD34+ cells. We have in two different studies proven that isolated CD34+ cells from baboon bone marrow and non-isolated BMMNCs from human bone marrow could be expanded with increase in percentage of CD34+ cells using a novel scaffold.

  19. Development of an ex vivo retention model simulating bioadhesion in the oral cavity using human saliva and physiologically relevant irrigation media

    DEFF Research Database (Denmark)

    Madsen, Katrine D.; Sander, Camilla; Baldursdottir, Stefania

    2013-01-01

    In recent years, there has been a particular interest in bioadhesive formulations for oromucosal drug delivery as this may promote prolonged local therapy and enhanced systemic effect. Saliva plays a vital role in oromucosal drug absorption by dissolving the drug and presenting it to the mucosal...... surface. However, the rheological, chemical, and interfacial properties of this complex biological fluid may strongly affect the adhesion of bioadhesive formulations. There is a need for well characterized in vitro models to assess the bioadhesive properties of oral dosage forms for administration...... in the oral cavity. Thus we aimed at developing an advanced ex vivo buccal retention model, with focus on choosing a physiologically relevant irrigation media closely resembling human saliva. Spray dried chitosan microparticles containing metformin hydrochloride as an example of a small hydrophilic drug, were...

  20. Consumption of selenium-enriched broccoli increases cytokine production in human peripheral blood mononuclear cells stimulated ex vivo, a preliminary human intervention study.

    Science.gov (United States)

    Bentley-Hewitt, Kerry L; Chen, Ronan K-Y; Lill, Ross E; Hedderley, Duncan I; Herath, Thanuja D; Matich, Adam J; McKenzie, Marian J

    2014-12-01

    Selenium (Se) is a micronutrient essential for human health, including immune function. Previous research indicates that Se supplementation may cause a shift from T helper (Th)1- to Th2-type immune responses. We aim to test the potential health promoting effects of Se-enriched broccoli. In a human trial, 18 participants consumed control broccoli daily for 3 days. After a 3-day wash-out period, the participants were provided with Se-enriched broccoli containing 200 μg of Se per serving for 3 days. Plasma and peripheral blood mononuclear cell (PBMC) samples were collected at the start and end of each broccoli feeding period for analysis of total Se and measurement of cytokine production from PBMC stimulated with antigens ex vivo. Plasma Se content remained consistent throughout the control broccoli feeding period and the baseline of the Se-enriched broccoli period (1.22 μmol/L) and then significantly increased following 3 days of Se-enriched broccoli feeding. Interleukin (IL-2, IL-4, IL-5, IL-13, and IL-22) production from PBMC significantly increased after 3 days of Se-enriched broccoli feeding compared with baseline. This study indicates that consumption of Se-enriched broccoli may increase immune responses toward a range of immune challenges. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Generation of Human Induced Pluripotent Stem Cell‐Derived Bona Fide Neural Stem Cells for Ex Vivo Gene Therapy of Metachromatic Leukodystrophy

    Science.gov (United States)

    Meneghini, Vasco; Sala, Davide; De Cicco, Silvia; Luciani, Marco; Cavazzin, Chiara; Paulis, Marianna; Mentzen, Wieslawa; Morena, Francesco; Giannelli, Serena; Sanvito, Francesca; Villa, Anna; Bulfone, Alessandro; Broccoli, Vania; Martino, Sabata

    2016-01-01

    Abstract Allogeneic fetal‐derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient‐specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene‐therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC‐derived neural stem cells (NSCs) showing a reliable “NSC signature” is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS‐NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a “gold standard” in a side‐by‐side comparison when validating the phenotype of hiPS‐NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS‐NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA‐overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA‐overexpressing iPSC‐derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient‐specific ARSA‐overexpressing hiPS‐NSCs may be used in autologous ex vivo gene therapy protocols to provide long‐lasting enzymatic

  2. Anti-tumor activity of the TRA-8 anti-DR5 antibody in combination with cisplatin in an ex vivo human cervical cancer model.

    Science.gov (United States)

    Kendrick, James E; Straughn, J Michael; Oliver, Patsy G; Wang, Wenquan; Nan, Li; Grizzle, William E; Stockard, Cecil R; Alvarez, Ronald D; Buchsbaum, Donald J

    2008-03-01

    To investigate the cytotoxicity of TRA-8, an antibody that specifically binds death receptor 5 (DR5), alone and in combination with cisplatin, using an ex vivo human cervical cancer model. Fifteen cervical cancer specimens were obtained at the time of radical hysterectomy and tumor slices were prepared with the Krumdieck tissue slicer. Tumor slices were exposed to varying concentrations of TRA-8, cisplatin, or the combination of TRA-8 and cisplatin. Using non-linear modeling, dose response curves and IC50 values were generated for each specimen treated with TRA-8. The additive cytotoxic effect of combination treatment was evaluated as well. In addition to ATP viability assays, treated and untreated slices were assessed by immunohistochemistry (IHC) and western blot analysis to confirm apoptosis induction via the extrinsic pathway. Eleven patient specimens yielded TRA-8-induced IC50 values. Sixty-four percent were found to be sensitive to TRA-8-induced cytotoxicity at IC50 doses less than 1000 ng/ml. Seven patient specimens underwent combination treatment with TRA-8 and cisplatin. Of these specimens, 86% exhibited additive cytotoxicity in comparison to treatment with either agent alone. IHC revealed an increase in DR5 expression in tumor slices treated with cisplatin for 24 h. IHC and Western blotting demonstrated TRA-8-induced cell death via apoptosis and activation of caspase 3 and 8. This study confirms the utility of an ex vivo human cervical cancer model, to evaluate the anti-tumor activity of TRA-8 and cisplatin. This model may be a useful pre-clinical tool to assess cytotoxicity and mechanistic properties of novel agents in cervical cancer.

  3. Effect of TRA-8 anti-death receptor 5 antibody in combination with chemotherapy in an ex vivo human ovarian cancer model.

    Science.gov (United States)

    Frederick, Peter J; Kendrick, James E; Straughn, J Michael; Della Manna, Debbie L; Oliver, Patsy G; Lin, Hui-Yi; Grizzle, William E; Stockard, Cecil R; Alvarez, Ronald D; Zhou, Tong; LoBuglio, Albert F; Buchsbaum, Donald J

    2009-07-01

    To investigate the cytotoxicity of TRA-8, an antibody that specifically binds death receptor 5, alone and in combination with chemotherapy, using an ex vivo human ovarian cancer model. Twenty-six ovarian cancer specimens were obtained during ovarian cancer debulking, and tumor slices were prepared with the Krumdieck tissue slicer. The tumor slices were exposed to varying concentrations of TRA-8, carboplatin/paclitaxel, or the combination of TRA-8 and chemotherapy. Using nonlinear modeling, dose-response curves and IC50 values were generated for specimens treated with TRA-8. The additive and synergistic cytotoxic effects of chemotherapy combination with TRA-8 were evaluated in specimens. In addition to adenosine triphosphate viability assays, the treated and untreated slices were assessed by immunohistochemistry to confirm apoptosis induction. Specimens from 13 patients yielded TRA-8-induced IC50 values. Of these specimens, 15% were found to be sensitive to TRA-8-induced cytotoxicity at IC50 doses less than 500 ng/mL. Specimens from 13 patients underwent combination treatment with TRA-8 and carboplatin/paclitaxel. Of these specimens, 77% exhibited additive cytotoxicity in comparison with those treated with either agent alone, whereas 15% exhibited synergistic cytotoxicity. Immunohistochemical analysis of terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling and cleaved caspase 3 staining demonstrated a dose-dependent increase in apoptosis with the combination treatment. This study demonstrates the efficacy of the death receptor monoclonal antibody TRA-8 in combination with conventional chemotherapy in an ex vivo human ovarian cancer model. This model can be used to assess cytotoxicity of novel agents in combination with chemotherapy in ovarian cancer.

  4. Removal process of prion and parvovirus from human platelet lysates used as clinical-grade supplement for ex vivo cell expansion.

    Science.gov (United States)

    Kao, Yu-Chun; Bailey, Andy; Samminger, Bernhard; Tanimoto, Junji; Burnouf, Thierry

    2016-07-01

    Pooled human platelet lysate (HPL) is becoming the new gold standard as supplement for ex vivo cell culture for clinical protocols. However, the risk of pathogen contamination of HPL increases with the platelet pool size. We hypothesized that hollow fiber anion exchange membrane chromatography using QyuSpeed D (QSD) could remove resistant and untested bloodborne pathogens, such as parvoviruses and prions, from HPL-supplemented growth media without substantially affecting their capacity to support ex vivo cell expansion. Frozen or thawed platelet concentrates were serum-converted and centrifuged for obtaining HPL that was added to various growth media (ca. 100 mL), filtered through a 0.6-mL QSD membrane and characterized for proteins, growth factors and chemical composition. Capacity to expand Chinese hamster ovary, periodontal ligament, gingival fibroblast cells and Wharton's jelly mesenchymal stromal cells was studied. Removal of porcine parvovirus (PPV) and of the 263K prion strain of hamster-adapted scrapie was studied by spiking experiments following international guidelines. QSD had minimal impact on HPL-supplemented medium composition in proteins, growth factors and chemical content, nor capacity to expand and differentiate cells. In addition, QSD could remove ≥5.58 log10 [TCID50/mL] and ≥3.72 log10 of PPV and the 263K prion, respectively. QSD hollow fiber chromatography can be used to improve the virus and prion safety of HPL-supplemented media to safely expand cells for clinical protocols. These data bring new perspectives for increasingly safer use of pooled HPL in cell therapy and regenerative medicine applications. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  5. Determinants in HIV-1 Nef for enhancement of virus replication and depletion of CD4+ T lymphocytes in human lymphoid tissue ex vivo

    Directory of Open Access Journals (Sweden)

    Sertel Serkan

    2009-01-01

    Full Text Available Abstract Background HIV-1 Nef critically contributes to AIDS in part by augmenting virus titers in infected individuals. Analyzing which of Nef's activities contribute to HIV pathogenesis has been hampered by the lack of a cell culture model in which Nef exerts pronounced effects on HIV replication. The human lymphoid aggregate culture (HLAC from tonsil maintains the cell populations and cytokine milieu found in vivo, supports a productive infection without exogenous stimulation, and Nef contributes to efficient HIV-1 replication as well as CD4+ T cell depletion in this experimental ex vivo-model. Results To identify determinants in Nef that mediate these activities, we infected HLAC with a panel of isogenic HIV-1NL4-3 strains that encode for well-characterized mutants of HIV-1SF2 Nef. Determination of HIV-1 replication revealed that enhancement of the virus spread by Nef is governed by a complex set of protein interaction surfaces. In contrast, increased CD4+ T lymphocyte depletion depended on only two protein interaction surfaces in Nef that mediate either downregulation of cell surface CD4 or interaction with the NAKC signalosome. Consistently, in HLAC from 9 out of 14 donors, Nef enhanced CD4+ T cell depletion in the absence of a significant effect on virus replication. Moreover, our results suggest that this Nef-dependent enhancement in depletion occurred predominately in uninfected bystander CD4+ T cells. Conclusion Our findings suggest that Nef facilitates depletion of CD4+ T lymphocytes in HIV-1-infected lymphoid tissue ex vivo by increasing the pool of productively infected cells and by sensitizing bystander cells for killing. This ability might contribute to Nef's pathogenic potential in vivo.

  6. Efficacy and Safety of the Collagenase of the Bacterium Clostridium Histolyticum for the Treatment of Capsular Contracture after Silicone Implants: Ex-Vivo Study on Human Tissue.

    Directory of Open Access Journals (Sweden)

    Sebastian Fischer

    Full Text Available The fibrotic capsule that surrounds silicone implants consists mainly of collagen. The FDA-approved collagenase of the bacterium clostridium histolyticum provides a reasonable treatment option. Safety and efficacy at the female breast site must be evaluated before clinical utilization.We incubated 20 samples of fibrotic capsule as well as 12 full thickness skin grafts harvested from the female breast site for 24 hours with different doses of collagenase. Outcome measures involved histological assessment of thickness and density of the capsule tissue as well as the skin grafts. Furthermore, we performed a collagen assay and immunohistochemistry staining for collagen subtypes.Collagenase treatment was able to degrade human capsule contracture tissue ex-vivo. The remaining collagen subtype after degradation was type 4 only. 0.3 mg/ml of collagenase was most effective in reducing capsule thickness when compared with higher concentrations. Of note, effectiveness was inversely related to capsule density, such that there was less reduction in thickness with higher capsule densities and vice versa. Furthermore, the application of 0.3mg/ml collagenase did not lead to thinning or perforation of full thickness skin grafts.Adjustment of collagenase dose will depend on thickness and density of the contracted capsule. A concentration of 0.3mg/ml seems to be safe and effective in an ex-vivo setting. The remaining collagen subtype 4 is suitable to serve as a neo-capsule/acellular tissue matrix. Collagenase treatment for capsular contracture may soon become a clinical reality.

  7. Evaluation of the Appearance of Nail Polish Following Daily Treatment of Ex Vivo Human Fingernails With Topical Solutions of Tavaborole or Efinaconazole.

    Science.gov (United States)

    Vlahovic, Tracey C; Coronado, Dina; Chanda, Sanjay; Merchant, Tejal; Zane, Lee T

    2016-01-01

    Patients with onychomycosis may mask infected nails with polish. Tavaborole topical solution, 5% is a boron-based, small-molecule pharmaceutical approved for the treatment of toenail onychomycosis caused by Trichophyton rubrum and Trichophyton mentagrophytes; efinaconazole topical solution, 10% is approved for the same indication. Nail polish appearance after application of tavaborole (dropper) or efinaconazole (brush); respective applicator appearance; presence of color transfer from respective applicators; and color transfer to remaining solutions after dosing of polished nails were evaluated. Twelve ex vivo human cadaver fingernails were cleaned, polished with two coats of L'Oréal® Nail Color, Devil Wears Red #420, and mounted on floral foam. Nails were treated with tavaborole or efinaconazole solutions once daily for 7 days. Dropper and brush applicators were applied to white watercolor paper immediately after dosing to evaluate color transfer from polished nails. On day 7, remaining solutions were transferred to clear glass vials to evaluate color transfer from applicators to solutions. Nails, applicators, and papers were photographed daily following application; remaining solutions were photographed after 7 days of dosing. Tavaborole-treated polished nails showed no polish discoloration, and tavaborole applicators did not change in appearance during treatment. No color transfer from polished nails was evident to applicator, paper, or remaining solution. Efinaconazole-treated polished nails showed substantial polish changes after the first day of treatment, with polish appearance and discoloration progressively worsening over 7 days of treatment. Color transfer from nails was evident to applicator, paper, and remaining solution. Daily dropper application of tavaborole to ex vivo polished nails did not alter polish appearance. Brush application of efinaconazole produced visible changes in polish appearance and color transfer to applicators, paper, and

  8. Modified Corneosurfametry as a new accelerated high-throughput ex vivo methodology for predicting cleanser effects towards human skin.

    Science.gov (United States)

    Liu, M; Mollica, L; Regan, J; Hawkins, S; McGuiness, H; Vetro, K; Garczarek, U; Shi, S; Vasudevan, V; Ananthapadmanabhan, K P

    2016-04-01

    Corneosurfametry (CSM) was originally developed as a tool to predict irritation potential of cleansers. In this method, surface skin stripped using cyanoacrylate is contacted with surfactants/products, rinsed and stained with toluidine blue and basic fuschin dyes. The intensity of staining increases with increases in irritation potential of surfactant. Our objective was to modify the CSM technique to achieve better control of the tape stripping process. Another objective was to correlate the modified CSM (MCSM) with a traditional in-vivo forearm controlled application test (FCAT) for mildness and to explore its utility to assess the state of corneum after a clinical test. Surface skin cells were tape stripped from forearms of volunteers with D-Squame Adhesive Discs. Discs were treated with a 10% solution of the product in a 96-plate well for 10 min, rinsed, dried and treated with basic fuschin-toluidine blue dye solution, rinsed and dried again. Forearm Controlled Application Test (FCAT) was based on a published protocol. Tape strips obtained after product treatment were also analyzed by the MCSM procedure without additional product treatment. Mildness/barrier damage assessed from in-vivo FCAT showed a similar ranking to the MCSM results. MCSM, TEWL and Erythema analysis of between-treatment differences showed a good correlation indicating that barrier damage seen in in-vivo studies can be predicted from ex-vivo MCSM studies. MCSM analysis of tape strips after the FCAT study showed that the damage decreased with increase in tape strip number. A moisturizing body wash (MBW) with mild surfactants showed the least damage in all layers. In contrast, harsh dish washing liquid showed significantly higher damage down to several layers. Another MBW with petrolatum in a harsher base showed damage almost similar to that of the harsh dish washing liquid in the surface layers. Thus, the MCSM was able to show underlying damage which would have been normally masked by the

  9. Human platelet lysate supports ex vivo expansion and enhances osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Xia, Wenjie; Li, Hui; Wang, Zhen; Xu, Ru; Fu, Yongshui; Zhang, Xiuming; Ye, Xin; Huang, Yingfeng; Xiang, Andy Peng; Yu, Weihua

    2011-06-01

    MSCs (mesenchymal stem cells) with their versatile growth and differentiation potential are ideal candidates for use in regenerative medicine and are currently making their way into clinical trials, which requires the development of xeno-free protocols for their culture. In this study, MSCs were cultured in 10% FCS or 7.5% HPL (human platelet lysate)-supplemented media. We found that both groups of MSCs showed a comparable morphology, phenotype and proliferation. The percentage of cells in the S- and G2-/M-phases, however, was slightly up-regulated (Pplatelet derived growth factor)-AB and IGF (insulin-like growth factor)-1. In addition, compared with FCS group, MSCs in HPL group showed an increase in osteogenic differentiation and a decrease in adipogenic differentiation. In conclusion, MSCs in HPL-supplemented media maintained similar growing potential and phenotype, while osteogenic potential was enhanced. HPL offers a promising alternative to FCS for MSC expansion for clinical application, especially in bone injury diseases.

  10. Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys.

    Science.gov (United States)

    Petersen, Björn; Ramackers, Wolf; Lucas-Hahn, Andrea; Lemme, Erika; Hassel, Petra; Queisser, Anna-Lisa; Herrmann, Doris; Barg-Kues, Brigitte; Carnwath, Joseph W; Klose, Johannes; Tiede, Andreas; Friedrich, Lars; Baars, Wiebke; Schwinzer, Reinhard; Winkler, Michael; Niemann, Heiner

    2011-01-01

    The major immunological hurdle to successful porcine-to-human xenotransplantation is the acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and perturbation of coagulation. Heme oxygenase-1 (HO-1) and its derivatives have anti-apoptotic, anti-inflammatory effects and protect against reactive oxygen species, rendering HO-1 a promising molecule to control AVR. Here, we report the production and characterization of pigs transgenic for human heme oxygenase-1 (hHO-1) and demonstrate significant protection in porcine kidneys against xenograft rejection in ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells (PAEC) in a TNF-α-mediated apoptosis assay. Transgenic and non-transgenic PAEC were tested in a TNF-α-mediated apoptosis assay. Expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) was measured by real-time PCR. hHO-1 transgenic porcine kidneys were perfused with pooled and diluted human AB blood in an ex vivo perfusion circuit. MHC class-II up-regulation after induction with IFN-γ was compared between wild-type and hHO-1 transgenic PAEC. Cloned hHO-1 transgenic pigs expressed hHO-1 in heart, kidney, liver, and in cultured ECs and fibroblasts. hHO-1 transgenic PAEC were protected against TNF-α-mediated apoptosis. Real-time PCR revealed reduced expression of adhesion molecules like ICAM-1, VCAM-1, and E-selectin. These effects could be abrogated by the incubation of transgenic PAECs with the specific HO-1 inhibitor zinc protoporphorine IX (Zn(II)PPIX, 20 μm). IFN-γ induced up-regulation of MHC class-II molecules was significantly reduced in PAECs from hHO-1 transgenic pigs. hHO-1 transgenic porcine kidneys could successfully be perfused with diluted human AB-pooled blood for a maximum of 240 min (with and without C1 inh), while in wild-type kidneys, blood flow ceased after ∼60 min. Elevated levels of d-Dimer and TAT were detected, but no significant consumption of fibrinogen and

  11. Impact of Cosmetic Lotions on Nanoparticle Penetration through ex Vivo C57BL/6 Hairless Mouse and Human Skin: A Comparison Study

    Directory of Open Access Journals (Sweden)

    Samreen Jatana

    2016-02-01

    Full Text Available Understanding the interactions of nanoparticles (NPs with skin is important from a consumer and occupational health and safety perspective, as well as for the design of effective NP-based transdermal therapeutics. Despite intense efforts to elucidate the conditions that permit NP penetration, there remains a lack of translatable results from animal models to human skin. The objectives of this study are to investigate the impact of common skin lotions on NP penetration and to quantify penetration differences of quantum dot (QD NPs between freshly excised human and mouse skin. QDs were mixed in seven different vehicles, including five commercial skin lotions. These were topically applied to skin using two exposure methods; a petri dish protocol and a Franz diffusion cell protocol. QD presence in the skin was quantified using Confocal Laser Scanning Microscopy. Results show that the commercial vehicles can significantly impact QD penetration in both mouse and human skin. Lotions that contain alpha hydroxyl acids (AHA facilitated NP penetration. Lower QD signal was observed in skin studied using a Franz cell. Freshly excised human skin was also studied immediately after the sub-cutaneous fat removal process, then after 24 h rest ex vivo. Resting human skin 24 h prior to QD exposure significantly reduced epidermal presence. This study exemplifies how application vehicles, skin processing and the exposure protocol can affect QD penetration results and the conclusions that maybe drawn between skin models.

  12. Impact of Cosmetic Lotions on Nanoparticle Penetration through ex vivo C57BL/6 Hairless Mouse and Human Skin: A Comparison Study

    Science.gov (United States)

    Jatana, Samreen; Callahan, Linda M.; Pentland, Alice P.; DeLouise, Lisa A.

    2016-01-01

    Understanding the interactions of nanoparticles (NPs) with skin is important from a consumer and occupational health and safety perspective, as well as for the design of effective NP-based transdermal therapeutics. Despite intense efforts to elucidate the conditions that permit NP penetration, there remains a lack of translatable results from animal models to human skin. The objectives of this study are to investigate the impact of common skin lotions on NP penetration and to quantify penetration differences of quantum dot (QD) NPs between freshly excised human and mouse skin. QDs were mixed in 7 different vehicles, including 5 commercial skin lotions. These were topically applied to skin using two exposure methods; a petri dish protocol and a Franz diffusion cell protocol. QD presence in the skin was quantified using Confocal Laser Scanning Microscopy. Results show that the commercial vehicles can significantly impact QD penetration in both mouse and human skin. Lotions that contain alpha hydroxyl acids (AHA) facilitated NP penetration. Lower QD signal was observed in skin studied using a Franz cell. Freshly excised human skin was also studied immediately after the sub-cutaneous fat removal process, then after 24 hours rest ex vivo. Resting human skin 24 hours prior to QD exposure significantly reduced epidermal presence. This study exemplifies how application vehicles, skin processing and the exposure protocol can affect QD penetration results and the conclusions that maybe drawn between skin models. PMID:27453793

  13. Impact of Cosmetic Lotions on Nanoparticle Penetration through ex vivo C57BL/6 Hairless Mouse and Human Skin: A Comparison Study.

    Science.gov (United States)

    Jatana, Samreen; Callahan, Linda M; Pentland, Alice P; DeLouise, Lisa A

    2016-03-01

    Understanding the interactions of nanoparticles (NPs) with skin is important from a consumer and occupational health and safety perspective, as well as for the design of effective NP-based transdermal therapeutics. Despite intense efforts to elucidate the conditions that permit NP penetration, there remains a lack of translatable results from animal models to human skin. The objectives of this study are to investigate the impact of common skin lotions on NP penetration and to quantify penetration differences of quantum dot (QD) NPs between freshly excised human and mouse skin. QDs were mixed in 7 different vehicles, including 5 commercial skin lotions. These were topically applied to skin using two exposure methods; a petri dish protocol and a Franz diffusion cell protocol. QD presence in the skin was quantified using Confocal Laser Scanning Microscopy. Results show that the commercial vehicles can significantly impact QD penetration in both mouse and human skin. Lotions that contain alpha hydroxyl acids (AHA) facilitated NP penetration. Lower QD signal was observed in skin studied using a Franz cell. Freshly excised human skin was also studied immediately after the sub-cutaneous fat removal process, then after 24 hours rest ex vivo. Resting human skin 24 hours prior to QD exposure significantly reduced epidermal presence. This study exemplifies how application vehicles, skin processing and the exposure protocol can affect QD penetration results and the conclusions that maybe drawn between skin models.

  14. Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation.

    Science.gov (United States)

    Zhang, Cheng Cheng; Kaba, Megan; Iizuka, Satoru; Huynh, HoangDinh; Lodish, Harvey F

    2008-04-01

    Hematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy, but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular, the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified angiopoietin-like proteins and IGF-binding protein 2 (IGFBP2) as new hormones that, together with other factors, can expand mouse bone marrow HSCs in culture. Here, we measure the activity of multipotent human severe combined immunodeficient (SCID)-repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum-free medium containing SCF, TPO, and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133+ cells. Addition of either angiopoietin-like 5 or IGF-binding protein 2 to the cultures led to a sizable expansion of HSC numbers, as assayed by NOD/SCID transplantation. A serum-free culture containing SCF, TPO, FGF-1, angiopoietin-like 5, and IGFBP2 supports an approximately 20-fold net expansion of repopulating human cord blood HSCs, a number potentially applicable to several clinical processes including HSC transplantation.

  15. Diagnostic Value of Ex-Vivo Three-Dimensional Micro-Computed Tomography Imaging of Primary Nonhematopoietic Human Bone Tumors: Osteosarcoma versus Chondrosarcoma

    Energy Technology Data Exchange (ETDEWEB)

    Langheinrich, A. C.; Stolle, C.; Kampschulte, M.; Lommel, D.; Rau, W. S.; Bassaly, B. (Depts. of Radiology and Pathology, Univ. of Giessen, Giessen (Germany))

    2008-10-15

    Background: Osteosarcoma and chondrosarcoma are the most common nonhematopoietic primary malignancies of bone. However, unusual radiographic appearances can lead to delay in diagnosis and confusion with benign diseases. Purpose: To evaluate the feasibility of micro-computed tomography (CT) for the analysis of primary, nonhematopoietic human bone tumors ex vivo. Material and Methods: Samples from 12 human bone specimens (osteosarcoma, n=6; chondrosarcoma, n=6) obtained for diagnostic purposes were scanned using industrial X-ray film without amplifier foil and scanned with micro-CT (7- and 12-mum-cubic voxels). Trabecular bone CT 'density' and tumor matrix CT 'density' were determined, and results were compared with those obtained from a detailed conventional histopathologic analysis of corresponding cross-sections. The significance of differences in grayscale measurements was tested with analysis of variance. Results: Micro-CT provided quantitative information on bone morphology equivalent to histopathological analysis. We established grayscale measurements by which tumor matrices of chondrosarcoma and osteosarcoma could be radiographically categorized following histological classifications (P<0.001). Conclusion: Micro-CT is feasible for the analysis and differentiation of human osteosarcoma and chondrosarcoma

  16. Assessing drug transport across the human placental barrier: from in vivo and in vitro measurements to the ex vivo perfusion method and in silico techniques.

    Science.gov (United States)

    Giaginis, Constantinos; Tsantili-Kakoulidou, Anna; Theocharis, Stamatios

    2011-05-01

    Assessing drug transport across the human placental barrier is of vital importance in order to guarantee drug safety during pregnancy. However, due to ethical reasons, in vivo fetal development risk assessment studies related to maternal drugs and chemicals exposure remain extremely limited. To overcome any ethical issues, several in vitro models applying primary trophoblastic cells, immortal cell lines and tissue explants of placental origin have recently been advanced. Alternatively, ex vivo human placental perfusion seems to be a more representative and highly informative method, which offers better insights into the different drug transporters, xenobiotic metabolism and tissue binding. Recently, in silico techniques have further been advanced as complementary tools to validate experimental placental transfer data, offering an attractive alternative for high throughput screening of potential fetotoxicity at the early stages of drug design. The present review scrutinizes, from a critical point of view, the current trends and perspectives in the emerging topic of drug transport across the human placental barrier. The special characteristics of the recently developed biopharmaceuticals on the transplacental transfer process are also discussed.

  17. Human hair growth ex vivo is correlated with in vivo hair growth: selective categorization of hair follicles for more reliable hair follicle organ culture.

    Science.gov (United States)

    Kwon, Oh Sang; Oh, Jun Kyu; Kim, Mi Hyang; Park, So Hyun; Pyo, Hyun Keol; Kim, Kyu Han; Cho, Kwang Hyun; Eun, Hee Chul

    2006-02-01

    Of the numerous assays used to assess hair growth, hair follicle organ culture model is one of the most popular and powerful in vitro systems. Changes in hair growth are commonly employed as a measurement of follicular activity. Hair cycle stage of mouse vibrissa follicles in vivo is known to determine subsequent hair growth and follicle behavior in vitro and it is recommended that follicles be taken at precisely the same cyclic stage. This study was performed to evaluate whether categorization of human hair follicles by the growth in vivo could be used to select follicles of the defined anagen stage for more consistent culture. Occipital scalp samples were obtained from three subjects, 2 weeks later after hair bleaching. Hair growth and follicle length of isolated anagen VI follicles were measured under a videomicroscope. Follicles were categorized into four groups according to hair growth and some were cultured ex vivo for 6 days. Follicles showed considerable variations with respect to hair growth and follicle length; however, these two variables were relatively well correlated. Hair growth in culture was closely related with hair growth rate in vivo. Moreover, minoxidil uniquely demonstrated a significant increase of hair growth in categorized hair follicles assumed at a similar early anagen VI stage of hair cycle. Selection of follicles at a defined stage based on hair-growth rate would permit a more reliable outcome in human hair follicle organ culture.

  18. Ex vivo permeation of carprofen from nanoparticles: A comprehensive study through human, porcine and bovine skin as anti-inflammatory agent.

    Science.gov (United States)

    Parra, Alexander; Clares, Beatriz; Rosselló, Ana; Garduño-Ramírez, María L; Abrego, Guadalupe; García, María L; Calpena, Ana C

    2016-03-30

    The purpose of this study was the development of poly(d,l-lactide-co-glycolide) acid (PLGA) nanoparticles (NPs) for the dermal delivery of carprofen (CP). The developed nanovehicle was then lyophilized using hydroxypropyl-β-cyclodextrin (HPβCD) as cryoprotectant. The ex vivo permeation profiles were evaluated using Franz diffusion cells using three different types of skin membranes: human, porcine and bovine. Furthermore, biomechanical properties of skin (trans-epidermal water loss and skin hydration) were tested. Finally, the in vivo skin irritation and the anti-inflammatory efficacy were also assayed. Results demonstrated the achievement of NPs 187.32 nm sized with homogeneous distribution, negatively charged surface (-23.39 mV) and high CP entrapment efficiency (75.38%). Permeation studies showed similar diffusion values between human and porcine skins and higher for bovine. No signs of skin irritation were observed in rabbits. Topically applied NPs significantly decreased in vivo inflammation compared to the reference drug in a TPA-induced mouse ear edema model. Thus, it was concluded that NPs containing CP may be a useful tool for the dermal treatment of local inflammation. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Sex differences in the pro-inflammatory cytokine response to endotoxin unfold in vivo but not ex vivo in healthy humans.

    Science.gov (United States)

    Wegner, Alexander; Benson, Sven; Rebernik, Laura; Spreitzer, Ingo; Jäger, Marcus; Schedlowski, Manfred; Elsenbruch, Sigrid; Engler, Harald

    2017-07-01

    Clinical data indicate that inflammatory responses differ across sexes, but the mechanisms remain elusive. Herein, we assessed in vivo and ex vivo cytokine responses to bacterial endotoxin in healthy men and women to elucidate the role of systemic and cellular factors underlying sex differences in inflammatory responses. Participants received an i.v. injection of low-dose endotoxin (0.4 ng/kg body mass), and plasma TNF-α and IL-6 responses were analyzed over a period of 6 h. In parallel, ex vivo cytokine production was measured in endotoxin-stimulated blood samples obtained immediately before in vivo endotoxin administration. As glucocorticoids (GCs) play an important role in the negative feedback regulation of the inflammatory response, we additionally analyzed plasma cortisol concentrations and ex vivo GC sensitivity of cytokine production. Results revealed greater in vivo pro-inflammatory responses in women compared with men, with significantly higher increases in plasma TNF-α and IL-6 concentrations. In addition, the endotoxin-induced rise in plasma cortisol was more pronounced in women. In contrast, no sex differences in ex vivo cytokine production and GC sensitivity were observed. Together, these findings demonstrate major differences in in vivo and ex vivo responses to endotoxin and underscore the importance of systemic factors underlying sex differences in the inflammatory response.

  20. 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.

  1. Inhibition of HIV-1 infection in ex vivo cervical tissue model of human vagina by palmitic acid; implications for a microbicide development.

    Directory of Open Access Journals (Sweden)

    Xudong Lin

    Full Text Available BACKGROUND: Approximately 80% of all new HIV-1 infections are acquired through sexual contact. Currently, there is no clinically approved microbicide, indicating a clear and urgent therapeutic need. We recently reported that palmitic acid (PA is a novel and specific inhibitor of HIV-1 fusion and entry. Mechanistically, PA inhibits HIV-1 infection by binding to a novel pocket on the CD4 receptor and blocks efficient gp120-to-CD4 attachment. Here, we wanted to assess the ability of PA to inhibit HIV-1 infection in cervical tissue ex vivo model of human vagina, and determine its effect on Lactobacillus (L species of probiotic vaginal flora. PRINCIPAL FINDINGS: Our results show that treatment with 100-200 µM PA inhibited HIV-1 infection in cervical tissue by up to 50%, and this treatment was not toxic to the tissue or to L. crispatus and jensenii species of vaginal flora. In vitro, in a cell free system that is independent of in vivo cell associated CD4 receptor; we determined inhibition constant (Ki to be ∼2.53 µM. SIGNIFICANCE: These results demonstrate utility of PA as a model molecule for further preclinical development of a safe and potent HIV-1 entry microbicide inhibitor.

  2. Development of an ex vivo retention model simulating bioadhesion in the oral cavity using human saliva and physiologically relevant irrigation media.

    Science.gov (United States)

    Madsen, Katrine D; Sander, Camilla; Baldursdottir, Stefania; Pedersen, Anne Marie L; Jacobsen, Jette

    2013-05-20

    In recent years, there has been a particular interest in bioadhesive formulations for oromucosal drug delivery as this may promote prolonged local therapy and enhanced systemic effect. Saliva plays a vital role in oromucosal drug absorption by dissolving the drug and presenting it to the mucosal surface. However, the rheological, chemical, and interfacial properties of this complex biological fluid may strongly affect the adhesion of bioadhesive formulations. There is a need for well characterized in vitro models to assess the bioadhesive properties of oral dosage forms for administration in the oral cavity. Thus we aimed at developing an advanced ex vivo buccal retention model, with focus on choosing a physiologically relevant irrigation media closely resembling human saliva. Spray dried chitosan microparticles containing metformin hydrochloride as an example of a small hydrophilic drug, were employed as bioadhesive formulations. Chewing-stimulated human whole saliva was collected and characterized for use in retention studies in comparison with four artificial irrigation media; phosphate buffer, Saliva Orthana(®), porcine gastric mucin base media (PGM3), and xanthan gum based media (XG2). Retention of metformin, applied as spray dried microparticles on porcine buccal mucosa, greatly depended on the characteristics of the irrigation media. When rheology of the irrigation media was examined, changes in retention profiles could be interpreted, as irrigation media containing mucin and xanthan gum possessed a higher viscosity than phosphate buffer, which led to longer retention of the drug due to better hydration of the mucosa and the spray dried microparticles. Metformin retention profiles were comparable when human saliva, Saliva Orthana(®), or PGM3 were used as irrigation media. Moreover, PGM3 displayed physico-chemical properties closest to those of human saliva with regard to pH, protein content and surface tension. Saliva Orthana(®) and PGM3 are therefore

  3. The Contributions of Human Mini-Intestines to the Study of Intestinal Physiology and Pathophysiology.

    Science.gov (United States)

    Yu, Huimin; Hasan, Nesrin M; In, Julie G; Estes, Mary K; Kovbasnjuk, Olga; Zachos, Nicholas C; Donowitz, Mark

    2017-02-10

    The lack of accessibility to normal and diseased human intestine and the inability to separate the different functional compartments of the intestine even when tissue could be obtained have held back the understanding of human intestinal physiology. Clevers and his associates identified intestinal stem cells and established conditions to grow "mini-intestines" ex vivo in differentiated and undifferentiated conditions. This pioneering work has made a new model of the human intestine available and has begun making contributions to the understanding of human intestinal transport in normal physiologic conditions and the pathophysiology of intestinal diseases. However, this model is reductionist and lacks many of the complexities of normal intestine. Consequently, it is not yet possible to predict how great the advances using this model will be for understanding human physiology and pathophysiology, nor how the model will be modified to include multiple other intestinal cell types and physical forces necessary to more closely approximate normal intestine. This review describes recent studies using mini-intestines, which have readdressed previously established models of normal intestinal transport physiology and newly examined intestinal pathophysiology. The emphasis is on studies with human enteroids grown either as three-dimensional spheroids or two-dimensional monolayers. In addition, comments are provided on mouse studies in cases when human studies have not yet been described.

  4. EX-VIVO MAGNETIC-RESONANCE-IMAGING OF PRETRANSPLANT HUMAN DONOR LIVER - CLINICAL-EXPERIENCE IN 66 CASES

    NARCIS (Netherlands)

    WOLF, RFE; MOOYAART, EL; KAMMAN, RL; DEKETH, HP; THIJN, CJP; SLOOFF, MJH

    Magnetic resonance imaging (MRI) was performed on 66 cold-stored human donor livers. Spin echo images were obtained with a clinical whole body MRI system. Various parenchymal and vascular abnormalities were found. An unexpected finding was the abundant presence of intrahepatic air. Although the

  5. Tribological behaviour of skin equivalents and ex-vivo human skin against the material components of artificial turf in sliding

    NARCIS (Netherlands)

    Morales Hurtado, Marina; Peppelman, P.; Zeng, Xiangqiong; van Erp, P.E.J.; van der Heide, Emile

    2016-01-01

    This research aims to analyse the interaction of three artificial skin equivalents and human skin against the main material components of artificial turf. The tribological performance of Lorica, Silicone Skin L7350 and a recently developed Epidermal Skin Equivalent (ESE) were studied and compared to

  6. Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology.

    Science.gov (United States)

    Zachos, Nicholas C; Kovbasnjuk, Olga; Foulke-Abel, Jennifer; In, Julie; Blutt, Sarah E; de Jonge, Hugo R; Estes, Mary K; Donowitz, Mark

    2016-02-19

    Identification of Lgr5 as the intestinal stem cell marker as well as the growth factors necessary to replicate adult intestinal stem cell division has led to the establishment of the methods to generate "indefinite" ex vivo primary intestinal epithelial cultures, termed "mini-intestines." Primary cultures developed from isolated intestinal crypts or stem cells (termed enteroids/colonoids) and from inducible pluripotent stem cells (termed intestinal organoids) are being applied to study human intestinal physiology and pathophysiology with great expectations for translational applications, including regenerative medicine. Here we discuss the physiologic properties of these cultures, their current use in understanding diarrhea-causing host-pathogen interactions, and potential future applications. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. ANALYSIS OF IMMEDIATE EX VIVO RELEASE OF NITRIC OXIDE FROM HUMAN COLONIC MUCOSA IN GASTROINTESTINALLY MEDIATED ALLERGY, INFLAMMATORY BOWEL DISEASE AND CONTROLS

    NARCIS (Netherlands)

    Raithel, M.; Hagel, A. F.; Zopf, Y.; Bijlsma, P. B.; de Rossi, T. M.; Gabriel, S.; Weidenhiller, M.; Kressel, J.; Hahn, E. G.; Konturek, P. C.

    2012-01-01

    Nitric oxide (NO) is a local mediator in inflammation and allergy. The aim of this study was to investigate whether live incubated colorectal mucosal tissue shows a direct NO response ex vivo to nonspecific and specific immunological stimuli and whether there are disease-specific differences between

  8. Soluble CD54 induces human endothelial cells ex vivo expansion useful for cardiovascular regeneration and tissue engineering application

    KAUST Repository

    Malara, N.M.

    2015-03-01

    Aim: Consistent expansion of primary human endothelial cells in vitro is critical in the development of engineered tissue. A variety of complex culture media and techniques developed from different basal media have been reported with alternate success. Incongruous results are further confounded by donor-to-donor variability and cellular source of derivation. Our results demonstrate how to overcome these limitations using soluble CD54 (sCD54) as additive to conventional culture medium. Methods and results: Isolated primary fragment of different vessel types was expanded in Ham\\'s F12 DMEM, enriched with growth factors, Fetal Calf Serum and conditioned medium of Human Umbilical Vein Endothelial Cells (HUVEC) collected at different passages. Cytokine content of culture media was analyzed in order to identify the soluble factors correlating with better proliferation profile. sCD54 was found to induce the in vitro expansion of human endothelial cells (HECs) independently from the vessels source and even in the absence of HUVEC-conditioned medium. The HECs cultivated in the presence of sCD54 (50 ng/ml), resulted positive for the expression of CD146 and negative for CD45, and lower fibroblast contamination. Cells were capable to proliferate with an S phase of 25%, to produce vascular endothelial growth factor, VEGF, (10 ng/ml) and to give origin to vessel-like tubule in vitro. Conclusion: Our results demonstrate that sCD54 is an essential factor for the in-vitro expansion of HECs without donor and vessel-source variability. Resulting primary cultures can be useful, for tissue engineering in regenerative medicine (e.g. artificial micro tissue generation, coating artificial heart valve etc.) and bio-nanotechnology applications. © 2015 The Authors. Published by Elsevier Ireland Ltd.

  9. Association between in vivo bone formation and ex vivo migratory capacity of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Andersen, Rikke K; Zaher, Walid; Larsen, Kenneth H

    2015-01-01

    INTRODUCTION: There is a clinical need for developing systemic transplantation protocols for use of human skeletal stem cells (also known bone marrow stromal stem cells) (hBMSC) in tissue regeneration. In systemic transplantation studies, only a limited number of hBMSC home to injured tissues...... vivo transwell migration and following intravenous injection, better in vivo homing ability to bone fracture when compared to LBF clones. Comparative microarray analysis of HBF versus LBF clones identified enrichment of gene categories of chemo-attraction, adhesion and migration associated genes. Among...

  10. Ex vivo human platelet aggregation induced by decompression during reduced barometric pressure, hydrostatic, and hydrodynamic (Bernoulli) effect.

    Science.gov (United States)

    Murayama, M

    1984-03-01

    Decompression of human platelet-rich plasma (PRP) in siliconized glass or plastic to 380 mm Hg for 3 hours at 38 degrees C produced platelet aggregation independent of pO2. Aggregation also took place when PRP was compressed to 8,000 PSI and then decompressed slowly to one atmosphere (14.7 PSI) without gas bubble formation. Platelets also aggregated when plasma was decompressed hydrodynamically (Bernoulli effect) at room temperature. It was also found that the drugs piracetam (2-oxypyrolidine acetamide) and pentoxifylline (1-(5-oxohexyl)-theobromine) at 0.5 and 1.0 mM prevent thrombocyte aggregation. Implications for mountain sickness are discussed.

  11. Integrating Dimension Reduction and Out-of-Sample Extension in Automated Classification of Ex Vivo Human Patellar Cartilage on Phase Contrast X-Ray Computed Tomography

    Science.gov (United States)

    Nagarajan, Mahesh B.; Coan, Paola; Huber, Markus B.; Diemoz, Paul C.; Wismüller, Axel

    2015-01-01

    Phase contrast X-ray computed tomography (PCI-CT) has been demonstrated as a novel imaging technique that can visualize human cartilage with high spatial resolution and soft tissue contrast. Different textural approaches have been previously investigated for characterizing chondrocyte organization on PCI-CT to enable classification of healthy and osteoarthritic cartilage. However, the large size of feature sets extracted in such studies motivates an investigation into algorithmic feature reduction for computing efficient feature representations without compromising their discriminatory power. For this purpose, geometrical feature sets derived from the scaling index method (SIM) were extracted from 1392 volumes of interest (VOI) annotated on PCI-CT images of ex vivo human patellar cartilage specimens. The extracted feature sets were subject to linear and non-linear dimension reduction techniques as well as feature selection based on evaluation of mutual information criteria. The reduced feature set was subsequently used in a machine learning task with support vector regression to classify VOIs as healthy or osteoarthritic; classification performance was evaluated using the area under the receiver-operating characteristic (ROC) curve (AUC). Our results show that the classification performance achieved by 9-D SIM-derived geometric feature sets (AUC: 0.96 ± 0.02) can be maintained with 2-D representations computed from both dimension reduction and feature selection (AUC values as high as 0.97 ± 0.02). Thus, such feature reduction techniques can offer a high degree of compaction to large feature sets extracted from PCI-CT images while maintaining their ability to characterize the underlying chondrocyte patterns. PMID:25710875

  12. Multiparametric analyses of human PBMCs loaded ex vivo with a candidate idiotype vaccine for HCV-related lymphoproliferative disorders.

    Directory of Open Access Journals (Sweden)

    Annacarmen Petrizzo

    Full Text Available Hepatitis C virus (HCV has been identified as one of the major risk factors for type II mixed cryoglobulinemia (MC, during the clinical evolution of chronic hepatitis, which may lead to development of B cell non-Hodgkin's lymphoma (NHL. We have previously shown that the candidate idiotype vaccine, based on the IGKV3-20 light chain protein, is able to induce activation and maturation of circulating antigen presenting cells (APCs in both HCV-positive and HCV-negative healthy control subjects, with production of Th2-type cytokines. Here, the effect of the recombinant IGKV3-20 protein on human peripheral blood mononuclear cells (PBMCs from HCV-positive subjects, with known blood levels of cryoglobulins, is shown via gene expression profiling analysis combined to multiparameter flow cytometry and multiplex analyses of cytokines.

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

    Science.gov (United States)

    Surjana, Devita; Halliday, Gary M; Damian, Diona L

    2013-05-01

    Nicotinamide (vitamin B3) protects from ultraviolet (UV) radiation-induced carcinogenesis in mice and from UV-induced immunosuppression in mice and humans. Recent double-blinded randomized controlled Phase 2 studies in heavily sun-damaged individuals have shown that oral nicotinamide significantly reduces premalignant actinic keratoses, and may reduce new non-melanoma skin cancers. Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD(+)), an essential coenzyme in adenosine triphosphate (ATP) production. Previously, we showed that nicotinamide prevents UV-induced ATP decline in HaCaT keratinocytes. Energy-dependent DNA repair is a key determinant of cellular survival after exposure to DNA-damaging agents such as UV radiation. Hence, in this study we investigated whether nicotinamide protection from cellular energy loss influences DNA repair. We treated HaCaT keratinocytes with nicotinamide and exposed them to low-dose solar-simulated UV (ssUV). Excision repair was quantified using an assay of unscheduled DNA synthesis. Nicotinamide increased both the proportion of cells undergoing excision repair and the repair rate in each cell. We then investigated ssUV-induced cyclobutane pyrimidine dimers (CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxoG) formation and repair by comet assay in keratinocytes and with immunohistochemistry in human skin. Nicotinamide reduced CPDs and 8oxoG in both models and the reduction appeared to be due to enhancement of DNA repair. These results show that nicotinamide enhances two different pathways for repair of UV-induced photolesions, supporting nicotinamide's potential as an inexpensive, convenient and non-toxic agent for skin cancer chemoprevention.

  14. Oral administration of pyridostigmine bromide and huperzine A protects human whole blood cholinesterases from ex vivo exposure to soman.

    Science.gov (United States)

    Gordon, Richard K; Haigh, Julian R; Garcia, Gregory E; Feaster, Shawn R; Riel, Michael A; Lenz, David E; Aisen, Paul S; Doctor, Bhupendra P

    2005-12-15

    Cholinesterases (ChEs) are classified as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) according to their substrate specificity and sensitivity to selected inhibitors. The activities of AChE in red blood cells (RBC-AChE) and BChE in serum can be used as potential biomarkers of suppressed and/or heightened activity in the central and peripheral nervous systems. Exposure to organophosphate (OP) chemical warfare agents (CWAs), pesticides, anesthetics, and a variety of drugs such as cocaine, as well as some neurodegenerative and liver disease states, selectively reduces AChE or BChE activity. In humans, the toxicity of pesticides is well documented. Therefore, blood cholinesterase activity can be exploited as a tool for confirming exposure to these agents and possible treatments. Current assays for measurement of RBC-AChE and serum BChE require several labor-intensive processing steps, suffer from wide statistical variation, and there is no inter-laboratory conversion between methods. These methods, which determine only the serum BChE or RBC-AChE but not both, include the Ellman, radiometric, and deltapH (modified Michel) methods. In contrast, the Walter Reed Army Institute of Research Whole Blood (WRAIR WB, US Patent #6,746,850) cholinesterase assay rapidly determines the activity of both AChE and BChE in unprocessed (uncentrifuged) whole blood, uses a minimally invasive blood sampling technique (e.g., blood from a finger prick), and is semi-automated for high-throughput using the Biomek 2000 robotic system. To date, the WRAIR whole blood assay was used to measure AChE and BChE activities in human blood from volunteers in FDA clinical trials. In the first FDA study, 24 human subjects were given either 30 mg PB orally (n = 19) or placebo (n = 5). Blood samples were obtained pre-dosing and 2.5, 5, 8, and 24 h post-dosing. The samples were analyzed for AChE and BChE activity using the WRAIR WB robotic system, and for PB concentration by HPLC. We found that

  15. Human prostate supports more efficient replication of HIV-1 R5 than X4 strains ex vivo

    Directory of Open Access Journals (Sweden)

    Denis Hélène

    2008-12-01

    Full Text Available Abstract Background In order to determine whether human prostate can be productively infected by HIV-1 strains with different tropism, and thus represent a potential source of HIV in semen, an organotypic culture of prostate from men undergoing prostatic adenomectomy for benign prostate hypertrophy (BPH was developed. The presence of potential HIV target cells in prostate tissues was investigated using immunohistochemistry. The infection of prostate explants following exposures with HIV-1 R5, R5X4 and X4 strains was analyzed through the measure of RT activity in culture supernatants, the quantification of HIV DNA in the explants and the detection of HIV RNA+ cells in situ. Results The overall prostate characteristics were retained for 21/2 weeks in culture. Numerous potential HIV-1 target cells were detected in the prostate stroma. Whilst HIV-1 R5SF162 strain consistently productively infected prostatic T lymphocytes and macrophages, the prototypic X4IIIB strain and a primary R5X4 strain showed less efficient replication in this organ. Conclusion The BPH prostate is a site of HIV-1 R5 replication that could contribute virus to semen. A limited spreading of HIV-1 X4 and R5X4 in this organ could participate to the preferential sexual transmission of HIV-1 R5 strains.

  16. Human prostate supports more efficient replication of HIV-1 R5 than X4 strains ex vivo.

    Science.gov (United States)

    Le Tortorec, Anna; Satie, Anne-Pascale; Denis, Hélène; Rioux-Leclercq, Nathalie; Havard, Laurence; Ruffault, Annick; Jégou, Bernard; Dejucq-Rainsford, Nathalie

    2008-12-31

    In order to determine whether human prostate can be productively infected by HIV-1 strains with different tropism, and thus represent a potential source of HIV in semen, an organotypic culture of prostate from men undergoing prostatic adenomectomy for benign prostate hypertrophy (BPH) was developed. The presence of potential HIV target cells in prostate tissues was investigated using immunohistochemistry. The infection of prostate explants following exposures with HIV-1 R5, R5X4 and X4 strains was analyzed through the measure of RT activity in culture supernatants, the quantification of HIV DNA in the explants and the detection of HIV RNA+ cells in situ. The overall prostate characteristics were retained for 21/2 weeks in culture. Numerous potential HIV-1 target cells were detected in the prostate stroma. Whilst HIV-1 R5SF162 strain consistently productively infected prostatic T lymphocytes and macrophages, the prototypic X4IIIB strain and a primary R5X4 strain showed less efficient replication in this organ. The BPH prostate is a site of HIV-1 R5 replication that could contribute virus to semen. A limited spreading of HIV-1 X4 and R5X4 in this organ could participate to the preferential sexual transmission of HIV-1 R5 strains.

  17. Reconstruction and Visualization of Fiber and Laminar Structure inthe Normal Human Heart from Ex Vivo DTMRI Data

    Energy Technology Data Exchange (ETDEWEB)

    Rohmer, Damien; Sitek, Arkadiusz; Gullberg, Grant T.

    2006-12-18

    Background - The human heart is composed of a helicalnetwork of muscle fibers. These fibers are organized to form sheets thatare separated by cleavage surfaces. This complex structure of fibers andsheets is responsible for the orthotropic mechanical properties ofcardiac muscle. The understanding of the configuration of the 3D fiberand sheet structure is important for modeling the mechanical andelectrical properties of the heart and changes in this configuration maybe of significant importance to understand the remodeling aftermyocardial infarction.Methods - Anisotropic least square filteringfollowed by fiber and sheet tracking techniques were applied to DiffusionTensor Magnetic Resonance Imaging (DTMRI) data of the excised humanheart. The fiber configuration was visualized by using thin tubes toincrease 3-dimensional visual perception of the complex structure. Thesheet structures were reconstructed from the DTMRI data, obtainingsurfaces that span the wall from the endo- to the epicardium. Allvisualizations were performed using the high-quality ray-tracing softwarePOV-Ray. Results - The fibers are shown to lie in sheets that haveconcave or convex transmural structure which correspond to histologicalstudies published in the literature. The fiber angles varied depending onthe position between the epi- and endocardium. The sheets had a complexstructure that depended on the location within the myocardium. In theapex region the sheets had more curvature. Conclusions - A high-qualityvisualization algorithm applied to demonstrated high quality DTMRI datais able to elicit the comprehension of the complex 3 dimensionalstructure of the fibers and sheets in the heart.

  18. Shear bond strength of Biodentine, ProRoot MTA, glass ionomer cement and composite resin on human dentine ex vivo.

    Science.gov (United States)

    Kaup, Markus; Dammann, Christoph Heinrich; Schäfer, Edgar; Dammaschke, Till

    2015-04-19

    The aim of this study was to compare the shear bond strength of Biodentine, ProRoot MTA (MTA), glass ionomer cement (GIC) and composite resin (CR) on dentine. 120 extracted human third molars were embedded in cold-cured-resin and grinned down to the dentine. For each material 30 specimens were produced in standardised height and width and the materials were applied according to manufacturers´ instructions on the dentine samples. Only in the CR group a self-etching dentine-adhesive was used. In all other groups the dentine was not pre-treated. All specimens were stored at 37.5 °C and 100% humidity for 2d, 7d and 14d. With a testing device the shear bond strength was determined (separation of the specimens from the dentine surface). The statistical evaluation was performed using ANOVA and Tukey-test (p Biodentine increased significantly compared to the 2d investigation period (p Biodentine showed a significantly higher shear bond strength than MTA (p Biodentine and GIC was not significant (p > 0.05). After 7d Biodentine showed comparable shear bond values than GIC, whereas the shear bond values for MTA were significantly lower even after 14d. The adhesion of Biodentine to dentine surface seams to be superior compared to that of MTA.

  19. The role of topically applied L-ascorbic acid in ex-vivo examination of burn-injured human skin

    Science.gov (United States)

    Pielesz, Anna; Biniaś, Dorota; Bobiński, Rafał; Sarna, Ewa; Paluch, Jadwiga; Waksmańska, Wioletta

    2017-10-01

    Wound treatment and healing is complex and is comprised of an elaborate set of processes including cellular, spectroscopic and biochemical ones as well as the ;reaction; of local tissue to thermal injury. Vitamin C as L-ascorbic acid (LA) prevents injurious effects of oxidants because it reduces reactive oxygen species to stable molecules, it becomes oxidized to the short-lived ascorbyl radical. As a result, antioxidant treatment may contribute to minimizing injury in burn patients. The aim of this study is to assess changes in molecular structure of collagen extracted from human epidermis burn wound scab during incubation of the epidermis in L-ascorbic acid solution. The study will be performed using FTIR and FT Raman spectroscopies. During this research it was observed that the intensity of Raman peaks increased where healing was being modified by LA. The intensity of the amide III band at 1247 cm- 1 relative to the intensity at 1326 cm- 1 was used to test tissue repair degree at the incision site. FTIR spectra were recorded from frozen specimens of serum modified by LA; an analysis of shifts in the amide I band position was conducted. The appearance of a new band for frozen samples modified by LA was observed around 1149-1220 cm- 1. The above conclusions confirmed the creation of hydrogen bonds between Nsbnd H stretch and Cdbnd O. Samples being incubated in solutions of L-ascorbic acid demonstrated the absence of electrophoretic bands of albumin. Alterations in the surface of the skin incubated in L-ascorbic acid were investigated with the use of Scanning Electron Microscopy (SEM). A decrease in external symptoms of burn injury was noted in the damaged epidermis incubated in L-ascorbic acid.

  20. Acute stress reduces wound-induced activation of microbicidal potential of ex vivo isolated human monocyte-derived macrophages.

    Directory of Open Access Journals (Sweden)

    Ulrike Kuebler

    Full Text Available BACKGROUND: Psychological stress delays wound healing but the precise underlying mechanisms are unclear. Macrophages play an important role in wound healing, in particular by killing microbes. We hypothesized that (a acute psychological stress reduces wound-induced activation of microbicidal potential of human monocyte-derived macrophages (HMDM, and (b that these reductions are modulated by stress hormone release. METHODS: Fourty-one healthy men (mean age 35 ± 13 years were randomly assigned to either a stress or stress-control group. While the stress group underwent a standardized short-term psychological stress task after catheter-induced wound infliction, stress-controls did not. Catheter insertion was controlled. Assessing the microbicidal potential, we investigated PMA-activated superoxide anion production by HMDM immediately before and 1, 10 and 60 min after stress/rest. Moreover, plasma norepinephrine and epinephrine and salivary cortisol were repeatedly measured. In subsequent in vitro studies, whole blood was incubated with norepinephrine in the presence or absence of phentolamine (norepinephrine blocker before assessing HMDM microbicidal potential. RESULTS: Compared with stress-controls, HMDM of the stressed subjects displayed decreased superoxide anion-responses after stress (p's <.05. Higher plasma norepinephrine levels statistically mediated lower amounts of superoxide anion-responses (indirect effect 95% CI: 4.14-44.72. Norepinephrine-treated HMDM showed reduced superoxide anion-production (p<.001. This effect was blocked by prior incubation with phentolamine. CONCLUSIONS: Our results suggest that acute psychological stress reduces wound-induced activation of microbicidal potential of HMDM and that this reduction is mediated by norepinephrine. This might have implications for stress-induced impairment in wound healing.

  1. A hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo expansion of human corneal epithelial stem cells.

    Science.gov (United States)

    Chen, D; Qu, Y; Hua, X; Zhang, L; Liu, Z; Pflugfelder, S C; Li, D-Q

    2017-06-01

    PurposeTo develop a hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo cultivation of human corneal epithelial stem cells (CESCs).Patients and MethodsCESCs were cultivated from donor limbal explants on the HyStem-C Hydrogel bio-scaffold in 12-well plates for 3 weeks. Group A used the traditional supplemented hormonal epidermal medium (SHEM) and group B used the defined SHEM (without fetal bovine serum and toxin A, adding 20% serum replacement). The growth and morphology of the cultured cells were assessed by phase contrast microscope. The expressions of specific cell markers were assessed by immunofluorescence staining and quantitative real-time PCR (qRT-PCR).ResultsSuccessful cultures of CESCs were obtained in both groups, resulting in multilayered stratified epithelia. Comparing to group A, the cells in group B was grown slightly slower and formed less cellular layers at the end of culture. The corneal specific cytokeratin (K) 12 and differentiation markers, involucrin, and connexin 43, were mainly expressed in the superficial cellular layers in both groups. Interestingly, certain basal cells were immune-positive to proposed stem cell markers such as K19, ABCG2, and integrin β1 in both groups. There was no significant difference between the two groups with regard to the gene expression levels of all these selected corneal markers (all P>0.05).ConclusionsThe hyaluronan hydrogel scaffold-based xeno-free culture system may support the expansion of regenerative CESCs without the risk of xeno component contamination. The regenerated epithelium maintains similar characteristics of native corneal epithelium.

  2. Human mesenchymal stem cells from the umbilical cord matrix: successful isolation and ex vivo expansion using serum-/xeno-free culture media.

    Science.gov (United States)

    Simões, Irina N; Boura, Joana S; dos Santos, Francisco; Andrade, Pedro Z; Cardoso, Carla M P; Gimble, Jeffrey M; da Silva, Cláudia L; Cabral, Joaquim M S

    2013-04-01

    Mesenchymal stem cells (MSC) could potentially be applied in therapeutic settings due to their multilineage differentiation ability, immunomodulatory properties, as well as their trophic activity. The umbilical cord matrix (UCM) represents a promising source of MSC for biomedical applications. The number of cells isloated per umbilical cord (UC) unit is limited and ex vivo expansion is imperative in order to reach clinically meaningful cell numbers. The limitations of poorly defined reagents (e.g. fetal bovine serum, which is commonly used as a supplement for human MSC expansion) make the use of serum-/xeno-free conditions mandatory. We demonstrated the feasibility of isolating UCM-MSC by plastic adherence using serum-/xeno-free culture medium following enzymatic digestion of UCs, with a 100% success rate. 2.6 ± 0.21 × 10(5) cells were isolated per UC unit, of which 1.9 ± 0.21 × 10(5) were MSC-like cells expressing CD73, CD90, and CD105. When compared to adult sources (bone marrow-derived MSC and adipose-derived stem/stromal cells), UCM-MSC displayed a similar immunophenotype and similar multilineage differentiation ability, while demonstrating a higher expansion potential (average fold increase of 7.4 for serum-containing culture medium and 11.0 for xeno-free culture medium (P3-P6)). The isolation and expansion of UCM-MSC under defined serum-/xeno-free conditions contributes to safer and more effective MSC cellular products, boosting the usefulness of MSC in cellular therapy and tissue engineering. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Enhancement of HIV-1 infection and intestinal CD4+ T cell depletion ex vivo by gut microbes altered during chronic HIV-1 infection.

    Science.gov (United States)

    Dillon, Stephanie M; Lee, Eric J; Donovan, Andrew M; Guo, Kejun; Harper, Michael S; Frank, Daniel N; McCarter, Martin D; Santiago, Mario L; Wilson, Cara C

    2016-01-14

    Early HIV-1 infection is characterized by high levels of HIV-1 replication and substantial CD4 T cell depletion in the intestinal mucosa, intestinal epithelial barrier breakdown, and microbial translocation. HIV-1-induced disruption of intestinal homeostasis has also been associated with changes in the intestinal microbiome that are linked to mucosal and systemic immune activation. In this study, we investigated the impact of representative bacterial species that were altered in the colonic mucosa of viremic HIV-1 infected individuals (HIV-altered mucosal bacteria; HAMB) on intestinal CD4 T cell function, infection by HIV-1, and survival in vitro. Lamina propria (LP) mononuclear cells were infected with CCR5-tropic HIV-1BaL or mock infected, exposed to high (3 gram-negative) or low (2 gram-positive) abundance HAMB or control gram-negative Escherichia coli and levels of productive HIV-1 infection and CD4 T cell depletion assessed. HAMB-associated changes in LP CD4 T cell activation, proliferation and HIV-1 co-receptor expression were also evaluated. The majority of HAMB increased HIV-1 infection and depletion of LP CD4 T cells, but gram-negative HAMB enhanced CD4 T cell infection to a greater degree than gram-positive HAMB. Most gram-negative HAMB enhanced T cell infection to levels similar to that induced by gram-negative E. coli despite lower induction of T cell activation and proliferation by HAMB. Both gram-negative HAMB and E. coli significantly increased expression of HIV-1 co-receptor CCR5 on LP CD4 T cells. Lipopolysaccharide, a gram-negative bacteria cell wall component, up-regulated CCR5 expression on LP CD4 T cells whereas gram-positive cell wall lipoteichoic acid did not. Upregulation of CCR5 by gram-negative HAMB was largely abrogated in CD4 T cell-enriched cultures suggesting an indirect mode of stimulation. Gram-negative commensal bacteria that are altered in abundance in the colonic mucosa of HIV-1 infected individuals have the capacity to enhance

  4. Variants of β-casofensin, a bioactive milk peptide, differently modulate the intestinal barrier: In vivo and ex vivo studies in rats.

    Science.gov (United States)

    Bruno, Jérémie; Nicolas, Aurélie; Pesenti, Sandra; Schwarz, Jessica; Simon, Jean-Luc; Léonil, Joëlle; Plaisancié, Pascale

    2017-05-01

    β-Casofensin is a bioactive milk peptide that modulates the intestinal barrier, particularly through its action on goblet cells. β-Casofensin corresponds to fragment (f) 94-123 of the bovine β-casein (β-CN) A2 variant. Fifteen genetic variants of bovine β-CN (A1-3, B-G, H1-2, I-L) are known, of which the A2, A1, and B forms are the most common. These variants differ from each other by the substitution of one or more amino acids, some of which are localized in f94 to 123. The aim of our study was to compare the intestinal effects of β-casofensin A2 and its 3 main variants: A1, A3, and B. For this purpose, a solution (0.1 µM; 10 μL/g of body weight, postnatal d 10-20) containing β-casofensin A2, one of its variants (A1, A3, or B), or drinking water (control; CT) was administered to rat pups orally. After euthanasia (postnatal d 20), intestinal segments were collected for biochemical and histochemical analysis and also used to determine paracellular permeability to fluorescein isothiocyanate-labeled 4-kDa dextran in an Ussing chamber. We also studied the direct effects of β-casofensin A2 and its A1 variant on the paracellular permeability of jejunum segments of adult rats. β-Casofensin A2 and its B variant significantly increased the population of goblet cells compared with the CT, A1, and A3 groups. The mucin 2 mRNA level was significantly higher in the β-casofensin A2 group than in the CT, A3, and B groups. Our results also revealed that the protein expression of zonula occludens-1 and occludin was reduced in the jejunum of rats in the A1, A3, and B groups compared with the CT group. However, the A1 variant was the only peptide to decrease jejunal permeability compared with the CT group. This variant, tested directly in the apical compartment of an Ussing chamber at a concentration of 0.1 nM, also reduced jejunal permeability. In conclusion, the substitution of a single amino acid alters the effect of β-CN sequence f94 to 123 on goblet cells and on

  5. Regulatory activity of azabisphosphonate-capped dendrimers on human CD4+ T cell proliferation enhances ex-vivo expansion of NK cells from PBMCs for immunotherapy

    Directory of Open Access Journals (Sweden)

    Caminade Anne-Marie

    2009-09-01

    specificity of the interaction of dendrimers with CD4+ T cell, we hypothesize that regulatory activity may signal through a specific receptor that remains to be indentified. Therefore phosphonate-capped dendrimers constitute not only tools for the ex-vivo expansion of NK cells in immunotherapy of cancers but their mode of action could also lead to further medical applications where T cell activation and proliferation need to be dampened.

  6. Micro-Scale Distribution of CA4+ in Ex vivo Human Articular Cartilage Detected with Contrast-Enhanced Micro-Computed Tomography Imaging

    Directory of Open Access Journals (Sweden)

    Sakari S. Karhula

    2017-08-01

    Full Text Available Contrast-enhanced micro-computed tomography (CEμCT with cationic and anionic contrast agents reveals glycosaminoglycan (GAG content and distribution in articular cartilage (AC. The advantage of using cationic stains (e.g., CA4+ compared to anionic stains (e.g., Hexabrix®, is that it distributes proportionally with GAGs, while anionic stain distribution in AC is inversely proportional to the GAG content. To date, studies using cationic stains have been conducted with sufficient resolution to study its distributions on the macro-scale, but with insufficient resolution to study its distributions on the micro-scale. Therefore, it is not known whether the cationic contrast agents accumulate in extra/pericellular matrix and if they interact with chondrocytes. The insufficient resolution has also prevented to answer the question whether CA4+ accumulation in chondrons could lead to an erroneous quantification of GAG distribution with low-resolution μCT setups. In this study, we use high-resolution μCT to investigate whether CA4+ accumulates in chondrocytes, and further, to determine whether it affects the low-resolution ex vivo μCT studies of CA4+ stained human AC with varying degree of osteoarthritis. Human osteochondral samples were immersed in three different concentrations of CA4+ (3 mgI/ml, 6 mgI/ml, and 24 mgI/ml and imaged with high-resolution μCT at several timepoints. Different uptake diffusion profiles of CA4+ were observed between the segmented chondrons and the rest of the tissue. While the X-ray -detected CA4+ concentration in chondrons was greater than in the rest of the AC, its contribution to the uptake into the whole tissue was negligible and in line with macro-scale GAG content detected from histology. The efficient uptake of CA4+ into chondrons and surrounding territorial matrix can be explained by the micro-scale distribution of GAG content. CA4+ uptake in chondrons occurred regardless of the progression stage of osteoarthritis

  7. Micro-Scale Distribution of CA4+ in Ex Vivo Human Articular Cartilage Detected with Contrast-Enhanced Micro-Computed Tomography Imaging

    Science.gov (United States)

    Karhula, Sakari S.; Finnilä, Mikko A.; Freedman, Jonathan D.; Kauppinen, Sami; Valkealahti, Maarit; Lehenkari, Petri; Pritzker, Kenneth P. H.; Nieminen, Heikki J.; Snyder, Brian D.; Grinstaff, Mark W.; Saarakkala, Simo

    2017-08-01

    Contrast-enhanced micro-computed tomography (CEµCT) with cationic and anionic contrast agents reveals glycosaminoglycan (GAG) content and distribution in articular cartilage (AC). The advantage of using cationic stains (e.g. CA4+) compared to anionic stains (e.g. Hexabrix®), is that it distributes proportionally with GAGs, while anionic stain distribution in AC is inversely proportional to the GAG content. To date, studies using cationic stains have been conducted with sufficient resolution to study its distributions on the macro-scale, but with insufficient resolution to study its distributions on the micro-scale. Therefore, it is not known whether the cationic contrast agents accumulate in extra/pericellular matrix and if they interact with chondrocytes. The insufficient resolution has also prevented to answer the question whether CA4+ accumulation in chondrons could lead to an erroneous quantification of GAG distribution with low-resolution µCT setups. In this study, we use high-resolution µCT to investigate whether CA4+ accumulates in chondrocytes, and further, to determine whether it affects the low-resolution ex vivo µCT studies of CA4+ stained human AC with varying degree of osteoarthritis. Human osteochondral samples were immersed in three different concentrations of CA4+ (3 mgI/ml, 6mgI/ml, and 24 mgI/ml) and imaged with high-resolution µCT at several timepoints. Different uptake diffusion profiles of CA4+ were observed between the segmented chondrons and the rest of the tissue. While the X-ray -detected CA4+ concentration in chondrons was greater than in the rest of the AC, its contribution to the uptake into the whole tissue was negligible and in line with macro-scale GAG content detected from histology. The efficient uptake of CA4+ into chondrons and surrounding territorial matrix can be explained by the micro-scale distribution of GAG content. CA4+ uptake in chondrons occurred regardless of the progression stage of osteoarthritis in the samples

  8. Kidneys From α1,3-Galactosyltransferase Knockout/Human Heme Oxygenase-1/Human A20 Transgenic Pigs Are Protected From Rejection During Ex Vivo Perfusion With Human Blood.

    Science.gov (United States)

    Ahrens, Hellen E; Petersen, Björn; Ramackers, Wolf; Petkov, Stoyan; Herrmann, Doris; Hauschild-Quintern, Janet; Lucas-Hahn, Andrea; Hassel, Petra; Ziegler, Maren; Baars, Wiebke; Bergmann, Sabine; Schwinzer, Reinhard; Winkler, Michael; Niemann, Heiner

    2015-07-01

    Multiple modifications of the porcine genome are required to prevent rejection after pig-to-primate xenotransplantation. Here, we produced pigs with a knockout of the α1,3-galactosyltransferase gene (GGTA1-KO) combined with transgenic expression of the human anti-apoptotic/anti-inflammatory molecules heme oxygenase-1 and A20, and investigated their xenoprotective properties. The GGTA1-KO/human heme oxygenase-1 (hHO-1)/human A20 (hA20) transgenic pigs were produced in a stepwise approach using zinc finger nuclease vectors targeting the GGTA1 gene and a Sleeping Beauty vector coding for hA20. Two piglets were analyzed by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and sequencing. The biological function of the genetic modifications was tested in a (51)Chromium release assay and by ex vivo kidney perfusions with human blood. Disruption of the GGTA1 gene by deletion of few basepairs was demonstrated in GGTA1-KO/hHO-1/hA20 transgenic pigs. The hHO-1 and hA20 mRNA expression was confirmed by quantitative reverse-transcription polymerase chain reaction. Ex vivo perfusion of 2 transgenic kidneys was feasible for the maximum experimental time of 240 minutes without symptoms of rejection. Results indicate that GGTA1-KO/hHO-1/hA20 transgenic pigs are a promising model to alleviate rejection and ischemia-reperfusion damage in porcine xenografts and could serve as a background for further genetic modifications toward the production of a donor pig that is clinically relevant for xenotransplantation.

  9. Trimethoprim-sulfamethoxazole exposure alters ex vivo function of B lymphocytes isolated from human immunodeficiency virus-infected patients receiving Zidovudine.

    Science.gov (United States)

    Venugopalan, Veena; Thornton, Alice C; Steinke, Douglass T; Rapp, Robert P; Romanelli, Frank; Feola, David J

    2009-04-01

    To determine if exposure to trimethoprim-sulfamethoxazole (TMP-SMX) causes a defect in peripheral B-cell function among patients with the human immunodeficiency virus (HIV) who are receiving zidovudine antiretroviral therapy. Prospective, single-center, single-group, case-crossover design with a 4-week exposure period. University-affiliated infectious diseases outpatient clinic. Fourteen HIV-infected adult men receiving zidovudine, who had CD4(+) cell counts above 350 cells/mm(3) and undetectable viral loads. Patients were administered a 28-day course of TMP 160 mg-SMX 800 mg/day (one double-strength tablet/day). Peripheral blood mononuclear cells (PBMCs) were obtained and isolated before and after exposure to TMP-SMX. Cells were cultured ex vivo with three mitogens of differing immunologic properties: pokeweed mitogen ([PWM] T-cell-dependent B-cell mitogen), Staphylococcus aureus Cowan ([SAC] T-cell-independent B-cell mitogen), and phytohemagglutinin A ([PHA] T-cell mitogen). Functionality of the B and T lymphocytes was then assessed. Proliferative capacity, cytokine secretion, and antibody production were measured and compared before and after TMP-SMX exposure. Reduced proliferative capacities of both PBMC and B cells stimulated with mitogens were observed at the 3-day culture time point in response to PWM, PHA, and SAC (p=0.029, 0.028, and 0.026, respectively). Proliferative capacity at day 7 of culture was not significantly different for any condition examined. Cytokine production was not altered by combination drug exposure after 10 days of culture when cells were stimulated with either PWM or PHA. Although antibody responses to PWM and PHA were similar, total immunoglobulin G concentration was lower in cells stimulated with SAC in samples obtained after TMP-SMX regimen completion compared with those obtained before exposure (p=0.005). Although these data were affected by limitations in power and study design, they suggest that peripheral B-lymphocyte function

  10. Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

    Science.gov (United States)

    Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A S; Zhou, Pengbo; Mesuraca, Maria; Bond, Heather Mandy; Morrone, Giovanni

    2017-07-04

    Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

  11. Effect of lung flooding and high-intensity focused ultrasound on lung tumours: an experimental study in an ex vivo human cancer model and simulated in vivo tumours in pigs.

    Science.gov (United States)

    Wolfram, Frank; Boltze, Carsten; Schubert, Harald; Bischoff, Sabine; Lesser, Thomas Günther

    2014-01-07

    High-intensity focused ultrasound is a valuable tool for minimally invasive tumour ablation. However, due to the air content in ventilated lungs, lung tumours have never been treated with high-intensity focused ultrasound. Lung flooding enables efficient lung sonography and tumour imaging in ex vivo human and in vivo porcine lung cancer models. The current study evaluates the effectiveness of lung flooding and sonography-guided high-intensity focused ultrasound for lung tumour ablation in ex vivo human and in vivo animal models. Lung flooding was performed in four human lung lobes which were resected from non-small cell lung cancers. B-mode imaging and temperature measurements were simultaneously obtained during high-intensity focused ultrasonography of centrally located lung cancers. The tumour was removed immediately following insonation and processed for nicotinamide adenine dinucleotide phosphate-diaphorase and H&E staining. In addition, the left lungs of three pigs were flooded. Purified BSA in glutaraldehyde was injected centrally into the left lower lung lobe to simulate a lung tumour. The ultrasound was focused transthoracically through the flooded lung into the simulated tumour with the guidance of sonography. The temperature of the tumour was simultaneously measured. The vital signs of the animal were monitored during the procedure. A well-demarcated lesion of coagulation necrosis was produced in four of four human lung tumours. There did not appear to be any damage to the surrounding lung parenchyma. After high-intensity focused ultrasound insonation, the mean temperature increase was 7.5-fold higher in the ex vivo human tumour than in the flooded lung tissue (52.1 K ± 8.77 K versus 7.1 K ± 2.5 K). The transthoracic high-intensity focused ultrasound of simulated tumours in the in vivo model resulted in a mean peak temperature increase up to 53.7°C (±4.5). All of the animals survived the procedure without haemodynamic complications. High

  12. Ex vivo tissue imaging of human glioblastoma using a small bore 7T MRI and correlation with digital pathology and proteomics profiling

    Science.gov (United States)

    Matsuda, Kant M.; Lopes-Calcas, Ana; Magyar, Thalia; O'Brien-Moran, Zoe; Buist, Richard; Martin, Melanie

    2017-03-01

    Recent advancement in MRI established multi-parametric imaging for in vivo characterization of pathologic changes in brain cancer, which is expected to play a role in imaging biomarker development. Diffusion Tensor Imaging (DTI) is a prime example, which has been deployed for assessment of therapeutic response via analysis of apparent diffusion coefficient (ADC) / mean diffusivity (MD) values. They have been speculated to reflect apoptosis/necrosis. As newer medical imaging emerges, it is essential to verify that apparent abnormal features in imaging correlate with histopathology. Furthermore, the feasibility of imaging correlation with molecular profile should be explored in order to enhance the potential of biomedical imaging as a reliable biomarker. We focus on glioblastoma, which is an aggressive brain cancer. Despite the increased number of studies involving DTI in glioblastoma; however, little has been explored to bridge the gap between the molecular biomarkers and DTI data. Due to spatial heterogeneity in, MRI signals, pathologic change and protein expression, precise correlation is required between DTI, pathology and proteomics data in a histoanatomically identical manner. The challenge is obtaining an identical plane from in vivo imaging data that exactly matches with histopathology section. Thus, we propose to incorporate ex vivo tissue imaging to bridge between in vivo imaging data and histopathology. With ex vivo scan of removed tissue, it is feasible to use high-field 7T MRI scanner, which can achieve microscopic resolution. Once histology section showing the identical plane, it is feasible to correlate protein expression by a unique technology, "multiplex tissue immunoblotting".

  13. Informing participants in clinical trials with ex vivo human tissue-engineered products: what to tell and how to tell it?

    Science.gov (United States)

    Trommelmans, Leen; Selling, Joseph; Dierickx, Kris

    2008-06-01

    Ex vivo tissue-engineered products are increasingly entered into clinical trials. To allow prospective participants to make a fully informed, autonomous decision on their participation, we have to adapt the informed consent process by taking the specific aspects of tissue engineering into consideration. New elements in ex vivo tissue engineering are the source and manipulation of the cells in the product, the implantation of the product and the additional risks and benefits due to the construction of the product and its activity in the body. They are the result of the delicate nature of some cell types and of the complexity of the tissue engineering process. The process of informing the participant should be designed in such a way that the participant's capacity to understand the intervention and its implications is enhanced. Crucial issues, such as the aim and procedure of the trial, the risks and benefits involved and the role of the investigator, have to be clarified. We suggest that participants' understanding of the trial can be enhanced through the use of audiovisual material, by developing a simple questionnaire to direct the information process further, and by the assistance of informed third parties to help participants in their decision-making processes. Copyright (c) 2008 John Wiley & Sons, Ltd.

  14. Enhanced Ex Vivo Expansion of Human Hematopoietic Progenitors on Native and Spin Coated Acellular Matrices Prepared from Bone Marrow Stromal Cells.

    Science.gov (United States)

    Wasnik, Samiksha; Kantipudi, Suma; Kirkland, Mark A; Pande, Gopal

    2016-01-01

    The extracellular microenvironment in bone marrow (BM) is known to regulate the growth and differentiation of hematopoietic stem and progenitor cells (HSPC). We have developed cell-free matrices from a BM stromal cell line (HS-5), which can be used as substrates either in native form or as tissue engineered coatings, for the enhanced ex vivo expansion of umbilical cord blood (UCB) derived HSPC. The physicochemical properties (surface roughness, thickness, and uniformity) of native and spin coated acellular matrices (ACM) were studied using scanning and atomic force microscopy (SEM and AFM). Lineage-specific expansion of HSPC, grown on these substrates, was evaluated by immunophenotypic (flow cytometry) and functional (colony forming) assays. Our results show that the most efficient expansion of lineage-specific HSPC occurred on spin coated ACM. Our method provides an improved protocol for ex vivo HSPC expansion and it offers a system to study the in vivo roles of specific molecules in the hematopoietic niche that influence HSPC expansion.

  15. Electrospun Zein/PCL Fibrous Matrices Release Tetracycline in a Controlled Manner, Killing Staphylococcus aureus Both in Biofilms and Ex Vivo on Pig Skin, and are Compatible with Human Skin Cells.

    Science.gov (United States)

    Alhusein, Nour; Blagbrough, Ian S; Beeton, Michael L; Bolhuis, Albert; De Bank, Paul A

    2016-01-01

    To investigate the destruction of clinically-relevant bacteria within biofilms via the sustained release of the antibiotic tetracycline from zein-based electrospun polymeric fibrous matrices and to demonstrate the compatibility of such wound dressing matrices with human skin cells. Zein/PCL triple layered fibrous dressings with entrapped tetracycline were electrospun. The successful entrapment of tetracycline in these dressings was validated. The successful release of bioactive tetracycline, the destruction of preformed biofilms, and the viability of fibroblast (FEK4) cells were investigated. The sustained release of tetracycline from these matrices led to the efficient destruction of preformed biofilms from Staphylococcus aureus MRSA252 in vitro, and of MRSA252 and ATCC 25923 bacteria in an ex vivo pig skin model using 1 × 1 cm square matrices containing tetracycline (30 μg). Human FEK4 cells grew normally in the presence of these matrices. The ability of the zein-based matrices to destroy bacteria within increasingly complex in vitro biofilm models was clearly established. An ex vivo pig skin assay showed that these matrices, with entrapped tetracycline, efficiently kill bacteria and this, combined with their compatibility with a human skin cell line suggest these matrices are well suited for applications in wound healing and infection control.

  16. Chemiluminescence response induced by mesenteric ischaemia/reperfusion: effect of antioxidative compounds ex vivo

    Science.gov (United States)

    Nosál'ová, Viera; Sotníková, Ružena; Drábiková, Katarína; Fialová, Silvia; Košťálová, Daniela; Banášová, Silvia; Navarová, Jana

    2010-01-01

    Ischaemia and reperfusion (I/R) play an important role in human pathophysiology as they occur in many clinical conditions and are associated with high morbidity and mortality. Interruption of blood supply rapidly damages metabolically active tissues. Restoration of blood flow after a period of ischaemia may further worsen cell injury due to an increased formation of free radicals. The aim of our work was to assess macroscopically the extent of intestinal pathological changes caused by mesenteric I/R, and to study free radical production by luminol enhanced chemiluminescence (CL) of ileal samples. In further experiments, the antioxidative activity of the drugs tested was evaluated spectrophotometrically by the use of the DPPH radical. We studied the potential protective ex vivo effect of the plant origin compound arbutin as well as of the pyridoindole stobadine and its derivative SMe1EC2. I/R induced pronounced haemorrhagic intestinal injury accompanied by increase of myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAGA) activity. Compared to sham operated (control) rats, there was only a slight increase of CL response after I/R, probably in association with neutrophil increase, indicated by enhanced MPO activity. All compounds significantly reduced the peak values of CL responses of the ileal samples ex vivo, thus reducing the I/R induced increase of free radical production. The antioxidants studied showed a similar inhibitory effect on the CL response influenced by mesenteric I/R. If proved in vivo, these compounds would represent potentially useful therapeutic antioxidants. PMID:21217883

  17. The Anti-Inflammatory Effects of Lipoxygenase and Cyclo-Oxygenase Inhibitors in Inflammation-Induced Human Fetal Glia Cells and the Aβ Degradation Capacity of Human Fetal Astrocytes in an Ex vivo Assay

    Directory of Open Access Journals (Sweden)

    Rea Pihlaja

    2017-05-01

    Full Text Available Chronic inflammation is a common phenomenon present in the background of multiple neurodegenerative diseases, including Alzheimer's disease (AD. The arachidonic acid pathway overproduces proinflammatory eicosanoids during these states and glial cells in the brain gradually lose their vital functions of protecting and supporting neurons. In this study, the role of different key enzymes of the eicosanoid pathway mediating inflammatory responses was examined in vitro and ex vivo using human fetal glial cells. Astrocytes and microglia were exposed to proinflammatory agents i.e., cytokines interleukin 1-β (IL-1β and tumor necrosis factor (TNF-α. ELISA assays were used to examine the effects of inhibitors of key enzymes in the eicosanoid pathway. Inhibitors for 5-lipoxygenase (5-LOX and cyclo-oxygenase 2 (COX-2 in both cell types and 5-, 12-, and 15-LOX-inhibitor in astrocytes reduced significantly IL-6 secretion, compared to exposed glial cells without inhibitors. The cytokine antibody array showed that especially treatments with 5, -12, and -15 LOX inhibitor in astrocytes, 5-LOX inhibitor in microglia and COX-2 inhibitor in both glial cell types significantly reduced the expression of multiple proinflammatory cytokines. Furthermore, human fetal astrocytes and microglia were cultured on top of AD-affected and control human brain sections for 30 h. According to the immunochemical evaluation of the level of total Aβ, astrocytes were very efficient at degrading Aβ from AD-affected brain sections ex vivo; simultaneously added enzyme inhibitors did not increase their Aβ degradation capabilities. Microglia were not able to reduce the level of total Aβ during the 30 h incubation time.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    . The subjects ingested rye bran enriched breads equivalent to similar to 10.2 mg ferulic acid per day. Results The urinary excretion of ferulic acid averaged similar to 4.8 mg per day during intervention with rye bran breads and similar to 1.9 mg per day on the control breads (P = 0.002). Rye bran intervention......Background Rye bread contributes an important part of the whole grain intake in the Scandinavian diet. Ferulic acid is the major phenolic compound in rye bran and is an antioxidant in vitro and may, therefore, contribute to cardioprotective effects of whole grain consumption. Aim of study Firstly...... women after a dietary intake of rye bran or an inert wheat bran (control) in a crossover study (2 x 6 weeks with 4 weeks washout). The potential antioxidative effect of the rye bran intervention was investigated by measuring low-density lipoprotein (LDL) susceptibility to copper oxidation ex vivo...

  19. Different Pathogenesis of CCR5-Using Primary HIV-1 Isolates from Non-Switch and Switch Virus Patients in Human Lymphoid Tissue Ex Vivo

    Science.gov (United States)

    Iarlsson, Ingrid; Grivel, Jean-Charles; Chen. Silvia; Karlsson, Anders; Albert, Jan; Fenyol, Eva Maria; Margolis, Leonid B.

    2005-01-01

    CCR5-utilizing HIV-1 variants (R5) typically transmit infection and dominate its early stages, whereas emergence of CXCR4-using (X4 or R5X4) HIV-1 is often associated with disease progression. However, such a switch in co-receptor usage can only be detected in approximately onehalf of HIV-infected patients (switch virus patients), and progression to immunodeficiency may also occur in patients without detectable switch in co-receptor usage (non-switch virus patients). Here, we used a system of ex vivo-infected tonsillar tissue to compare the pathogenesis of sequential primary R5 HIV-1 isolates from the switch and non-switch patients. Inoculation of ex vivo tissue with these R5 isolates resulted in viral replication and CCR5(+)CD4(+) T cell depletion. The levels of such depletion by HIV-1 isolated from non-switch virus patients were significantly higher than those by R5 HIV-1 isolates from switch virus patients. T cell depletion seemed to be controlled by viral factors and did not significantly vary between tissues from different donors. In contrast, viral replication did not correlate with the switch status of the patients; in tissues fiom different donors it varied 30-fold and seemed to be controlled by a combination of viral and tissue factors. Nevertheless, replication-level hierarchy among sequential isolates remained constant in tissues from various donors. Viral load in vivo was higher in switch virus patients compared to non-switch virus patients. The high cytopathogenicity of CCR5(+)CD4(+) T cells by R5 HIV-1 isolates from non-switch virus patients may explain the steady decline of CD4(+) T cells in the absence of CXCR4 using virus; elimination of target cells by these isolates may limit their own replication in vivo.

  20. Effects of a skin-massaging device on the ex-vivo expression of human dermis proteins and in-vivo facial wrinkles.

    Science.gov (United States)

    Caberlotto, Elisa; Ruiz, Laetitia; Miller, Zane; Poletti, Mickael; Tadlock, Lauri

    2017-01-01

    Mechanical and geometrical cues influence cell behaviour. At the tissue level, almost all organs exhibit immediate mechanical responsiveness, in particular by increasing their stiffness in direct proportion to an applied mechanical stress. It was recently shown in cultured-cell models, in particular with fibroblasts, that the frequency of the applied stress is a fundamental stimulating parameter. However, the influence of the stimulus frequency at the tissue level has remained elusive. Using a device to deliver an oscillating torque that generates cyclic strain at different frequencies, we studied the effect(s) of mild skin massage in an ex vivo model and in vivo. Skin explants were maintained ex vivo for 10 days and massaged twice daily for one minute at various frequencies within the range of 65-85 Hz. Biopsies were analysed at D0, D5 and D10 and processed for immuno-histological staining specific to various dermal proteins. As compared to untreated skin explants, the massaging procedure clearly led to higher rates of expression, in particular for decorin, fibrillin, tropoelastin, and procollagen-1. The mechanical stimulus thus evoked an anti-aging response. Strikingly, the expression was found to depend on the stimulus frequency with maximum expression at 75Hz. We then tested whether this mechanical stimulus had an anti-aging effect in vivo. Twenty Caucasian women (aged 65-75y) applied a commercial anti-aging cream to the face and neck, followed by daily treatments using the anti-aging massage device for 8 weeks. A control group of twenty-two women, with similar ages to the first group, applied the cream alone. At W0, W4 and W8, a blinded evaluator assessed the global facial wrinkles, skin texture, lip area, cheek wrinkles, neck sagging and neck texture using a clinical grading scale. We found that combining the massaging device with a skin anti-aging formulation amplified the beneficial effects of the cream.

  1. Effects of a skin-massaging device on the ex-vivo expression of human dermis proteins and in-vivo facial wrinkles.

    Directory of Open Access Journals (Sweden)

    Elisa Caberlotto

    Full Text Available Mechanical and geometrical cues influence cell behaviour. At the tissue level, almost all organs exhibit immediate mechanical responsiveness, in particular by increasing their stiffness in direct proportion to an applied mechanical stress. It was recently shown in cultured-cell models, in particular with fibroblasts, that the frequency of the applied stress is a fundamental stimulating parameter. However, the influence of the stimulus frequency at the tissue level has remained elusive. Using a device to deliver an oscillating torque that generates cyclic strain at different frequencies, we studied the effect(s of mild skin massage in an ex vivo model and in vivo. Skin explants were maintained ex vivo for 10 days and massaged twice daily for one minute at various frequencies within the range of 65-85 Hz. Biopsies were analysed at D0, D5 and D10 and processed for immuno-histological staining specific to various dermal proteins. As compared to untreated skin explants, the massaging procedure clearly led to higher rates of expression, in particular for decorin, fibrillin, tropoelastin, and procollagen-1. The mechanical stimulus thus evoked an anti-aging response. Strikingly, the expression was found to depend on the stimulus frequency with maximum expression at 75Hz. We then tested whether this mechanical stimulus had an anti-aging effect in vivo. Twenty Caucasian women (aged 65-75y applied a commercial anti-aging cream to the face and neck, followed by daily treatments using the anti-aging massage device for 8 weeks. A control group of twenty-two women, with similar ages to the first group, applied the cream alone. At W0, W4 and W8, a blinded evaluator assessed the global facial wrinkles, skin texture, lip area, cheek wrinkles, neck sagging and neck texture using a clinical grading scale. We found that combining the massaging device with a skin anti-aging formulation amplified the beneficial effects of the cream.

  2. Aflatoxin B1 binding by a mixture of Lactobacillus and Propionibacterium: in vitro versus ex vivo.

    Science.gov (United States)

    Gratz, S; Mykkänen, H; El-Nezami, H

    2005-11-01

    Aflatoxin B1 (AFB) is a well-known carcinogen and reducing its bioavailability is of great interest for human and animal health. Several probiotic bacteria are able to bind AFB1 in vitro, including Lactobacillus rhamnosus LC-705 and Propionibacterium freudenreichii subsp. shermanii JS. A mixture of these two probiotics is used by the food and feed industry as biopreservative (Bioprofit), making it a promising candidate for future applications. Consequently, this study aims to investigate the in vitro and ex vivo ability of this probiotic mixture to bind AFB1. For in vitro experiments, probiotic mixture was suspended in an AFB1 solution (5 microM), incubated for 1 to 30 min, centrifuged, and AFB1 residues were quantitated in supernatant and pellet. For ex vivo experiments, duodenal loops of chicks were ligated and injected with either AFB1 solution alone or probiotic mixture suspension and AFB1 solution. Lumen content was centrifuged and AFB1 was quantitated in supernatant and pellet. Additionally, AFB1 was extracted from duodenal tissue to calculate tissue uptake. In vitro, 57 to 66% of AFB1 was removed from the solution by the probiotic mixture, but only 38 to 47% could be extracted from the bacterial surface. In ex vivo experiments, only up to 25% of AFB1 was bound by bacteria, and tissue uptake of AFB1 was significantly reduced when probiotic bacteria were present in the duodenal loop. Furthermore, the effect of intestinal mucus on the bacterial binding ability was investigated in vitro and was found to significantly reduce AFB1 binding by the probiotic mixture. However, probiotic mixture could only retard but not prevent AFB1 absorption in duodenal loops. Further work needs to assess the potential of probiotics in different experimental setups.

  3. Alpha tocopherol loaded chitosan oleate nanoemulsions for wound healing. Evaluation on cell lines and ex vivo human biopsies, and stabilization in spray dried Trojan microparticles.

    Science.gov (United States)

    Bonferoni, M C; Riva, F; Invernizzi, A; Dellera, E; Sandri, G; Rossi, S; Marrubini, G; Bruni, G; Vigani, B; Caramella, C; Ferrari, F

    2017-11-15

    An amphiphilic chitosan salt, chitosan oleate (CS-OA), was previously proposed for the physical stabilization of lemongrass antimicrobial nanoemulsions (NE) through a mild spontaneous emulsification process. As both chitosan and oleic acid are described in the literature for their positive effects in wound healing, in the present study CS-OA has been proposed to encapsulate alpha tocopherol (αTph) in NEs aimed to skin wounds. A NE formulation was developed showing about 220 nm dimensions, 36% drug loading, and αTph concentration up to 1 mg/ml. Both CS-OA and αTph NE stimulated cell proliferation on keratinocytes and fibroblast cell cultures, and in ex vivo skin biopsies, suggesting the suitability of CS-OA and of the antioxidant agent for topical application in wound healing. αTph stability, was further improved with respect of encapsulation, by spray drying the NE into a powder (up to about 90% αTph residual after 3 months). The spray drying process was optimized, to improve powder yield and αTph recovery, by a design of experiments approach. The powder obtained was easily re-suspended to deliver the NE and resulted able to completely release αTph. Copyright © 2017. Published by Elsevier B.V.

  4. Ebola Virus Persistence in Semen Ex Vivo.

    Science.gov (United States)

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

    2016-02-01

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

  5. Coagulation Management in Jersey Calves: An ex vivo Study.

    Science.gov (United States)

    Gröning, Sabine; Maas, Judith; van Geul, Svenja; Rossaint, Rolf; Steinseifer, Ulrich; Grottke, Oliver

    2017-01-01

    Jersey calves are frequently used as an experimental animal model for in vivo testing of cardiac assist devices or orthopedic implants. In this ex vivo study, we analyzed the coagulation system of the Jersey calves and the potential of human-based coagulation management to circumvent perioperative bleeding complications during surgery. Experimental Procedure: Blood from 7 Jersey calves was subjected to standard laboratory tests and thromboelastometry analysis. An ex vivo model of dilutional coagulopathy was used to study the effects of fibrinogen or prothrombin complex concentrate supplementation. Fibrinolysis was induced with tissue plasminogen activator to identify potential therapeutic strategies involving tranexamic acid or aprotinin. Furthermore, anticoagulation strategies were evaluated by incubating the blood samples with dabigatran or rivaroxaban. Baseline values for thromboelastometry and standard laboratory parameters, including prothrombin time, activated partial thromboplastin time, fibrinogen, antithrombin III, and D-dimers, were established. Fifty percent diluted blood showed a statistically significant impairment of hemostasis. The parameters significantly improved after the administration of fibrinogen or prothrombin complex concentrate. Tranexamic acid and aprotinin ameliorated tissue plasminogen activator-induced fibrinolysis. Both dabigatran and rivaroxaban significantly prolonged the coagulation parameters. In this ex vivo study, coagulation factors, factor concentrate, antifibrinolytic reagents, and anticoagulants regularly used in the clinic positively impacted coagulation parameters in Jersey calf blood. © 2017 S. Karger AG, Basel.

  6. In vitro and ex vivo corneal penetration and absorption models.

    Science.gov (United States)

    Agarwal, Priyanka; Rupenthal, Ilva D

    2016-12-01

    Topical drug administration is the preferred route of drug delivery to the eye despite the poor bioavailability. To develop more efficient drug carriers, reliable in vitro or ex vivo models are required in the early stages of formulation development, with such methods being faster, cheaper, and more ethical alternatives to in vivo studies. In vitro cell culture models are increasingly being used for transcorneal penetration studies, with primary cell cultures and immortalized cell lines now giving way to the development of organotypic corneal constructs for studying ocular drug bioavailability. Artificially cultured human corneal equivalents are still in the early stages of development, but present tremendous potential for corneal penetration studies. Ex vivo models using excised animal tissue are also being used to study corneal penetration with promising results, although significant inter-species variations need to be considered. This review discusses the in vitro and ex vivo models currently being used to study corneal penetration and evaluates their advantages and limitations with a focus on diffusion cell assemblies. In addition to the tissue used, the diffusion cell set-up can significantly influence the penetration profile and should be cautiously adjusted to simulate clinical conditions.

  7. Ex-vivo dynamic 3-D culture of human tissues in the RCCS™ bioreactor allows the study of Multiple Myeloma biology and response to therapy.

    Science.gov (United States)

    Ferrarini, Marina; Steimberg, Nathalie; Ponzoni, Maurilio; Belloni, Daniela; Berenzi, Angiola; Girlanda, Stefania; Caligaris-Cappio, Federico; Mazzoleni, Giovanna; Ferrero, Elisabetta

    2013-01-01

    Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients'sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo. Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy.

  8. 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

  9. Maternal plasma or human serum albumin in wash buffer enhances enrichment and ex vivo expansion of human umbilical cord blood CD34+ cells.

    Science.gov (United States)

    Kwok, Yvonne K; Tang, Mary H Y; Law, Helen K W; Ngai, Cora S; Lau, Yu Lung; Lau, Elizabeth T

    2007-06-01

    Umbilical cord blood is a valuable source of haemopoietic stem/progenitor cells (HSC) for transplantation. This study explored the effect of maternal plasma/human serum albumin (HSA) in the purification and culture conditions of CD34+ cells derived from human umbilical cord blood. During CD34+ cell enrichment, including maternal plasma or HSA instead of fetal bovine serum (FBS) in the wash buffer, significantly increased the purity and the fold expansion of CD34+ cells. The increase in fold expansion of CD34+ cells was independent of CD34+ cell purity before expansion. With FBS, the mean fold expansion of CD34+ cells and total nucleated cells on day 7 was 9.7 +/- 5.5 and 39.7 +/- 13.7 respectively. The use of maternal plasma increased the mean fold expansion of CD34+ cells and total nucleated cells on day 7 to 28.2 +/- 6.7 and 71.5 +/- 15.4 respectively. When HSA was added to wash buffer, the mean fold expansion of CD34+ cells and total nucleated cells were 30.4 +/- 10.5 and 83.5 +/- 24.8 respectively. No statistical significance was found between using HSA and maternal plasma on total cell and CD34+ cell expansion. We propose that HSA in maternal plasma was responsible for the positive effect on CD34+ cell enrichment and expansion.

  10. Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Bernardi, Martina; Albiero, Elena; Alghisi, Alberta; Chieregato, Katia; Lievore, Chiara; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2013-08-01

    A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  11. Effects of lipid-lowering drugs on irisin in human subjects in vivo and in human skeletal muscle cells ex vivo.

    Directory of Open Access Journals (Sweden)

    Ioanna Gouni-Berthold

    Full Text Available The myokine irisin has been proposed to regulate energy homeostasis. Little is known about its association with metabolic parameters and especially with parameters influencing pathways of lipid metabolism. In the context of a clinical trial, an exploratory post hoc analysis has been performed in healthy subjects to determine whether simvastatin and/or ezetimibe influence serum irisin levels. The direct effects of simvastatin on irisin were also examined in primary human skeletal muscle cells (HSKMCs.A randomized, parallel 3-group study was performed in 72 men with mild hypercholesterolemia and without apparent cardiovascular disease. Each group of 24 subjects received a 14-day treatment with either simvastatin 40 mg, ezetimibe 10 mg, or their combination.Baseline irisin concentrations were not significantly correlated with age, BMI, estimated GFR, thyroid parameters, glucose, insulin, lipoproteins, non-cholesterol sterols, adipokines, inflammation markers and various molecular markers of cholesterol metabolism. Circulating irisin increased significantly in simvastatin-treated but not in ezetimibe-treated subjects. The changes were independent of changes in LDL-cholesterol and were not correlated with changes in creatine kinase levels. In HSKMCs, simvastatin significantly increased irisin secretion as well as mRNA expression of its parent peptide hormone FNDC5. Simvastatin significantly induced cellular reactive oxygen species levels along with expression of pro- and anti-oxidative genes such as Nox2, and MnSOD and catalase, respectively. Markers of cellular stress such as atrogin-1 mRNA and Bax protein expression were also induced by simvastatin. Decreased cell viability and increased irisin secretion by simvastatin was reversed by antioxidant mito-TEMPO, implying in part that irisin is secreted as a result of increased mitochondrial oxidative stress and subsequent myocyte damage.Simvastatin increases irisin concentrations in vivo and in vitro

  12. Ex vivo expanded human regulatory T cells delay islet allograft rejection via inhibiting islet-derived monocyte chemoattractant protein-1 production in CD34+ stem cells-reconstituted NOD-scid IL2rγnull mice.

    Directory of Open Access Journals (Sweden)

    Fang Xiao

    Full Text Available Type 1 diabetes mellitus (T1DM is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo.

  13. A Wearable Goggle Navigation System for Dual-Mode Optical and Ultrasound Localization of Suspicious Lesions: Validation Studies Using Tissue-Simulating Phantoms and an Ex Vivo Human Breast Tissue Model.

    Directory of Open Access Journals (Sweden)

    Zeshu Zhang

    Full Text Available Surgical resection remains the primary curative treatment for many early-stage cancers, including breast cancer. The development of intraoperative guidance systems for identifying all sites of disease and improving the likelihood of complete surgical resection is an area of active ongoing research, as this can lead to a decrease in the need of subsequent additional surgical procedures. We develop a wearable goggle navigation system for dual-mode optical and ultrasound imaging of suspicious lesions. The system consists of a light source module, a monochromatic CCD camera, an ultrasound system, a Google Glass, and a host computer. It is tested in tissue-simulating phantoms and an ex vivo human breast tissue model. Our experiments demonstrate that the surgical navigation system provides useful guidance for localization and core needle biopsy of simulated tumor within the tissue-simulating phantom, as well as a core needle biopsy and subsequent excision of Indocyanine Green (ICG-fluorescing sentinel lymph nodes. Our experiments support the contention that this wearable goggle navigation system can be potentially very useful and fully integrated by the surgeon for optimizing many aspects of oncologic surgery. Further engineering optimization and additional in vivo clinical validation work is necessary before such a surgical navigation system can be fully realized in the everyday clinical setting.

  14. Bioengineering of functional human induced pluripotent stem cell-derived intestinal grafts.

    Science.gov (United States)

    Kitano, Kentaro; Schwartz, Dana M; Zhou, Haiyang; Gilpin, Sarah E; Wojtkiewicz, Gregory R; Ren, Xi; Sommer, Cesar A; Capilla, Amalia V; Mathisen, Douglas J; Goldstein, Allan M; Mostoslavsky, Gustavo; Ott, Harald C

    2017-10-10

    Patients with short bowel syndrome lack sufficient functional intestine to sustain themselves with enteral intake alone. Transplantable vascularized bioengineered intestine could restore nutrient absorption. Here we report the engineering of humanized intestinal grafts by repopulating decellularized rat intestinal matrix with human induced pluripotent stem cell-derived intestinal epithelium and human endothelium. After 28 days of in vitro culture, hiPSC-derived progenitor cells differentiate into a monolayer of polarized intestinal epithelium. Human endothelial cells seeded via native vasculature restore perfusability. Ex vivo isolated perfusion testing confirms transfer of glucose and medium-chain fatty acids from lumen to venous effluent. Four weeks after transplantation to RNU rats, grafts show survival and maturation of regenerated epithelium. Systemic venous sampling and positron emission tomography confirm uptake of glucose and fatty acids in vivo. Bioengineering intestine on vascularized native scaffolds could bridge the gap between cell/tissue-scale regeneration and whole organ-scale technology needed to treat intestinal failure patients.There is a need for humanised grafts to treat patients with intestinal failure. Here, the authors generate intestinal grafts by recellularizing native intestinal matrix with human induced pluripotent stem cell-derived epithelium and human endothelium, and show nutrient absorption after transplantation in rats.

  15. Ex vivo lung perfusion review of a revolutionary technology.

    Science.gov (United States)

    Makdisi, George; Makdisi, Tony; Jarmi, Tambi; Caldeira, Christiano C

    2017-09-01

    Donor lung shortage has been the main reason to the increasing number of patients waiting for lung transplant. Ex vivo lung perfusion (EVLP) is widely expanding technology to assess and prepare the lungs who are considered marginal for transplantation. the outcomes are encouraging and comparable to the lungs transplanted according to the standard criteria. in this article, we will discuss the history of development, the techniques and protocols of ex vivo, and the logics and rationales for ex vivo use.

  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. In vivo and ex vivo methods of growing a liver bud through tissue connection.

    Science.gov (United States)

    Yanagi, Yusuke; Nakayama, Koichi; Taguchi, Tomoaki; Enosawa, Shin; Tamura, Tadashi; Yoshimaru, Koichiro; Matsuura, Toshiharu; Hayashida, Makoto; Kohashi, Kenichi; Oda, Yoshinao; Yamaza, Takayoshi; Kobayashi, Eiji

    2017-10-26

    Cell-based therapy has been proposed as an alternative to orthotopic liver transplantation. The novel transplantation of an in vitro-generated liver bud might have therapeutic potential. In vivo and ex vivo methods for growing a liver bud are essential for paving the way for the clinical translation of liver bud transplantation. We herein report a novel transplantation method for liver buds that are grown in vivo involving orthotopic transplantation on the transected parenchyma of the liver, which showed long engraftment and marked growth in comparison to heterotopic transplantation. Furthermore, this study demonstrates a method for rapidly fabricating scalable liver-like tissue by fusing hundreds of liver bud-like spheroids using a 3D bioprinter. Its system to fix the shape of the 3D tissue with the needle-array system enabled the fabrication of elaborate geometry and the immediate execution of culture circulation after 3D printing-thereby avoiding an ischemic environment ex vivo. The ex vivo-fabricated human liver-like tissue exhibited self-tissue organization ex vivo and engraftment on the liver of nude rats. These achievements conclusively show both in vivo and ex vivo methods for growing in vitro-generated liver buds. These methods provide a new approach for in vitro-generated liver organoids transplantation.

  18. In Vitro and Ex Vivo Approaches to Evaluate Next-Generation Tobacco and Non-Tobacco Products on Human Blood Platelets.

    Science.gov (United States)

    Spinelli, Sherry L; Lannan, Katie L; Loelius, Shannon G; Phipps, Richard P

    2017-03-01

    Human blood platelets are major hemostatic regulators in the circulation and important in the mediation of chronic inflammation and immunomodulation. They are key elements that promote cardiovascular pathogenesis that leads to atherosclerosis, thrombosis, myocardial infarction, and stroke. New information on tobacco use and platelet dysregulation shows that these highly understudied vascular cells are dysregulated by tobacco smoke. Thus, platelet function studies should be an important consideration for the evaluation of existing and next-generation tobacco and non-tobacco products. Novel in vitro approaches are being sought to investigate these products and their influence on platelet function. Platelets are ideally suited for product assessment, as robust and novel in vitro translational methods are available to assess platelet function. Furthermore, the use of human biological systems has the advantage that risk predictions will better reflect the human condition.

  19. Three-dimensional Integrated Functional, Structural, and Computational Mapping to Define the Structural "Fingerprints" of Heart-Specific Atrial Fibrillation Drivers in Human Heart Ex Vivo.

    Science.gov (United States)

    Zhao, Jichao; Hansen, Brian J; Wang, Yufeng; Csepe, Thomas A; Sul, Lidiya V; Tang, Alan; Yuan, Yiming; Li, Ning; Bratasz, Anna; Powell, Kimerly A; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul M L; Weiss, Raul; Simonetti, Orlando P; Hummel, John D; Fedorov, Vadim V

    2017-08-22

    Structural remodeling of human atria plays a key role in sustaining atrial fibrillation (AF), but insufficient quantitative analysis of human atrial structure impedes the treatment of AF. We aimed to develop a novel 3-dimensional (3D) structural and computational simulation analysis tool that could reveal the structural contributors to human reentrant AF drivers. High-resolution panoramic epicardial optical mapping of the coronary-perfused explanted intact human atria (63-year-old woman, chronic hypertension, heart weight 608 g) was conducted during sinus rhythm and sustained AF maintained by spatially stable reentrant AF drivers in the left and right atrium. The whole atria (107×61×85 mm 3 ) were then imaged with contrast-enhancement MRI (9.4 T, 180×180×360-μm 3 resolution). The entire 3D human atria were analyzed for wall thickness (0.4-11.7 mm), myofiber orientations, and transmural fibrosis (36.9% subendocardium; 14.2% midwall; 3.4% subepicardium). The 3D computational analysis revealed that a specific combination of wall thickness and fibrosis ranges were primarily present in the optically defined AF driver regions versus nondriver tissue. Finally, a 3D human heart-specific atrial computer model was developed by integrating 3D structural and functional mapping data to test AF induction, maintenance, and ablation strategies. This 3D model reproduced the optically defined reentrant AF drivers, which were uninducible when fibrosis and myofiber anisotropy were removed from the model. Our novel 3D computational high-resolution framework may be used to quantitatively analyze structural substrates, such as wall thickness, myofiber orientation, and fibrosis, underlying localized AF drivers, and aid the development of new patient-specific treatments. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  20. Ultrasound can detect macroscopically undetectable changes in osteoarthritis reflecting the superficial histological and biochemical degeneration: ex vivo study of rabbit and human cartilage.

    Directory of Open Access Journals (Sweden)

    Kohei Nishitani

    Full Text Available Recognizing subtle cartilage changes in the preclinical stage of osteoarthritis (OA is essential for early diagnosis. To this end, the ability of the ultrasound signal intensity to detect macroscopically undetectable cartilage change was investigated. In this study, cartilage of rabbit OA model and human OA samples was examined by macroscopic evaluation, ultrasound signal intensity, histology with Mankin scores, and Fourier transform infrared imaging (FTIRI analysis. Rabbit OA was induced by anterior cruciate ligament transection and evaluated at 1, 2, 4 and 12 weeks. Twenty human samples were harvested during total knee arthroplasty from OA patients who had macroscopically normal human cartilage (ICRS grade 0 on the lateral femoral condyle. In the animal study, there was no macroscopic OA change at 2 weeks, but histology detected degenerative changes at this time point. Ultrasound signal intensity also detected degeneration at 2 weeks. In human samples, all samples were obtained from macroscopically intact site, however nearly normal (0 ≤ Mankin score <2, early OA (2 ≤ Mankin score <6, and moderate OA (6 ≤ Mankin score <10 samples were actually intermixed. Ultrasound signal intensity was significantly different among these 3 stages and was well correlated with Mankin scores (R = -0.80 and FTIR parameters related to collagen and proteoglycan content in superficial zone. In conclusion, ultrasound can detect microscopic cartilage deterioration when such changes do not exist macroscopically, reflecting superficial histological and biochemical changes.

  1. Chromosome copy number variation in telomerized human bone marrow stromal cells; insights for monitoring safe ex-vivo expansion of adult stem cells

    DEFF Research Database (Denmark)

    Burns, Jorge S.; Harkness, Linda; Aldahmash, Abdullah

    2017-01-01

    Adult human bone marrow stromal cells (hBMSC) cultured for cell therapy require evaluation of potency and stability for safe use. Chromosomal aberrations upsetting genomic integrity in such cells have been contrastingly described as "Limited" or "Significant". Previously reported stepwise...

  2. Impact of multiple genetic polymorphisms on effects of a 4-week blueberry juice intervention on ex vivo induced lymphocytic DNA damage in human volunteers

    NARCIS (Netherlands)

    Wilms, Lonneke C.; Boots, Agnes W.; de Boer, Vincent C. J.; Maas, Lou M.; Pachen, Daniëlle M. F. A.; Gottschalk, Ralph W. H.; Ketelslegers, Hans B.; Godschalk, Roger W. L.; Haenen, Guido R. M. M.; van Schooten, Frederik J.; Kleinjans, Jos C. S.

    2007-01-01

    Consumption of fruits and vegetables has been associated with a decrease in cancer incidence and cardiovascular disease, presumably caused by antioxidants. We designed a human intervention study to assess antioxidative and possible anti-genotoxic properties of fruit-borne antioxidants. We

  3. Chromosome copy number variation in telomerized human bone marrow stromal cells; insights for monitoring safe ex-vivo expansion of adult stem cells

    DEFF Research Database (Denmark)

    Burns, Jorge S.; Harkness, Linda; Aldahmash, Abdullah

    2017-01-01

    Adult human bone marrow stromal cells (hBMSC) cultured for cell therapy require evaluation of potency and stability for safe use. Chromosomal aberrations upsetting genomic integrity in such cells have been contrastingly described as "Limited" or "Significant". Previously reported stepwise acquisi...

  4. Enhanced Ex Vivo Stimulation of Mycobacterium tuberculosis-Specific T Cells in Human Immunodeficiency Virus-Infected Persons via Antigen Delivery by the Bordetella pertusis Adenylate Cyclase Vector

    Czech Academy of Sciences Publication Activity Database

    Connell, T. G.; Shey, M. S.; Seldon, R.; Rangaka, M. X.; van Cutsem, G.; Šimšová, Marcela; Marčeková, Zuzana; Šebo, Peter; Curtis, N.; Diwakar, L.; Meintjes, G. A.; Leclerc, C.; Wilkinson, R. J.; Wilkinson, K. A.

    2007-01-01

    Roč. 14, č. 7 (2007), s. 847-854 ISSN 1556-6811 R&D Projects: GA MŠk 2B06161 Institutional research plan: CEZ:AV0Z50200510 Keywords : mycobacterium tuberculosis * bordetella pertusis * human immunodeficiency virus Subject RIV: EE - Microbiology, Virology Impact factor: 1.995, year: 2007

  5. Tumor necrosis factor-α-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo

    DEFF Research Database (Denmark)

    Ågren, Magnus S; Schnabel, Reinhild; Christensen, Lise H

    2015-01-01

    Tumor necrosis factor (TNF)-α induces matrix metalloproteinases (MMPs) that may disrupt skin integrity. We have investigated the effects and mechanisms of exogenous TNF-α on collagen degradation by incubating human skin explants in defined serum-free media with or without TNF-α (10ng/ml) in the a......Tumor necrosis factor (TNF)-α induces matrix metalloproteinases (MMPs) that may disrupt skin integrity. We have investigated the effects and mechanisms of exogenous TNF-α on collagen degradation by incubating human skin explants in defined serum-free media with or without TNF-α (10ng....../ml) in the absence or presence of the nonselective MMP inhibitor GM6001 for 8 days. The basal culture conditions promoted type I collagen catabolism that was accelerated by TNF-α (p... were associated with increased collagen degradation. TNF-α increased secretion of MMP-1 (p

  6. Radiological-Pathological Correlations Following Blast-Related Traumatic Brain Injury in the Whole Human Brain Using ex Vivo Diffusion Tensor Imaging

    Science.gov (United States)

    2014-01-01

    injuries caused by non-blast related trauma (e.g. falls, motor vehicle accidents, etc.), post-mortem pathological analyses have revealed that...part of these advances is the use of high resolution imaging of blocks of human brain at 4.7 Tesla . This high resolution imaging has allowed...to noise Stereological methods: StereoInvestigator software coupled to a motorized stage on a Nikon 80i upright microscope. All tissue

  7. An examination of resveratrol's mechanisms of action in human tissue: impact of a single dose in vivo and dose responses in skeletal muscle ex vivo.

    Directory of Open Access Journals (Sweden)

    Cameron B Williams

    Full Text Available The current study tested the hypothesis that a single, moderate dose of RSV would activate the AMPK/SIRT1 axis in human skeletal muscle and adipose tissue. Additionally, the effects of RSV on mitochondrial respiration in PmFBs were examined. Eight sedentary men (23.8±2.4 yrs; BMI: 32.7±7.1 reported to the lab on two occasions where they were provided a meal supplemented with 300 mg of RSV or a placebo. Blood samples, and a muscle biopsy were obtained in the fasted state and again, with the addition of an adipose tissue biopsy, two hours post-prandial. The effect of RSV on mitochondrial respiration was examined in PmFBs taken from muscle biopsies from an additional eight men (23.4±5.4 yrs; BMI: 24.4±2.8. No effect of RSV was observed on nuclear SIRT1 activity, acetylation of p53, or phosphorylation of AMPK, ACC or PKA in either skeletal muscle or adipose tissue. A decrease in post absorptive insulin levels was accompanied by elevated skeletal muscle phosphorylation of p38 MAPK, but no change in either skeletal muscle or adipose tissue insulin signalling. Mitochondrial respiration in PmFBs was rapidly inhibited by RSV at 100-300 uM depending on the substrate examined. These results question the efficacy of a single dose of RSV at altering skeletal muscle and adipose tissue AMPK/SIRT1 activity in humans and suggest that RSV mechanisms of action in humans may be associated with altered cellular energetics resulting from impaired mitochondrial ATP production.

  8. Selective Inhibition of ADAM17 Efficiently Mediates Glycoprotein Ibα Retention During Ex Vivo Generation of Human Induced Pluripotent Stem Cell-Derived Platelets.

    Science.gov (United States)

    Hirata, Shinji; Murata, Takahiko; Suzuki, Daisuke; Nakamura, Sou; Jono-Ohnishi, Ryoko; Hirose, Hidenori; Sawaguchi, Akira; Nishimura, Satoshi; Sugimoto, Naoshi; Eto, Koji

    2016-10-05

    : Donor-independent platelet concentrates for transfusion can be produced in vitro from induced pluripotent stem cells (iPSCs). However, culture at 37°C induces ectodomain shedding on platelets of glycoprotein Ibα (GPIbα), the von Willebrand factor receptor critical for adhesive function and platelet lifetime in vivo, through temperature-dependent activation of a disintegrin and metalloproteinase 17 (ADAM17). The shedding can be suppressed by using inhibitors of panmetalloproteinases and possibly of the upstream regulator p38 mitogen-activated protein kinase (p38 MAPK), but residues of these inhibitors in the final platelet products may be accompanied by harmful risks that prevent clinical application. Here, we optimized the culture conditions for generating human iPSC-derived GPIbα(+) platelets, focusing on culture temperature and additives, by comparing a new and safe selective ADAM17 inhibitor, KP-457, with previous inhibitors. Because cultivation at 24°C (at which conventional platelet concentrates are stored) markedly diminished the yield of platelets with high expression of platelet receptors, 37°C was requisite for normal platelet production from iPSCs. KP-457 blocked GPIbα shedding from iPSC platelets at a lower half-maximal inhibitory concentration than panmetalloproteinase inhibitor GM-6001, whereas p38 MAPK inhibitors did not. iPSC platelets generated in the presence of KP-457 exhibited improved GPIbα-dependent aggregation not inferior to human fresh platelets. A thrombus formation model using immunodeficient mice after platelet transfusion revealed that iPSC platelets generated with KP-457 exerted better hemostatic function in vivo. Our findings suggest that KP-457, unlike GM-6001 or p38 MAPK inhibitors, effectively enhances the production of functional human iPSC-derived platelets at 37°C, which is an important step toward their clinical application. This study is a tip for overcoming a practical but critical barrier for manufacturing human

  9. Patterns of Human Immunodeficiency Virus type 1 recombination ex vivo provide evidence for coadaptation of distant sites, resulting in purifying selection for intersubtype recombinants during replication

    DEFF Research Database (Denmark)

    Galli, Andrea; Kearney, Mary; Nikolaitchik, Olga A

    2010-01-01

    in human populations. We hypothesize that sequence diversity affects the emergence of viable recombinants by decreasing recombination events and reducing the ability of the recombinants to replicate. To test our hypothesis, we compared recombination between two viruses containing subtype B pol genes (B....../F than in B/B viruses, and the overall distribution of crossover junctions in pol was similar for the two classes of recombinants. We then examined the emergence of recombinants in a multiple cycle assay, so that functional pol gene products were selected. We found that the emerging B/B recombinants had...

  10. Application of two segmentation protocols during the processing of virtual images in rapid prototyping: ex vivo study with human dry mandibles.

    Science.gov (United States)

    Ferraz, Eduardo Gomes; Andrade, Lucio Costa Safira; dos Santos, Aline Rode; Torregrossa, Vinicius Rabelo; Rubira-Bullen, Izabel Regina Fischer; Sarmento, Viviane Almeida

    2013-12-01

    The aim of this study was to evaluate the accuracy of virtual three-dimensional (3D) reconstructions of human dry mandibles, produced from two segmentation protocols ("outline only" and "all-boundary lines"). Twenty virtual three-dimensional (3D) images were built from computed tomography exam (CT) of 10 dry mandibles, in which linear measurements between anatomical landmarks were obtained and compared to an error probability of 5 %. The results showed no statistically significant difference among the dry mandibles and the virtual 3D reconstructions produced from segmentation protocols tested (p = 0,24). During the designing of a virtual 3D reconstruction, both "outline only" and "all-boundary lines" segmentation protocols can be used. Virtual processing of CT images is the most complex stage during the manufacture of the biomodel. Establishing a better protocol during this phase allows the construction of a biomodel with characteristics that are closer to the original anatomical structures. This is essential to ensure a correct preoperative planning and a suitable treatment.

  11. Effects of UV Rays and Thymol/Thymus vulgaris L. Extract in an ex vivo Human Skin Model: Morphological and Genotoxicological Assessment.

    Science.gov (United States)

    Cornaghi, Laura; Arnaboldi, Francesca; Calò, Rossella; Landoni, Federica; Baruffaldi Preis, William Franz; Marabini, Laura; Donetti, Elena

    2016-01-01

    Ultraviolet (UV) radiation is the major environmental factor affecting functions of the skin. Compounds rich in polyphenols, such as Thymus vulgaris leaf extract and thymol, have been proposed for the prevention of UV-induced skin damage. We compared the acute effects induced by UVA and UVB rays on epidermal morphology and proliferation, cytotoxicity, and genotoxicity. Normal human skin explants were obtained from young healthy women (n = 7) after informed consent and cultured at the air-liquid interface overnight. After 24 h, the samples were divided in 2 groups: the former exposed to UVA (16 or 24 J/cm2) and the latter irradiated with UVB (0.24 or 0.72 J/cm2). One hour after the end of irradiation, supernatants were collected for evaluation of the lactate dehydrogenase activity. Twenty-four hours after UVB exposure, biopsies were processed for light and transmission electron microscopy analysis, proliferation, cytotoxicity, and genotoxicity. UVB and UVA rays induced early inhibition of cell proliferation and DNA damage compared to controls. In particular, UVB rays were always more cytotoxic and genotoxic than UVA ones. For this reason, we evaluated the effect of either T. vulgaris L. extract (1.82 µg/ml) or thymol (1 µg/ml) on all samples treated for 1 h before UVB irradiation. While Thymus had a protective action for all of the endpoints evaluated, the action of the extract was less pronounced on epidermal proliferation and morphological features. The results presented in this study could be the basis for investigating the mechanism of thymol and T. vulgaris L. extract against the damage induced by UV radiation. © 2016 S. Karger AG, Basel.

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

    Directory of Open Access Journals (Sweden)

    Paulo Manuel Pêgo-Fernandes

    2010-12-01

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

  13. Comparison of ex vivo stability of copeptin and vasopressin

    NARCIS (Netherlands)

    Heida, Judith E; Boesten, Lianne S M; Ettema, Esmée M; Muller Kobold, Anneke C.; Franssen, Casper F M; Gansevoort, Ron T; Zittema, Debbie

    BACKGROUND: Copeptin, part of the vasopressin precursor, is increasingly used as marker for vasopressin and is claimed to have better ex vivo stability. However, no study has directly compared the ex vivo stability of copeptin and vasopressin. METHODS: Blood of ten healthy volunteers was collected

  14. Comparison of two in vivo and two ex vivo tests to assess the antibacterial activity of several antiseptics.

    Science.gov (United States)

    Messager, S; Goddard, P A; Dettmar, P W; Maillard, J-Y

    2004-10-01

    An ex vivo test was adapted to mimic the in vivo conditions of testing antiseptic activity on human forearms and in the European Standard Hygienic Handwash Test (BSEN 1499). The study was to validate the ex vivo protocols using 4.8% (w/v) para-chloro-meta-xylenol (PCMX, neat Dettol), 0.5% (w/v) triclosan in 70% (v/v) isopropanol, and 2% (v/v) povidone-iodine against a high bacterial inoculum (>10(8) cfu/mL) of Escherichia coli NCTC 10538. Two ex vivo tests using human skin samples, including one introducing a mechanical rubbing effect, were compared with two corresponding in vivo tests (the forearm test and the BSEN handwashing test). All antiseptics assessed in vivo (forearm and handwash tests) produced reductions in bacterial counts that were significantly greater than those for the non-medicated soft soap control. When assessed ex vivo without rubbing, only PCMX and povidone-iodine achieved reductions significantly greater than soft soap. When assessed ex vivo with mechanical rubbing, only PCMX and triclosan achieved reductions significantly greater than soft soap. Overall, the antiseptics at the concentrations tested were more active when tested in vivo than ex vivo. The addition of a mechanical effect, either in vivo by the volunteers washing their hands or ex vivo by a drill rubbing two skin samples against each other, produced a significantly greater reduction in bacterial concentrations. The ex vivo tests were easily adapted to mimic in vivo protocols. The value of such tests, particularly the one that includes a rubbing effect, may be significant as they avoid the need for human volunteers.

  15. Physiologic assessment of the ex vivo donor lung for transplantation.

    Science.gov (United States)

    Yeung, Jonathan C; Cypel, Marcelo; Machuca, Tiago N; Koike, Terumoto; Cook, Douglas J; Bonato, Riccardo; Chen, Manyin; Sato, Masaaki; Waddell, Thomas K; Liu, Mingyao; Slutsky, Arthur S; Keshavjee, Shaf

    2012-10-01

    The evaluation of donor lungs by normothermic ex vivo acellular perfusion has improved the safety of organ utilization. However, this strategy requires a critical re-evaluation of the parameters used to assess lungs during ex vivo perfusion compared with those traditionally used to evaluate the donor lung in vivo. Using a porcine model, we studied the physiology of acellular lung perfusion with the aim of improving the accuracy of clinical ex vivo evaluation. Porcine lungs after 10 hours of brain death and 24 hours of cold ischemia and uninjured control lungs were perfused for 12 hours and then transplanted. PaO2, compliance, airway pressure and pulmonary vascular resistance were measured. Ventilation with 100% nitrogen and addition of red blood cells to the perfusate were used to clarify the physiologic disparities between in vivo blood perfusion and ex vivo acellular perfusion. During 12 hours of ex vivo perfusion, injured lungs developed edema with decreased compliance and increased airway pressure, but ex vivo PO2 remained stable. After transplantation, injured lungs demonstrated high vascular resistance and poor PaO2. A reduced effect of shunt on ex vivo lung perfusion PO2 was found to be attributable to the linearization of the relationship between oxygen content and PO2, which occurs with acellular perfusate. Ex vivo PO2 may not be the first indication of lung injury and, taken alone, may be misleading in assessing the ex vivo lung. Thus, evaluation of other physiologic parameters takes on greater importance. Copyright © 2012 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

  16. Ultrasound elasticity imaging for detecting intestinal fibrosis and inflammation in rats and humans with Crohn's disease.

    Science.gov (United States)

    Stidham, Ryan W; Xu, Jingping; Johnson, Laura A; Kim, Kang; Moons, David S; McKenna, Barbara J; Rubin, Jonathan M; Higgins, Peter D R

    2011-09-01

    Intestinal fibrosis causes many complications of Crohn's disease (CD). Available biomarkers and imaging modalities lack sufficient accuracy to distinguish intestinal inflammation from fibrosis. Transcutaneous ultrasound elasticity imaging (UEI) is a promising, noninvasive approach for measuring tissue mechanical properties. We hypothesized that UEI could differentiate inflammatory from fibrotic bowel wall changes in both animal models of colitis and humans with CD. Female Lewis rats underwent weekly trinitrobenzene sulfonic acid enemas yielding models of acute inflammatory colitis (n = 5) and chronic intestinal fibrosis (n = 6). UEI scanning used a novel speckle-tracking algorithm to estimate tissue strain. Resected bowel segments were evaluated for evidence of inflammation and fibrosis. Seven consecutive patients with stenotic CD were studied with UEI and their resected stenotic and normal bowel segments were evaluated by ex vivo elastometry and histopathology. Transcutaneous UEI normalized strain was able to differentiate acutely inflamed (-2.07) versus chronic fibrotic (-1.10) colon in rat models of inflammatory bowel disease (IBD; P = .037). Transcutaneous UEI normalized strain also differentiated stenotic (-0.87) versus adjacent normal small bowel (-1.99) in human CD (P = .0008), and this measurement also correlated well with ex vivo elastometry (r = -0.81). UEI can differentiate inflammatory from fibrotic intestine in rat models of IBD and can differentiate between fibrotic and unaffected intestine in a pilot study in humans with CD. UEI represents a novel technology with potential to become a new objective measure of progression of intestinal fibrosis. Prospective clinical studies in CD are needed. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  17. 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.

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

    Science.gov (United States)

    Galozzi, P; Negm, O; Greco, E; Alkhattabi, N; Gava, A; Sfriso, P; Fairclough, L; Todd, I; Tighe, P; Punzi, L

    2015-03-31

    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.

  19. Ex vivo regenerative effects of a spring water.

    Science.gov (United States)

    Nicoletti, Giovanni; Saler, Marco; Pellegatta, Tommaso; Tresoldi, Marco Mario; Bonfanti, Viola; Malovini, Alberto; Faga, Angela; Riva, Federica

    2017-12-01

    Previous experiments by our group have indicated the regenerative effects of a spring water (Comano), which was possibly associated with the native non-pathogenic bacterial flora. The present study aimed to confirm these regenerative properties in a human ex vivo experimental model in the context of physiological wound healing. Human 6-mm punch skin biopsies harvested during plastic surgery sessions were injured in their central portion to induce skin loss and were cultured in either conventional medium (controls) or medium powder reconstituted with filtered Comano spring water (treated samples). At 24, 48 and 72 h the specimens were observed following staining with hematoxylin and eosin, Picrosirius Red, orcein and anti-proliferating cell nuclear antigen. Compared with the controls, the treated samples exhibited reduced overall cell infiltration, evidence of fibroblasts, stimulation of cell proliferation and collagen and elastic fiber regeneration. In the spring water, in addition to 12 resident non-pathogenic bacterial strains exhibiting favorable metabolic activities, more unknown non-pathogenic species are being identified by genomic analysis. In the present study, the efficacy of this 'germ-free', filtered spring water in wound regeneration was indicated. Thus, the Comano spring water microbiota should be acknowledged for its regenerative properties.

  20. 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. Copyright © 2010 John Wiley & Sons, Ltd.

  1. Ex Vivo Lung Perfusion: Establishment and Operationalization in Iran.

    Science.gov (United States)

    Shafaghi, Shadi; Abbasi Dezfuli, Azizollah; Ansari Aval, Zahra; Sheikhy, Kambiz; Farzanegan, Behrooz; Mortaz, Esmaeil; Emami, Habib; Aigner, Clemens; Hosseini-Baharanchi, Fatemeh Sadat; Najafizadeh, Katayoun

    2017-02-01

    Although the number of lung transplants is limited because of general shortage of organ donors, ex vivo lung perfusion is a novel method with 2 main benefits, including better evaluation of lung potential and recovery of injured lungs. The main aim of this study was to establish and operationalize ex vivo lung perfusion as the first experience in Iran. This was a prospective operational research study on 5 cases, including 1 pig from Vienna Medical University and 4 patients from Masih Daneshvari Hospital. All organ donations from brain dead donors were evaluated according to lung transplant or ex vivo lung perfusion criteria from May 2013 to July 2015 in Tehran, Iran. If a donor did not have any sign of severe chest trauma or pneumonia but had poor oxygenation due to possible atelectasis or neurogenic pulmonary edema, their lungs were included for ex vivo lung perfusion. A successful trend in the difference between the pulmonary arterial Po2 and the left atrial Po2 was observed, as well as an increasing pattern in other functional parameters, including dynamic lung compliance and a decreasing trend in pulmonary vascular resistance. These initial trials indicate that ex vivo lung perfusion can lead to remarkable progress in lung transplant in Iran. They also provide several important pieces of guidance for successful ex vivo lung perfusion, including the necessity of following standard lung retrieval procedures and monitoring temperature and pressure precisely. The development of novel methods can provide opportunities for further research studies on lungs of deceased donors and lead to undiscovered findings. By keeping this science up to date in Iran and developing such new and creative methods, we can reveal effective strategies to promote the quality of donor lungs to support patients on transplant wait lists.

  2. Ex Vivo (Fluorescence) Confocal Microscopy in Surgical Pathology: State of the Art.

    Science.gov (United States)

    Ragazzi, Moira; Longo, Caterina; Piana, Simonetta

    2016-05-01

    First developed in 1957, confocal microscopy is a powerful imaging tool that can be used to obtain near real-time reflected light images of untreated human tissue with nearly histologic resolution. Besides its research applications, in the last decades, confocal microscopy technology has been proposed as a useful device to improve clinical diagnosis, especially in ophthalmology, dermatology, and endomicroscopy settings, thanks to advances in instrument development. Compared with the wider use of the in vivo tissue assessment, ex vivo applications of confocal microscopy are not fully explored. A comprehensive review of the current literature was performed here, focusing on the reliable applications of ex vivo confocal microscopy in surgical pathology and on some potential evolutions of this new technique from pathologists' viewpoint.

  3. Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

    Science.gov (United States)

    Klöß, Stephan; Oberschmidt, Olaf; Morgan, Michael; Dahlke, Julia; Arseniev, Lubomir; Huppert, Volker; Granzin, Markus; Gardlowski, Tanja; Matthies, Nadine; Soltenborn, Stephanie; Schambach, Axel; Koehl, Ulrike

    2017-10-01

    The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56 + CD3 - ) was carried out with the CliniMACS Prodigy ® in a single process, starting with approximately 1.2 × 10 9 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 10 6 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO ™ 10, CellGro ® , TexMACS ™ , and NK MACS ® ). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56 + CD3 - target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3

  4. Antimicrobial Blue Light Therapy for Infectious Keratitis: Ex Vivo and In Vivo Studies.

    Science.gov (United States)

    Zhu, Hong; Kochevar, Irene E; Behlau, Irmgard; Zhao, Jie; Wang, Fenghua; Wang, Yucheng; Sun, Xiaodong; Hamblin, Michael R; Dai, Tianhong

    2017-01-01

    To investigate the effectiveness of antimicrobial blue light (aBL) as an alternative or adjunctive therapeutic for infectious keratitis. We developed an ex vivo rabbit model and an in vivo mouse model of infectious keratitis. A bioluminescent strain of Pseudomonas aeruginosa was used as the causative pathogen, allowing noninvasive monitoring of the extent of infection in real time via bioluminescence imaging. Quantitation of bacterial luminescence was correlated to colony-forming units (CFU). Using the ex vivo and in vivo models, the effectiveness of aBL (415 nm) for the treatment of keratitis was evaluated as a function of radiant exposure when aBL was delivered at 6 or 24 hours after bacterial inoculation. The aBL exposures calculated to reach the retina were compared to the American National Standards Institute standards to estimate aBL retinal safety. Pseudomonas aeruginosa keratitis fully developed in both the ex vivo and in vivo models at 24 hours post inoculation. Bacterial luminescence in the infected corneas correlated linearly to CFU (R2 = 0.921). Bacterial burden in the infected corneas was rapidly and significantly reduced (>2-log10) both ex vivo and in vivo after a single exposure of aBL. Recurrence of infection was observed in the aBL-treated mice at 24 hours after aBL exposure. The aBL toxicity to the retina is largely dependent on the aBL transmission of the cornea. Antimicrobial blue light is a potential alternative or adjunctive therapeutic for infectious keratitis. Further studies of corneal and retinal safety using large animal models, in which the ocular anatomies are similar to that of humans, are warranted.

  5. Paediatric laparoscopic hernia repair: Ex vivo skills in the reduced ...

    African Journals Online (AJOL)

    With repeated attempts trainee's timings and accuracy improved until by the 10th repair they were no different from benchmark consultant scores. Conclusion: A simple, procedure specifi c ex-vivo training model has been evaluated for laparoscopic hernia training in paediatric surgery. The results suggest improvements in ...

  6. Ex vivo exposure to gaseous pollutants of air passages and human and animal pulmonary vessels: effect on the reactivity and cell signalling; Exposition ex vivo aux polluants gazeux des voies aeriennes et de vaisseaux pulmonaires humains et animaux: effet sur la reactivite et la signalisation cellulaire

    Energy Technology Data Exchange (ETDEWEB)

    Hyvelin, J.M.

    2000-07-01

    The objectives of this work have been to characterize effects of exposure to several pollutants on the reactivity of air passages, to study the action mechanisms of pollutants on the smooth muscle cell of air passages, to characterize the calcic signalling of the human bronchi smooth muscle in order to identify the pollutants cell targets, to look for others pollutants cell targets. This work contributes to a better knowledge of air pollution effects, by underlining the additive character of pollutants. It allows a better knowledge of cell mechanisms implied in the pollutants effects. Then, it notices the existence of other cell types, the pulmonary arterial myocyte, sensitive to pollutants exposure and that can be implied in the respiratory health degradation. (N.C.)

  7. Ex Vivo Restimulation of Human PBMC Expands a CD3+CD4−CD8− γδ + T Cell Population That Can Confound the Evaluation of CD4 and CD8 T Cell Responses to Vaccination

    Science.gov (United States)

    Sedgmen, B. J.; Papalia, L.; Wang, L.; Dyson, A. R.; McCallum, H. A.; Simson, C. M.; Pearse, M. J.; Maraskovsky, E.; Hung, D.; Eomois, P. P.; Hartel, G.; Barnden, M. J.; Rockman, S. P.

    2013-01-01

    The measurement of vaccine-induced humoral and CD4+ and CD8+ cellular immune responses represents an important correlate of vaccine efficacy. Accurate and reliable assays evaluating such responses are therefore critical during the clinical development phase of vaccines. T cells play a pivotal role both in coordinating the adaptive and innate immune responses and as effectors. During the assessment of cell-mediated immunity (CMI) in subjects participating in a large-scale influenza vaccine trial, we identified the expansion of an IFN-γ-producing CD3+CD4−CD8− γδ + T cell population in the peripheral blood of 90/610 (15%) healthy subjects. The appearance of CD3+CD4−CD8− γδ + T cells in the blood of subjects was transient and found to be independent of the study cohort, vaccine group, subject gender and ethnicity, and ex vivo restimulation conditions. Although the function of this population and relevance to vaccination are unclear, their inclusion in the total vaccine-specific T-cell response has the potential to confound data interpretation. It is thus recommended that when evaluating the induction of IFN-γ-producing CD4+ and CD8+ immune responses following vaccination, the CD3+CD4−CD8− γδ + T cells are either excluded or separately enumerated from the overall frequency determination. PMID:24066003

  8. Ex Vivo Restimulation of Human PBMC Expands a CD3+CD4-CD8-γδ+ T Cell Population That Can Confound the Evaluation of CD4 and CD8 T Cell Responses to Vaccination

    Directory of Open Access Journals (Sweden)

    B. J. Sedgmen

    2013-01-01

    Full Text Available The measurement of vaccine-induced humoral and CD4+ and CD8+ cellular immune responses represents an important correlate of vaccine efficacy. Accurate and reliable assays evaluating such responses are therefore critical during the clinical development phase of vaccines. T cells play a pivotal role both in coordinating the adaptive and innate immune responses and as effectors. During the assessment of cell-mediated immunity (CMI in subjects participating in a large-scale influenza vaccine trial, we identified the expansion of an IFN-γ-producing CD3+CD4-CD8-γδ+ T cell population in the peripheral blood of 90/610 (15% healthy subjects. The appearance of CD3+CD4-CD8-γδ+ T cells in the blood of subjects was transient and found to be independent of the study cohort, vaccine group, subject gender and ethnicity, and ex vivo restimulation conditions. Although the function of this population and relevance to vaccination are unclear, their inclusion in the total vaccine-specific T-cell response has the potential to confound data interpretation. It is thus recommended that when evaluating the induction of IFN-γ-producing CD4+ and CD8+ immune responses following vaccination, the CD3+CD4-CD8-γδ+ T cells are either excluded or separately enumerated from the overall frequency determination.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    Fish oil (FO) is considered antiinflammatory, but evidence regarding its effect on human cytokine production is conflicting. High linoleic acid (LA) intake may impair any effects of FO. The aim of this study was to investigate how FO combined with high or low LA intake affected ex vivo cytokine p...

  10. 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...

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

    NARCIS (Netherlands)

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

    2005-01-01

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

  12. The effect of artificial antigen-presenting cells with preclustered anti-CD28/-CD3/-LFA-1 monoclonal antibodies on the induction of ex vivo expansion of functional human antitumor T cells.

    Science.gov (United States)

    Zappasodi, Roberta; Di Nicola, Massimo; Carlo-Stella, Carmelo; Mortarini, Roberta; Molla, Alessandra; Vegetti, Claudia; Albani, Salvatore; Anichini, Andrea; Gianni, Alessandro M

    2008-10-01

    Adoptive cell therapy with ex vivo expanded autologous antitumor cytotoxic T lymphocytes represents an important therapeutic option as an anticancer strategy. In order to identify a reliable method for producing adequate amounts of functional antitumor cytotoxic T lymphocytes with a potentially long in vivo lifespan, we tested the T-cell expansion efficiency of a new artificial antigen-presenting cell-based system. Our artificial antigen-presenting cells were generated with activating (anti-CD3), co-stimulating (anti-CD28) and adhesion (anti-LFA-1) biotinylated monoclonal antibodies preclustered in microdomains held on a liposome scaffold by neutravidin rafts. The co-localization of T-cell ligands in microdomains and the targeting of an adhesion protein, increasing the efficiency of immunological synapse formation, represent the novelties of our system. The activity of our artificial antigen-presenting cells was compared with that of anti-CD3/-CD28 coated immunomagnetic microbeads and immobilized anti-CD3 monoclonal antibody (OKT3 clone), the only two commercially available artificial systems. Our artificial antigen-presenting cells expanded both polyclonal T cells and MART-1-specific CD8(+) T cells in a more efficient manner than the other systems. Stimulation with artificial antigen-presenting cells allows for the generation of viable T cells displaying an immunophenotype consistent with in vivo potential for persistence, without increasing the frequency of regulatory T cells. The starting specificity of anti MART-1 CD8(+) T cells was preserved after stimulation with artificial antigen-presenting cells and it was statistically greater when compared to the activity of the same cells expanded with the other systems. Finally, our artificial antigen-presenting cells proved to be suitable for large-scale application, minimizing the volume and the costs of T-cell expansion. Our artificial antigen-presenting cells might represent an efficient tool to rapidly obtain a

  13. 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.

  14. 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.

  15. Comparison of in vivo and ex vivo imaging of the microvasculature with 2-photon fluorescence microscopy

    Science.gov (United States)

    Steinman, Joe; Koletar, Margaret; Stefanovic, Bojana; Sled, John G.

    2016-03-01

    This study evaluates 2-Photon fluorescence microscopy of in vivo and ex vivo cleared samples for visualizing cortical vasculature. Four mice brains were imaged with in vivo 2PFM. Mice were then perfused with a FITC gel and cleared in fructose. The same regions imaged in vivo were imaged ex vivo. Vessels were segmented automatically in both images using an in-house developed algorithm that accounts for the anisotropic and spatially varying PSF ex vivo. Through non-linear warping, the ex vivo image and tracing were aligned to the in vivo image. The corresponding vessels were identified through a local search algorithm. This enabled comparison of identical vessels in vivo/ex vivo. A similar process was conducted on the in vivo tracing to determine the percentage of vessels perfused. Of all the vessels identified over the four brains in vivo, 98% were present ex vivo. There was a trend towards reduced vessel diameter ex vivo by 12.7%, and the shrinkage varied between specimens (0% to 26%). Large diameter surface vessels, through a process termed 'shadowing', attenuated in vivo signal from deeper cortical vessels by 40% at 300 μm below the cortical surface, which does not occur ex vivo. In summary, though there is a mean diameter shrinkage ex vivo, ex vivo imaging has a reduced shadowing artifact. Additionally, since imaging depths are only limited by the working distance of the microscope objective, ex vivo imaging is more suitable for imaging large portions of the brain.

  16. 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

    2015-04-02

    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 methodology in applied ethnopharmacological research. 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. 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 silybin A, silybin B and hydrastine and berberine on CYP2C9 and CYP3A4/5, respectively, results which more

  17. Effect of implanted brachytherapy seeds on optical fluence distribution: preliminary ex vivo study

    Science.gov (United States)

    Hetzel, Fred W.; Chen, Qun; Ding, Meisong; Newman, Francis; Dole, Kenneth C.; Huang, Zheng; Blanc, Dominique

    2007-02-01

    Photodynamic therapy (PDT) has gradually found its place in the treatment of malignant and non-malignant human diseases. Currently, interstitial PDT is being explored as an alternative modality for newly diagnosed and recurrent organ-confined prostate cancer. The interstitial PDT for the treatment of prostate cancer might be considered to treat prostates with permanent radioactive seeds implantation. However, the effect of implanted brachytherapy seeds on the optical fluence distribution of PDT light has not been studied before. This study investigated, for the first time, the effect of brachytherapy seed on the optical fluence distribution of 760 nm light in ex vivo models (meat and canine prostate).

  18. Evaluation of the Doxycycline Release from AH26 Sealer-Doxycycline Combination: An ex vivo Study

    OpenAIRE

    Ashofteh Yazdi, Kazem; Shokouhinejad, Noushin; Moazeni, Esmaeil; Mirzayi Rad, Sina

    2011-01-01

    INTRODUCTION The purpose of this ex vivo study was to determine the releasing characteristics and doxycycline dentinal diffusion of AH26 sealer-doxycycline combination from apical 3mm of tooth root and apical foramen. MATERIALS AND METHODS One-hundred and two recently extracted single-rooted human teeth were decoronated and prepared with #3 and #4 Gates-Glidden drills and rotary Mtwo files. Smear layer was removed; all surfaces except for apical 3mm of each root were sealed with two coats of ...

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

    Science.gov (United States)

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

    2017-08-24

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

  20. Ex vivo virotherapy with myxoma virus does not impair hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Villa, Nancy Y; Bais, Swarna; Chan, Winnie M; Meacham, Amy M; Wise, Elizabeth; Rahman, Masmudur M; Moreb, Jan S; Rosenau, Emma H; Wingard, John R; McFadden, Grant; Cogle, Christopher R

    2016-03-01

    Relapsing disease is a major challenge after hematopoietic cell transplantation for hematological malignancies. Myxoma virus (MYXV) is an oncolytic virus that can target and eliminate contaminating cancer cells from auto-transplant grafts. The aims of this study were to examine the impact of MYXV on normal hematopoietic stem and progenitor cells and define the optimal treatment conditions for ex vivo virotherapy. Bone marrow (BM) and mobilized peripheral blood stem cells (mPBSCs) from patients with hematologic malignancies were treated with MYXV at various time, temperature and incubation media conditions. Treated BM cells from healthy normal donors were evaluated using flow cytometry for MYXV infection, long-term culture-initiating cell (LTC-IC) assay and colony-forming cell (CFC) assay. MYXV initiated infection in up to 45% of antigen-presenting monocytes, B cells and natural killer cells; however, these infections were uniformly aborted in >95% of all cells. Fresh graft sources showed higher levels of MYXV infection initiation than cryopreserved specimens, but in all cases less than 10% of CD34(+) cells could be infected after ex vivo MYXV treatment. MYXV did not impair LTC-IC colony numbers compared with mock treatment. CFC colony types and numbers were also not impaired by MYXV treatment. MYXV incubation time, temperature or culture media did not significantly change the percentage of infected cells, LTC-IC colony formation or CFC colony formation. Human hematopoietic cells are non-permissive for MYXV. Human hematopoietic stem and progenitor cells were not infected and thus unaffected by MYXV ex vivo treatment. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. 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

  3. Identification of differentiation-stage specific markers that define the ex vivo osteoblastic phenotype.

    Science.gov (United States)

    Twine, Natalie A; Chen, Li; Pang, Chi N; Wilkins, Marc R; Kassem, Moustapha

    2014-10-01

    The phenotype of osteoblastic (OB) cells in culture is currently defined using a limited number of markers of low sensitivity and specificity. For the clinical use of human skeletal (stromal, mesenchymal) stem cells (hMSC) in therapy, there is also a need to identify a set of gene markers that predict in vivo bone forming capacity. Thus, we used RNA sequencing to examine changes in expression for a set of skeletally-related genes across 8 time points between 0 and 12days of ex vivo OB differentiation of hMSC. We identified 123 genes showing significant temporal expression change. Hierarchical clustering and Pearson's correlation generated 4 groups of genes: early stage differentiation genes (peak expression: 0-24h, n=28) which were enriched for extracellular matrix organisation, e.g. COL1A1, LOX, and SERPINH1; middle stage differentiating genes (peak expression days: 3 and 6, n=20) which were enriched for extracellular matrix/skeletal system development e.g. BMP4, CYP24A1, and TGFBR2; and late stage differentiation genes (peak expression days: 9 and 12, n=27) which were enriched for bone development/osteoblast differentiation, e.g. BMP2 and IGF2. In addition, we identified 13 genes with bimodal temporal expression (2 peaks of expression: days 0 and 12) including VEGFA, PDGFA and FGF2. We examined the specificity of the 123 genes' expression in skeletal tissues and thus propose a set of ex vivo differentiation-stage-specific markers (n=21). In an independent analysis, we identified a subset of genes (n=20, e.g. ELN, COL11A1, BMP4) to predict the bone forming capacity of hMSC and another set (n=20, e.g. IGF2, TGFB2, SMAD3) associated with the ex vivo phenotype of hMSC obtained from osteoporotic patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Sustained function of genetically modified porcine lungs in an ex vivo model of pulmonary xenotransplantation.

    Science.gov (United States)

    Westall, Glen P; Levvey, Browyn J; Salvaris, Evelyn; Gooi, Julian; Marasco, Sylvana; Rosenfeldt, Frank; Egan, Chris; McEgan Ccp, Robin; Mennen, Mark; Russell, Prue; Robson, Simon C; Nottle, Mark B; Dwyer, Karen M; Snell, Greg I; Cowan, Peter J

    2013-11-01

    Xenotransplantation could provide a solution to the donor shortage that is currently the major barrier to solid-organ transplantation. The ability to breed pigs with multiple genetic modifications provides a unique opportunity to explore the immunologic challenges of pulmonary xenotransplantation. Explanted lungs from wild-type and 3 groups of genetically modified pigs were studied: (i) α1,3-galactosyltransferase gene knockout (GTKO); (ii) GTKO pigs expressing the human complementary regulatory proteins CD55 and CD59 (GTKO/CD55-59); and (iii) GTKO pigs expressing both CD55-59 and CD39 (GTKO/CD55-59/CD39). The physiologic, immunologic and histologic properties of porcine lungs were evaluated on an ex vivo rig after perfusion with human blood. Lungs from genetically modified pigs demonstrated stable pulmonary vascular resistance and better oxygenation of the perfusate, and survived longer than wild-type lungs. Physiologic function was inversely correlated with the degree of platelet sequestration into the xenograft. Despite superior physiologic profiles, lungs from genetically modified pigs still showed evidence of intravascular thrombosis and coagulopathy after perfusion with human blood. The ability to breed pigs with multiple genetic modifications, and to evaluate lung physiology and histology in real-time on an ex vivo rig, represent significant advances toward better understanding the challenges inherent to pulmonary xenotransplantation. © 2013 International Society for Heart and Lung Transplantation. All rights reserved.

  5. Interferon-α Subtypes in an Ex Vivo Model of Acute HIV-1 Infection: Expression, Potency and Effector Mechanisms

    Science.gov (United States)

    Harper, Michael S.; Guo, Kejun; Gibbert, Kathrin; Lee, Eric J.; Dillon, Stephanie M.; Barrett, Bradley S.; McCarter, Martin D.; Hasenkrug, Kim J.; Dittmer, Ulf; Wilson, Cara C.; Santiago, Mario L.

    2015-01-01

    HIV-1 is transmitted primarily across mucosal surfaces and rapidly spreads within the intestinal mucosa during acute infection. The type I interferons (IFNs) likely serve as a first line of defense, but the relative expression and antiviral properties of the 12 IFNα subtypes against HIV-1 infection of mucosal tissues remain unknown. Here, we evaluated the expression of all IFNα subtypes in HIV-1-exposed plasmacytoid dendritic cells by next-generation sequencing. We then determined the relative antiviral potency of each IFNα subtype ex vivo using the human intestinal Lamina Propria Aggregate Culture model. IFNα subtype transcripts from the centromeric half of the IFNA gene complex were highly expressed in pDCs following HIV-1 exposure. There was an inverse relationship between IFNA subtype expression and potency. IFNα8, IFNα6 and IFNα14 were the most potent in restricting HIV-1 infection. IFNα2, the clinically-approved subtype, and IFNα1 were both highly expressed but exhibited relatively weak antiviral activity. The relative potencies correlated with binding affinity to the type I IFN receptor and the induction levels of HIV-1 restriction factors Mx2 and Tetherin/BST-2 but not APOBEC3G, F and D. However, despite the lack of APOBEC3 transcriptional induction, the higher relative potency of IFNα8 and IFNα14 correlated with stronger inhibition of virion infectivity, which is linked to deaminase-independent APOBEC3 restriction activity. By contrast, both potent (IFNα8) and weak (IFNα1) subtypes significantly induced HIV-1 GG-to-AG hypermutation. The results unravel non-redundant functions of the IFNα subtypes against HIV-1 infection, with strong implications for HIV-1 mucosal immunity, viral evolution and IFNα-based functional cure strategies. PMID:26529416

  6. Experimental Nanopulse Ablation of Multiple Membrane Parasite on Ex Vivo Hydatid Cyst

    Directory of Open Access Journals (Sweden)

    Xinhua Chen

    2018-01-01

    Full Text Available The impact of ultrashort nanopulse on cellular membrane is of biological significance and thus has been studied intensively. Different from cell study, this ex vivo study aims to investigate the biological effects of nanosecond pulsed electric field (nsPEF on an independent multimembrane parasite, human hydatid cyst, to observe the unique influence of nanopulse on macromembrane structure, permeabilization, and biochemistry. The 300 ns nsPEF was delivered on an experimental model of single human hydatid cyst ex vivo with eight different parameters. Then pathological changes during 7 days of 48 parasite cysts were followed up after nsPEF. The laminated layer, the germinal layer, the protoscolex, and cyst fluid were evaluated by the morphological, pathological, and biochemical measurements. The parameter screening found that nsPEF can damage hydatid cyst effectively when the field strength is higher than 14 kV/cm. When nsPEF is higher than 29 kV/cm, nsPEF destroy hydatid cyst completely by collapsing the germinal layer, destructing protoscolices, and exhausting the nutrition.

  7. Bioequivalence Methodologies for Topical Drug Products: In Vitro and Ex Vivo Studies with a Corticosteroid and an Anti-Fungal Drug.

    Science.gov (United States)

    Leal, Leila Bastos; Cordery, Sarah F; Delgado-Charro, M Begoña; Bunge, Annette L; Guy, Richard H

    2017-04-01

    To examine whether in vitro and ex vivo measurements of topical drug product performance correlate with in vivo outcomes, such that more efficient experimental approaches can be reliably and reproducibly used to establish (in)equivalence between formulations for skin application. In vitro drug release through artificial membranes, and drug penetration into porcine skin ex vivo, were compared with published human in vivo studies. Two betamethasone valerate (BMV) formulations, and three marketed econazole nitrate (EN) creams were assessed. For BMV, the stratum corneum (SC) uptake of drug in 6 h closely matched data observed in vivo in humans, and distinguished between inequivalent formulations. SC uptake of EN from the 3 creams mirrored the in vivo equivalence in man (both clinically and via similar tape-stripping experiments). However, EN clearance from SC ex vivo did not parallel that in vivo, presumably due to the absence of a functioning microcirculation. In vitro release of BMV from the different formulations did not overlap with either ex vivo or in vivo tape-stripping data whereas, for EN, a good correlation was observed. No measurable permeation of either BMV or EN was detected in a 6-h in vitro skin penetration experiment. In vitro and ex vivo methods for topical bioequivalence determination can show correlation with in vivo outcomes. However, these surrogates have understandable limitations. A "one-size-fits-all" approach for topical bioequivalence evaluation may not always be successful, therefore, and the judicious use of complementary methods may prove a more effective and reliable strategy.

  8. Ex vivo and in vivo label-free imaging of lymphatic vessels using OCT lymphangiography (Conference Presentation)

    Science.gov (United States)

    Gong, Peijun; Es'haghian, Shaghayegh; Karnowski, Karol; Rea, Suzanne; Wood, Fiona M.; Yu, Dao-Yi; McLaughlin, Robert A.; Sampson, David D.

    2017-02-01

    We have been developing an automated method to image lymphatic vessels both ex vivo and in vivo with optical coherence tomography (OCT), using their optical transparency. Our method compensates for the OCT signal attenuation for each A-scan in combination with the correction of the confocal function and sensitivity fall-off, enabling reliable thresholding of lymphatic vessels from the OCT scans. Morphological image processing with a segment-joining algorithm is also incorporated into the method to mitigate partial-volume artifacts, which are particularly evident with small lymphatic vessels. Our method is demonstrated for two different clinical application goals: the monitoring of conjunctival lymphatics for surgical guidance and assessment of glaucoma treatment; and the longitudinal monitoring of human burn scars undergoing laser ablation treatment. We present examples of OCT lymphangiography ex vivo on porcine conjunctivas and in vivo on human burn scars, showing the visualization of the lymphatic vessel network and their longitudinal changes due to treatment.

  9. Characterising the tumour morphological response to therapeutic intervention: an ex vivo model

    Directory of Open Access Journals (Sweden)

    Anne Savage

    2013-01-01

    In cancer, morphological assessment of histological tissue samples is a fundamental part of both diagnosis and prognosis. Image analysis offers opportunities to support that assessment through quantitative metrics of morphology. Generally, morphometric analysis is carried out on two-dimensional tissue section data and so only represents a small fraction of any tumour. We present a novel application of three-dimensional (3D morphometrics for 3D imaging data obtained from tumours grown in a culture model. Minkowski functionals, a set of measures that characterise geometry and topology in n-dimensional space, are used to quantify tumour topology in the absence of and in response to therapeutic intervention. These measures are used to stratify the morphological response of tumours to therapeutic intervention. Breast tumours are characterised by estrogen receptor (ER status, human epidermal growth factor receptor (HER2 status and tumour grade. Previously, we have shown that ER status is associated with tumour volume in response to tamoxifen treatment ex vivo. Here, HER2 status is found to predict the changes in morphology other than volume as a result of tamoxifen treatment ex vivo. Finally, we show the extent to which Minkowski functionals might be used to predict tumour grade. Minkowski functionals are generalisable to any 3D data set, including in vivo and cellular systems. This quantitative topological analysis can provide a valuable link among biomarkers, drug intervention and tumour morphology that is complementary to existing, non-morphological measures of tumour response to intervention and could ultimately inform patient treatment.

  10. White Blood Cell-Based Detection of Asymptomatic Scrapie Infection by Ex Vivo Assays

    Science.gov (United States)

    Halliez, Sophie; Jaumain, Emilie; Huor, Alvina; Douet, Jean-Yves; Lugan, Séverine; Cassard, Hervé; Lacroux, Caroline; Béringue, Vincent; Andréoletti, Olivier; Vilette, Didier

    2014-01-01

    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. PMID:25122456

  11. A novel ex-vivo porcine renal xenotransplantation model using a pulsatile machine preservation system.

    Science.gov (United States)

    Guarrera, James V; Stone, Jonathan; Tulipan, Jacob; Jhang, Jeffrey; Arrington, Ben; Boykin, Jason; Markowitz, Glen; Ratner, Lloyd E

    2011-01-01

    Animal models to investigate pathophysiology and xenotransplantation require complex techniques and significant animal utilization. The aim of the study was to develop a reliable ex-vivo technique to test xenotransplant interventions. Miniature Swine being utilized for a nonsurvival study acted as donor animals. Kidneys were flushed and rapidly explanted and chilled to 4°C. Kidneys were assigned to be the control (CK) (n=3) and the mate were used as a Xenograft Kidneys (XK) (n=3). Kidneys were perfused on separate Waters RM 3 perfusion devices. Perfusion temperature was 35-37°C and pressure was 100-110/60-70 mmHg at 60 pulses per minute. CKs were reperfused with autologous blood collected at the time of organ procurement. XKs were reperfused using freshly donated whole human blood. Physical characteristics, urine output were recorded. Core needle biopsies were obtained and examined by a blinded pathologist for evidence of antibody mediated rejection. XK kidneys demonstrated homogenous reperfusion which rapidly became patchy at 5-7 minutes. XK kidneys had become complete black and thrombosed by 60-70 minutes. XK biopsies demonstrated peritubular capillaritis. CK kidneys demonstrated homogenous reperfusion and urine production. H&E stain of CKs only demonstrated nonspecific inflammation. Our ex-vivo porcine xenotransplant model shows early promise as a tool for studying Xeno- associated hyperacute rejection. This technique saves resources and animal utilization.

  12. Survival of cord blood haematopoietic stem cells in a hyaluronan hydrogel for ex vivo biomimicry.

    Science.gov (United States)

    Demange, Elise; Kassim, Yusra; Petit, Cyrille; Buquet, Catherine; Dulong, Virginie; Cerf, Didier Le; Buchonnet, Gérard; Vannier, Jean-Pierre

    2013-11-01

    Haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs) grow in a specified niche in close association with the microenvironment, the so-called 'haematopoietic niche'. Scaffolds have been introduced to overcome the liquid culture limitations, mimicking the presence of the extracellular matrix (ECM). In the present study the hyaluronic acid scaffold, already developed in the laboratory, has been used for the first time to maintain long-term cultures of CD34⁺ haematopoietic cells obtained from human cord blood. One parameter investigated was the impact on ex vivo survival of CD34⁺ cord blood cells (CBCs) on the hyaluronic acid surface, immobilized with peptides containing the RGD motif. This peptide was conjugated by coating the hyaluronan hydrogel and cultured in serum-free liquid phase complemented with stem cell factor (SCF), a commonly indispensable cytokine for haematopoiesis. Our work demonstrated that these hyaluronan hydrogels were superior to traditional liquid cultures by maintaining and expanding the HPCs without the need for additional cytokines, and a colonization of 280-fold increment in the hydrogel compared with liquid culture after 28 days of ex vivo expansion. Copyright © 2012 John Wiley & Sons, Ltd.

  13. Passive cavitation detection during pulsed HIFU exposures of ex vivo tissues and in vivo mouse pancreatic tumors.

    Science.gov (United States)

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

    2014-07-01

    Pulsed high-intensity focused ultrasound (pHIFU) has been shown to enhance vascular permeability, disrupt tumor barriers and enhance drug penetration into tumor tissue through acoustic cavitation. Monitoring of cavitation activity during pHIFU treatments and knowing the ultrasound pressure levels sufficient to reliably induce cavitation in a given tissue are therefore very important. Here, three metrics of cavitation activity induced by pHIFU and evaluated by confocal passive cavitation detection were introduced: cavitation probability, cavitation persistence and the level of the broadband acoustic emissions. These metrics were used to characterize cavitation activity in several ex vivo tissue types (bovine tongue and liver and porcine adipose tissue and kidney) and gel phantoms (polyacrylamide and agarose) at varying peak-rare factional focal pressures (1-12 MPa) during the following pHIFU protocol: frequency 1.1 MHz, pulse duration 1 ms and pulse repetition frequency 1 Hz. To evaluate the relevance of the measurements in ex vivo tissue, cavitation metrics were also investigated and compared in the ex vivo and in vivo murine pancreatic tumors that develop spontaneously in transgenic KrasLSL.G12 D/+; p53 R172 H/+; PdxCretg/+ (KPC) mice and closely re-capitulate human disease in their morphology. The cavitation threshold, defined at 50% cavitation probability, was found to vary broadly among the investigated tissues (within 2.5-10 MPa), depending mostly on the water-lipid ratio that characterizes the tissue composition. Cavitation persistence and the intensity of broadband emissions depended both on tissue structure and lipid concentration. Both the cavitation threshold and broadband noise emission level were similar between ex vivo and in vivo pancreatic tumor tissue. The largest difference between in vivo and ex vivo settings was found in the pattern of cavitation occurrence throughout pHIFU exposure: it was sporadic in vivo, but it decreased rapidly and stopped

  14. The Human Milk Oligosaccharide 2'-Fucosyllactose Quenches Campylobacter jejuni-Induced Inflammation in Human Epithelial Cells HEp-2 and HT-29 and in Mouse Intestinal Mucosa.

    Science.gov (United States)

    Yu, Zhuo-Teng; Nanthakumar, N Nanda; Newburg, David S

    2016-10-01

    Campylobacter jejuni causes diarrhea worldwide; young children are most susceptible. Binding of virulent C. jejuni to the intestinal mucosa is inhibited ex vivo by α1,2-fucosylated carbohydrate moieties, including human milk oligosaccharides (HMOSs). The simplest α1,2-fucosylated HMOS structure, 2'-fucosyllactose (2'-FL), can be predominant at ≤5 g/L milk. Although 2'-FL inhibits C. jejuni binding ex vivo and in vivo, the effects of 2'FL on the cell invasion central to C. jejuni pathogenesis have not been tested. Clinical isolates of C. jejuni infect humans, birds, and ferrets, limiting studies on its mammalian pathobiology. Human epithelial cells HEp-2 and HT-29 infected with the virulent C. jejuni strain 81-176 human isolate were treated with 5 g 2'-FL/L, and the degree of infection and inflammatory response was measured. Four-week-old male wild-type C57BL/6 mice were fed antibiotics to reduce their intestinal microbiota and were inoculated with C. jejuni strain 81-176. The sensitivity of the resulting acute transient enteric infection and immune response to inhibition by 2'-FL ingestion was tested. In HEp-2 and HT-29 cells, 2'-FL attenuated 80% of C. jejuni invasion (P jejuni colonization by 80%, weight loss by 5%, histologic features of intestinal inflammation by 50-70%, and induction of inflammatory signaling molecules of the acute-phase mucosal immune response by 50-60% (P jejuni clinical disease, 2'-FL inhibited pathogenesis and its sequelae. These data strongly support the hypothesis that 2'-FL represents a new class of oral agent for prevention, and potentially for treatment, of specific enteric infectious diseases. © 2016 American Society for Nutrition.

  15. 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

  16. 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

    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...... (with skin) and skin-removed tissues. To identify the spectral differences between normal and cancer breast tissue, the paired t-test was carried out for each wavenumber using the whole spectral range from both groups. Quadratic discriminate analysis based on principal component analysis (PCA) was also...... used to determine and evaluate differences in the Raman spectra for the various samples as a basis for diagnostic purposes. The differences in the Raman spectra of the samples were due to biochemical changes at the molecular, cellular and tissue levels. The sensitivity and specificity...

  17. Epidermal protection with cryogen spray cooling during high fluence pulsed dye laser irradiation: an ex vivo study.

    Science.gov (United States)

    Tunnell, J W; Nelson, J S; Torres, J H; Anvari, B

    2000-01-01

    Higher laser fluences than currently used in therapy (5-10 J/cm(2)) are expected to result in more effective treatment of port wine stain (PWS) birthmarks. However, higher incident fluences increase the risk of epidermal damage caused by absorption of light by melanin. Cryogen spray cooling offers an effective method to reduce epidermal injury during laser irradiation. The objective of this study was to determine whether high laser incident fluences (15-30 J/cm(2)) could be used while still protecting the epidermis in ex vivo human skin samples. Non-PWS skin from a human cadaver was irradiated with a Candela ScleroPlus Laser (lambda = 585 nm; pulse duration = 1.5 msec) by using various incident fluences (8-30 J/cm(2)) without and with cryogen spray cooling (refrigerant R-134a; spurt durations: 40-250 msec). Assessment of epidermal damage was based on histologic analysis. Relatively short spurt durations (40-100 msec) protected the epidermis for laser incident fluences comparable to current therapeutic levels (8-10 J/cm(2)). However, longer spurt durations (100-250 msec) increased the fluence threshold for epidermal damage by a factor of three (up to 30 J/cm(2)) in these ex vivo samples. Results of this ex vivo study show that epidermal protection from high laser incident fluences can be achieved by increasing the cryogen spurt duration immediately before pulsed laser exposure. Copyright 2000 Wiley-Liss, Inc.

  18. Comparison of ex vivo stability of copeptin and vasopressin.

    Science.gov (United States)

    Heida, Judith E; Boesten, Lianne S M; Ettema, Esmée M; Muller Kobold, Anneke C; Franssen, Casper F M; Gansevoort, Ron T; Zittema, Debbie

    2017-06-27

    Copeptin, part of the vasopressin precursor, is increasingly used as marker for vasopressin and is claimed to have better ex vivo stability. However, no study has directly compared the ex vivo stability of copeptin and vasopressin. Blood of ten healthy volunteers was collected in EDTA tubes. Next, we studied the effect of various pre-analytical conditions on measured vasopressin and copeptin levels: centrifugation speed, short-term storage temperature and differences between whole blood and plasma, long-term storage temperature and repeated freezing and thawing. The acceptable change limit (ACL), indicating the maximal percentage change that can be explained by assay variability, was used as cut-off to determine changes in vasopressin and copeptin. The ACL was 25% for vasopressin and 19% for copeptin. Higher centrifugation speed resulted in lower vasopressin levels, whereas copeptin concentration was unaffected. In whole blood, vasopressin was stable up to 2 h at 25°C and 6 h at 4°C. In plasma, vasopressin was stable up to 6 h at 25°C and 24 h at 4°C. In contrast, copeptin was stable in whole blood and plasma for at least 24h at both temperatures. At -20°C, vasopressin was stable up to 1 month and copeptin for at least 4 months. Both vasopressin and copeptin were stable after 4 months when stored at -80°C and -150°C. Vasopressin concentration decreased after four freeze-thaw cycles, whereas copeptin concentration was unaffected. Vasopressin levels were considerably affected by pre-analytical conditions, while copeptin levels were stable. Therefore, a strict sample handling protocol for measurement of vasopressin is recommended.

  19. 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.

  20. Spectroscopic optical coherence tomography for ex vivo brain tumor analysis

    Science.gov (United States)

    Lenz, Marcel; Krug, Robin; Dillmann, Christopher; Gerling, Alexandra; Gerhardt, Nils C.; Welp, Hubert; Schmieder, Kirsten; Hofmann, Martin R.

    2017-02-01

    For neurosurgeries precise tumor resection is essential for the subsequent recovery of the patients since nearby healthy tissue that may be harmed has a huge impact on the life quality after the surgery. However, so far no satisfying methodology has been established to assist the surgeon during surgery to distinguish between healthy and tumor tissue. Optical Coherence Tomography (OCT) potentially enables non-contact in vivo image acquisition at penetration depths of 1-2 mm with a resolution of approximately 1-15 μm. To analyze the potential of OCT for distinction between brain tumors and healthy tissue, we used a commercially available Thorlabs Callisto system to measure healthy tissue and meningioma samples ex vivo. All samples were measured with the OCT system and three dimensional datasets were generated. Afterwards they were sent to the pathology for staining with hematoxylin and eosin and then investigated with a bright field microscope to verify the tissue type. This is the actual gold standard for ex vivo analysis. The images taken by the OCT system exhibit variations in the structure for different tissue types, but these variations may not be objectively evaluated from raw OCT images. Since an automated distinction between tumor and healthy tissue would be highly desirable to guide the surgeon, we applied Spectroscopic Optical Coherence Tomography to further enhance the differences between the tissue types. Pattern recognition and machine learning algorithms were applied to classify the derived spectroscopic information. Finally, the classification results are analyzed in comparison to the histological analysis of the samples.

  1. Ex vivo confocal imaging with contrast agents for the detection of oral potentially malignant lesions.

    Science.gov (United States)

    El Hallani, S; Poh, C F; Macaulay, C E; Follen, M; Guillaud, M; Lane, P

    2013-06-01

    We investigated the potential use of real-time confocal microscopy in the non-invasive detection of occult oral potentially malignant lesions. Our objectives were to select the best fluorescence contrast agent for cellular morphology enhancement, to build an atlas of confocal microscopic images of normal human oral mucosa, and to determine the accuracy of confocal microscopy to recognize oral high-grade dysplasia lesions on live human tissue. Five clinically used fluorescent contrast agents were tested in vitro on cultured human cells and validated ex vivo on human oral mucosa. Images acquired ex vivo from normal and diseased human oral biopsies with bench-top fluorescent confocal microscope were compared to conventional histology. Image analyzer software was used as an adjunct tool to objectively compare high-grade dysplasia versus low-grade dysplasia and normal epithelium. Acriflavine Hydrochloride provided the best cellular contrast by preferentially staining the nuclei of the epithelium. Using topical application of Acriflavine Hydrochloride followed by confocal microscopy, we could define morphological characteristics of each cellular layer of the normal human oral mucosa, building an atlas of histology-like images. Applying this technique to diseased oral tissue specimen, we were also able to accurately diagnose the presence of high-grade dysplasia through the increased cellularity and changes in nuclear morphological features. Objective measurement of cellular density by quantitative image analysis was a strong discriminant to differentiate between high-grade dysplasia and low-grade dysplasia lesions. Pending clinical investigation, real-time confocal microscopy may become a useful adjunct to detect precancerous lesions that are at high risk of cancer progression, direct biopsy and delineate excision margins. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Eviscerated Corneas as Tissue Source for Ex Vivo Expansion of Limbal Epithelial Cells on Platelet-Rich Plasma Gels.

    Science.gov (United States)

    Heydenrych, Leonard G; du Toit, Donald F; Aldous, Colleen M

    2016-12-01

    Purpose/Aim of the study: To assess if corneal epithelium can be cultured ex vivo from corneas eviscerated due to irretrievable trauma, according to a cell culture method that made use of autologous platelet-rich plasma (A-PRP) as culture substrate. To compare corneal epithelium cultured ex vivo from corneas eviscerated following trauma using A-PRP combined with Dulbecco's modified Eagles medium (DMEM), versus DMEM alone. This was a laboratory case-controlled study of human corneal cells cultured in a mixture of A-PRP and DMEM, versus DMEM alone from six eviscerated corneas. A 100 explants were created, of which 50 explants were plated on A-PRP-gel construct combined with DMEM and 50 controls were placed in serum-free DMEM alone. Donor patients received systemic antibiotics prior to evisceration. Confluent epithelium in monolayers could be cultured when donor limbal biopsies were placed in a mixture of A-PRP culture medium and DMEM. No growth was observed when corneas were placed in serum-free DMEM medium only (p < 0.05). No bacterial infection was observed in cultures. This study demonstrated that A-PRP is a viable and effective alternative to bovine serum for the ex vivo expansion of limbal epithelial cells. It also shows that eviscerated corneas are a viable source of donor tissue for this purpose in South Africa where access to tissue banks is limited.

  5. Ex-vivo training model for laparoendoscopic single-site surgery

    Directory of Open Access Journals (Sweden)

    Kommu Sashi

    2011-01-01

    Full Text Available Background: Laparoendoscopic single-site surgery (LESS has recently been applied successfully in the performance of a host of surgical procedures. Preliminary consensus from the experts is that this mode of surgery is technically challenging and requires expertise. The transition from trainee to practicing surgeon, especially in complex procedures with challenging learning curves, takes time and mentor-guided nurturing. However, the trainee needs to use platforms of training to gain the skills that are deemed necessary for undertaking the live human case. Objective: This article aims to demonstrate a step-by-step means of how to acquire the necessary instrumentation and build a training model for practicing steeplechase exercises in LESS for urological surgeons and trainees. The tool built as a result of this could set the platform for performance of basic and advanced skills uptake using conventional, bent and articulated instruments. A preliminary construct validity of the platform was conducted. Materials and Methods: A box model was fitted with an R-Port™ and camera. Articulated and conventional instruments were used to demonstrate basic exercises (e.g. glove pattern cutting, loop stacking and suturing and advanced exercises (e.g. pyeloplasty. The validation included medical students (M, final year laparoscopic fellows (F and experienced consultant laparoscopic surgeons (C with at least 50 LESS cases experience in total, were tested on eight basic skill tasks (S including manipulation of the flexible cystoscope (S1, hand eye coordination (S2, cutting with flexible scissors (S3, grasping with flexible needle holders (S4, two-handed maneuvers (S5, object translocation (S6, cross hand suturing with flexible instruments (S7 and conduction of an ex-vivo pyeloplasty. Results: The successful application of the box model was demonstrated by trainee based exercises. The cost of the kit with circulated materials was less than £150 (Pounds Sterling

  6. Correlation of in vivo and ex vivo 1H-MRI with histology in two severities of mouse spinal cord injury

    Directory of Open Access Journals (Sweden)

    Harun Najib NORISTANI

    2015-03-01

    Full Text Available 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 spinal cord injury (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 epicentre 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.

  7. Ricin crosses polarized human intestinal cells and intestines of ricin-gavaged mice without evident damage and then disseminates to mouse kidneys.

    Directory of Open Access Journals (Sweden)

    Alyssa D Flora

    Full Text Available Ricin is a potent toxin found in the beans of Ricinus communis and is often lethal for animals and humans when aerosolized or injected and causes significant morbidity and occasional death when ingested. Ricin has been proposed as a bioweapon because of its lethal properties, environmental stability, and accessibility. In oral intoxication, the process by which the toxin transits across intestinal mucosa is not completely understood. To address this question, we assessed the impact of ricin on the gastrointestinal tract and organs of mice after dissemination of toxin from the gut. We first showed that ricin adhered in a specific pattern to human small bowel intestinal sections, the site within the mouse gut in which a variable degree of damage has been reported by others. We then monitored the movement of ricin across polarized human HCT-8 intestinal monolayers grown in transwell inserts and in HCT-8 cell organoids. We observed that, in both systems, ricin trafficked through the cells without apparent damage until 24 hours post intoxication. We delivered a lethal dose of purified fluorescently-labeled ricin to mice by oral gavage and followed transit of the toxin from the gastrointestinal tracts to the internal organs by in vivo imaging of whole animals over time and ex vivo imaging of organs at various time points. In addition, we harvested organs from unlabeled ricin-gavaged mice and assessed them for the presence of ricin and for histological damage. Finally, we compared serum chemistry values from buffer-treated versus ricin-intoxicated animals. We conclude that ricin transverses human intestinal cells and mouse intestinal cells in situ prior to any indication of enterocyte damage and that ricin rapidly reaches the kidneys of intoxicated mice. We also propose that mice intoxicated orally with ricin likely die from distributive shock.

  8. Ricin crosses polarized human intestinal cells and intestines of ricin-gavaged mice without evident damage and then disseminates to mouse kidneys.

    Science.gov (United States)

    Flora, Alyssa D; Teel, Louise D; Smith, Mark A; Sinclair, James F; Melton-Celsa, Angela R; O'Brien, Alison D

    2013-01-01

    Ricin is a potent toxin found in the beans of Ricinus communis and is often lethal for animals and humans when aerosolized or injected and causes significant morbidity and occasional death when ingested. Ricin has been proposed as a bioweapon because of its lethal properties, environmental stability, and accessibility. In oral intoxication, the process by which the toxin transits across intestinal mucosa is not completely understood. To address this question, we assessed the impact of ricin on the gastrointestinal tract and organs of mice after dissemination of toxin from the gut. We first showed that ricin adhered in a specific pattern to human small bowel intestinal sections, the site within the mouse gut in which a variable degree of damage has been reported by others. We then monitored the movement of ricin across polarized human HCT-8 intestinal monolayers grown in transwell inserts and in HCT-8 cell organoids. We observed that, in both systems, ricin trafficked through the cells without apparent damage until 24 hours post intoxication. We delivered a lethal dose of purified fluorescently-labeled ricin to mice by oral gavage and followed transit of the toxin from the gastrointestinal tracts to the internal organs by in vivo imaging of whole animals over time and ex vivo imaging of organs at various time points. In addition, we harvested organs from unlabeled ricin-gavaged mice and assessed them for the presence of ricin and for histological damage. Finally, we compared serum chemistry values from buffer-treated versus ricin-intoxicated animals. We conclude that ricin transverses human intestinal cells and mouse intestinal cells in situ prior to any indication of enterocyte damage and that ricin rapidly reaches the kidneys of intoxicated mice. We also propose that mice intoxicated orally with ricin likely die from distributive shock.

  9. 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

  10. Synovitis biomarkers: ex vivo characterization of three biomarkers for identification of inflammatory osteoarthritis.

    Science.gov (United States)

    Kjelgaard-Petersen, Cecilie; Siebuhr, Anne Sofie; Christiansen, Thorbjørn; Ladel, Christoph; Karsdal, Morten; Bay-Jensen, Anne-Christine

    2015-01-01

    Characterize biomarkers measuring extracellular matrix turnover of inflamed osteoarthritis synovium. Human primary fibroblast-like synoviocytes and synovial membrane explants (SMEs) treated with various cytokines and growth factors were assessed by C1M, C3M, and acMMP3 in the conditioned medium. TNFα significantly increased C1M up to seven-fold (p = 0.0002), C3M up to 24-fold (p = 0.0011), and acMMP3 up to 14-fold (p biomarkers C1M, C3M, and acMMP-3 were synovitis biomarkers ex vivo and provide a translational tool together with the SME model.

  11. 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.

  12. TNF-alpha promoter, Nod2 and toll-like receptor-4 polymorphisms and the in vivo and ex vivo response to endotoxin

    NARCIS (Netherlands)

    Schippers, Emile F.; van 't Veer, Cornelis; van Voorden, Sjaak; Martina, Cerithsa A. E.; le Cessie, Saskia; van Dissel, Jaap T.

    2004-01-01

    Humans exhibit substantial inter-individual differences in TNF-alpha production upon endotoxin stimulation. To determine to what extent the lipopolysaccharide-induced TNF-alpha production capacity in vivo and ex vivo is determined by polymorphisms in toll-like receptor-4 (TLR4), the TNF-alpha

  13. Novel 11β-hydroxysteroid dehydrogenase 1 inhibitors reduce cortisol levels in keratinocytes and improve dermal collagen content in human ex vivo skin after exposure to cortisone and UV.

    Directory of Open Access Journals (Sweden)

    Stéphanie M Boudon

    Full Text Available Activity and selectivity assessment of new bi-aryl amide 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1 inhibitors, prepared in a modular manner via Suzuki cross-coupling, are described. Several compounds inhibiting 11β-HSD1 at nanomolar concentrations were identified. Compounds 2b, 3e, 7b and 12e were shown to selectively inhibit 11β-HSD1 over 11β-HSD2, 17β-HSD1 and 17β-HSD2. These inhibitors also potently inhibited 11β-HSD1 activity in intact HEK-293 cells expressing the recombinant enzyme and in intact primary human keratinocytes expressing endogenous 11β-HSD1. Moreover, compounds 2b, 3e and 12e were tested for their activity in human skin biopsies. They were able to prevent, at least in part, both the cortisone- and the UV-mediated decreases in collagen content. Thus, inhibition of 11β-HSD1 by these compounds can be further investigated to delay or prevent UV-mediated skin damage and skin aging.

  14. Residual Antimicrobial Activity of MTAD(® in Human Dentin After Obturation with Gutta-Percha/AH26 and Resilon/RealSeal SE at Different Time Intervals; An Ex Vivo Study.

    Directory of Open Access Journals (Sweden)

    Behnam Bolhari

    2014-02-01

    Full Text Available To eliminate microorganisms that are responsible for pulpal and periapical infections and to prevent reinfection of the root canal system an effective chemomechanical preparation by irrigants with sustained antimicrobial activity is beneficial. Hereby, we evaluated the residual antibacterial activity of MTAD after canal obturation at different time intervals.A total of 120 human single-canalled anterior teeth were selected. The root canals were instrumented to a standardized apical size. Among all, 90 teeth received final irrigation with MTAD and were divided into three groups according to their obturation materials; i.e. gutta-percha/AH26, Resilon/RealSeal SE and positive controls. All these groups were divided into three 1-, 3- and 6-week time interval subgroups. Thirty teeth as negative control had no final irrigation with MTAD, but were obturated with gutta-percha/AH26 or Resilon/RealSealSE. Dentin powder was prepared after 1, 3 and 6 weeks. Dentin powder was exposed to Enterococcus faecalis for 24h and then cultured. Colony Forming Unit (CFU was counted.Residual antimicrobial activity of MTAD in the teeth obturated with guttapercha/AH26 was significantly higher than the teeth obturated with Resilon/RealSeal SE (p<0.001. It also showed a time dependent decrease in MTAD antimicrobial activity for all groups. The highest antimicrobial activity of MTAD was found in the 1-week positive control and 1-week gutta-percha/AH26 specimens. The lowest antimicrobial activity of MTAD was found in 6-week Resilon/RealSeal SE samples and then the negative controls.MTAD had antimicrobial activity even at the sixth week, although it had a time-dependent decrease. Resilon/Epiphany SE significantly decreased antimicrobial activity of MTAD at all time points.

  15. Photoacoustic tomography of ex vivo mouse hearts with myocardial infarction

    Science.gov (United States)

    Holotta, Markus; Grossauer, Harald; Kremser, Christian; Torbica, Pavle; Völkl, Jakob; Degenhart, Gerald; Esterhammer, Regina; Nuster, Robert; Paltauf, Günther; Jaschke, Werner

    2011-03-01

    In the present study, we evaluated the applicability of ex vivo photoacoustic imaging (PAI) on small animal organs. We used photoacoustic tomography (PAT) to visualize infarcted areas within murine hearts and compared these data to other imaging techniques [magnetic resonance imaging (MRI), micro-computed tomography] and histological slices. In order to induce ischemia, an in vivo ligation of the left anterior descending artery was performed on nine wild-type mice. After varying survival periods, the hearts were excised and fixed in formaldehyde. Samples were illuminated with nanosecond laser pulses delivered by a Nd:YAG pumped optical parametric oscillator. Ultrasound detection was achieved using a Mach-Zehnder interferometer (MZI) working as an integrating line detector. The voxel data were computed using a Fourier-domain based reconstruction algorithm, followed by inverse Radon transforms. The results clearly showed the capability of PAI to visualize myocardial infarction and to produce three-dimensional images with a spatial resolution of approximately 120 μm. Regions of affected muscle tissue in PAI corresponded well with the results of MRI and histology. Photoacoustic tomography utilizing a MZI for ultrasound detection allows for imaging of small tissue samples. Due to its high spatial resolution, good soft tissue contrast and comparatively low cost, PAT offers great potentials for imaging.

  16. Effects of Ex Vivo y-Tocopherol on Airway Macrophage ...

    Science.gov (United States)

    Elevated inflammation and altered immune responses are features found in atopic asthmatic airways. Recent studies indicate y-tocopherol (GT) supplementation can suppress airway inflammation in allergic asthma. We studied the effects of in vitro GT supplementation on receptor-mediated phagocytosis and expression of cell surface molecules associated with innate and adaptive immunity on sputum-derived macrophages. Cells from nonsmoking healthy (n = 6)and mild house dust mite-sensitive allergic asthmatics (n =6) were treated ex vivo with GT (300 uM) or saline (control). Phagocytosis of opsonized zymosan A bioparticles (Saccharomyces cerevisiae) and expression of surface molecules associated with innate and adaptive immunity were assessed using flow cytometry. GT caused significantly decreased (p innate and adaptive immune response elements, and atopic status appears to be an important factor. Recent studies on the effects of the fat-soluble steriod hormone vitamins D and E suggest that dietary suplementation with these vitamins may be helpful for the prevention or in the treatment of inflammatory and immune-mediated diseases, including atopic asthma.

  17. Laser welding and syncristallization techniques comparison: "Ex vivo" study.

    Science.gov (United States)

    Fornaini, Carlo; Meleti, Marco; Vescovi, Paolo; Merigo, Elisabetta; Rocca, Jean-Paul

    2013-12-30

    Stabilization of implant abutments through electric impulses at high voltage for a very short time (electrowelding) was developed in the Eighties. In 2009, the same procedure was performed through the use of laser (laser welding) The aim of this study is to compare electrowelding and laser welding for intra-oral implant abutments stabilization on "ex vivo models" (pig jaws). Six bars were welded with two different devices (Nd:YAG laser and Electrowelder) to eighteen titanium implant abutment inserted in three pig jaws. During the welding process, thermal increase was recorded, through the use of k-thermocouples, in the bone close to the implants. The strength of the welded joints was evaluated by a traction test after the removal of the implants. For temperature measurements a descriptive analysis and for traction test "values unpaired t test with Welch's correction" were performed: the significance level was set at Pwelding gives a lower thermal increase than Electrowelding at the bone close to implants (Mean: 1.97 and 5.27); the strength of laser welded joints was higher than that of Electrowelding even if nor statistically significant. (Mean: 184.75 and 168.29) CONCLUSION: Electrowelding seems to have no advantages, in term of thermal elevation and strength, while laser welding may be employed to connect titanium implants for immediate load without risks of thermal damage at surrounding tissues.

  18. Ex vivo thickness measurement of cartilage covering the temporomandibular joint.

    Science.gov (United States)

    Mirahmadi, Fereshteh; Koolstra, Jan Harm; Lobbezoo, Frank; van Lenthe, G Harry; Everts, Vincent

    2017-02-08

    Articular cartilage covers the temporomandibular joint (TMJ) and provides smooth and nearly frictionless articulation while distributing mechanical loads to the subchondral bone. The thickness of the cartilage is considered to be an indicator of the stage of development, maturation, aging, loading history, and disease. The aim of our study was to develop a method for ex vivo assessment of the thickness of the cartilage that covers the TMJ and to compare that with two other existing methods. Eight porcine TMJ condyles were used to measure cartilage thickness. Three different methods were employed: needle penetration, micro-computed tomography (micro-CT), and histology; the latter was considered the gold standard. Histology and micro-CT scanning results showed no significant differences between thicknesses throughout the condyle. Needle penetration produced significantly higher values than histology, in the lateral and anterior regions. All three methods showed the anterior region to be thinner than the other regions. We concluded that overestimated thickness by the needle penetration is caused by the penetration of the needle through the first layer of subchondral bone, in which mineralization is less than in deeper layers. Micro-CT scanning method was found to be a valid method to quantify the thickness of the cartilage, and has the advantage of being non-destructive. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Ex vivo expansion of hematopoietic progenitor cells and mature cells.

    Science.gov (United States)

    McNiece, I; Briddell, R

    2001-01-01

    Hematopoietic cells have the potential for providing benefit in a variety of clinical settings. These include cells for support of patients undergoing high-dose chemotherapy, as a target for replacement gene therapy, and as a source of cells for immunotherapy. The limitation to many of these applications has been the total absolute number of defined target cells. Therefore many investigators have explored methods to culture hematopoietic cells in vitro to increase the numbers of these cells. Studies attempting to expand hematopoietic stem cells, progenitor cells, and mature cells in vitro have become possible over the past decade due to the availability of recombinant growth factors and cell selection technologies. To date, no studies have demonstrated convincing data on the expansion of true stem cells, and so the focus of this review is the expansion of committed progenitor cells and mature cells. A number of clinical studies have been preformed using a variety of culture conditions, and several studies are currently in progress that explore the use of ex vivo expanded cells. These studies will be discussed in this review. There are evolving data that suggest that there are real clinical benefits associated with the use of the expanded cells; however, we are still at the early stages of understanding how to optimally culture different cell populations. The next decade should determine what culture conditions and what cell populations are needed for a range of clinical applications.

  20. Impact of Hydration Media on Ex Vivo Corneal Elasticity Measurements.

    Science.gov (United States)

    Dias, Janice; Ziebarth, Noël M

    2015-09-01

    To determine the effect of hydration media on ex vivo corneal elasticity. Experiments were conducted on 40 porcine eyes retrieved from an abattoir (10 eyes each for phosphate-buffered saline (PBS), balanced salt solution, Optisol, 15% dextran). The epithelium was removed, and the cornea was excised with an intact scleral rim and placed in 20% dextran overnight to restore its physiological thickness. For each hydration media, corneas were evenly divided into two groups: one with an intact scleral rim and the other without. Corneas were mounted onto a custom chamber and immersed in a hydration medium for elasticity testing. Although in each medium, corneal elasticity measurements were performed for 2 hr: at 5-min intervals for the first 30 min and then 15-min intervals for the remaining 90 min. Elasticity testing was performed using nanoindentation with spherical indenters, and Young modulus was calculated using the Hertz model. Thickness measurements were taken before and after elasticity testing. The percentage change in corneal thickness and elasticity was calculated for each hydration media group. Balanced salt solution, PBS, and Optisol showed an increase in thickness and Young moduli for corneas with and without an intact scleral rim. Fifteen percent dextran exhibited a dehydrating effect on corneal thickness and provided stable maintenance of corneal elasticity for both groups. Hydration media affects the stability of corneal thickness and elasticity measurements over time. Fifteen percent dextran was most effective in maintaining corneal hydration and elasticity, followed by Optisol.

  1. 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.

  2. Effective Single Photodynamic Treatment of ex Vivo Onychomycosis Using a Multifunctional Porphyrin Photosensitizer and Green Light

    Science.gov (United States)

    den Hollander, Chelsea; Visser, Jasper; de Haas, Ellen; Incrocci, Luca; Smijs, Threes

    2015-01-01

    Onychomycosis is predominantly caused by the dermatophytes Trichophyton rubrum, Trichophyton mentagrophytes and Trichophyton tonsurans. The main treatment obstacle concerns low nail-plate drug permeability. In vitro antifungal photodynamic treatment (PDT) and nail penetration enhancing effectiveness have been proven for multifunctional photosensitizer 5,10,15-tris(4-N-methylpyridinium)-20-(4-(butyramido-methylcysteinyl)-hydroxyphenyl)-[21H,23H]-porphine trichloride (PORTHE). This study investigates single PORTHE green laser/LED PDT of varying degrees of ex vivo onychomycoses in a human nail model. T. mentagrophytes, T. rubrum, T. tonsurans onychomycoses were ex vivo induced on nail pieces at 28 °C (normal air) and 37 °C (6.4% CO2) during 3 to 35 days and PDTs applied to the 37 °C infections. All dermatophytes showed increasingly nail plate invasion at 37 °C between 7 and 35 days; arthroconidia were observed after 35 days for T. mentagrophytes and T. tonsurans. Using 81 J/cm2 (532 nm) 7-day T. mentagrophytes onychomycoses were cured (92%) with 80 µM PORTHE (pH 8) after 24 h propylene glycol (PG, 40%) pre-treatment and 35-day onychomycoses (52%–67%) with 24 h PORTHE (40–80 µM)/40% PG treatment (pH 5). 28 J/cm2 LED light (525 ± 37 nm) improved cure rates to 72%, 83% and 73% for, respectively, T. mentagrophytus, T. rubrum and T. tonsurans 35-day onychomycoses and to 100% after double PDT. Data indicate PDT relevance for onychomycosis. PMID:29376905

  3. The effect of different root canal medicaments on the elimination of Enterococcus faecalis ex vivo

    Science.gov (United States)

    Dammaschke, Till; Jung, Nina; Harks, Inga; Schafer, Edgar

    2013-01-01

    Objective: The aim of this study was to evaluate the antimicrobial effect of chlorhexidine gel (CHX-G) 2%, chlorhexidine powder (CHX-P) 1%, povidone-iodine (PVP-I), polyhexanide and camphorated-and-mentholated chlorophenol (ChKM) ex vivo. Materials and Methods: For every medicament group 10 root segments (15 mm long) of extracted human teeth were prepared to ISO-size 45 and sterilized (n = 50). The root segments were then inoculated with Enterococcus faecalis and aerobically incubated at 37°C. After 1 week, ten root canals were filled with one of the medicaments, respectively and aerobically incubated at 37°C for another week. Ten teeth served as positive controls and were filled with sterile saline solution. After 7 days, the medicaments were inactivated and all root canals were instrumented to ISO-size 50. The obtained dentin samples were dispersed in Ringer solution followed by the preparation of serial dilutions. 10 μl per sample were applied to an agar plate and incubated at 37°C for 48 h. The colony forming units were counted and the reduction factors (RFs) were calculated and statistically analyzed. Results: Compared with the positive controls all medicaments exhibited an antibacterial effect against E. faecalis. The RFs for CHX-G, CHX-P and ChKM were significantly higher compared to PVP-I and polyhexanide (P faecalis from all dentin samples. Conclusions: Within the limitations of this ex vivo investigation, 2% CHX-G and CHX-P were as effective as ChKM against E. faecalis. Thus, when choosing a root canal medicament the better biocompatibility of CHX compared with ChKM should be taken in consideration. PMID:24932119

  4. Morphological and cytofluorimetric analysis of adult mesenchymal stem cells expanded ex vivo from periodontal ligament.

    Science.gov (United States)

    Trubiani, O; Di Primio, R; Traini, T; Pizzicannella, J; Scarano, A; Piattelli, A; Caputi, S

    2005-01-01

    Many adult tissues contain a population of stem cells that have the ability of regeneration after trauma, disease or aging. Recently, there has been great interest in mesenchymal stem cells and their roles in maintaining the physiological structure of tissues, and their studies have been considered very important and intriguing, after having shown that this cell population can be expanded ex vivo to regenerate tissues not only of the mesenchymal lineage, such as intervertebral disc cartilage, bone, tooth-associated tissue, cardiomyocytes, but also to differentiate into cells derived from other embryonic layers, including neurons. Currently, different efforts have been focused on the identification of odontogenic progenitors from oral tissues. In this study we isolated and characterized a population of homogeneous human mesenchymal stem cells proliferating in culture with an attached well-spread morphology derived from periodontal ligament, a tissue of ectomesenchymal origin, with the ability to form a specialized joint between alveolar bone and tooth. The adherent cells were harvested and expanded ex vivo under specific conditions and analysed by FACScan flow cytometer and morphological analysis was carried out by light, scanning and transmission electron microscopy. Our results displayed highly evident cells with a fibroblast-like morphology and a secretory apparatus, probably indicating that the enhanced function of the secretory apparatus of the mesenchymal stem cells may be associated with the secretion of molecules that are required to survive and proliferate. Moreover, the presence in periodontal ligament of CD90, CD29, CD44,CD166, CD 105, CD13 positive cells, antigens that are also identified as stromal precursors of the bone marrow, indicate that the periodontal ligament may turn out to be a new efficient source of the cells with intrinsic capacity to self-renewal, high ability to proliferate and differentiate, that can be utilized for a new approach to

  5. 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.

  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. Biomechanical characterization of keratoconus corneas ex vivo with Brillouin microscopy.

    Science.gov (United States)

    Scarcelli, Giuliano; Besner, Sebastien; Pineda, Roberto; Yun, Seok Hyun

    2014-06-17

    Loss of corneal strength is a central feature of keratoconus progression. However, it is currently difficult to measure corneal mechanical changes noninvasively. The objective of this study is to evaluate if Brillouin optical microscopy can differentiate the mechanical properties of keratoconic corneas versus healthy corneas ex vivo. We obtained eight tissue samples from healthy donor corneas used in Descemet's stripping endothelial keratoplasty (DSEK) and 10 advanced keratoconic corneas from patients undergoing deep anterior lamellar keratoplasty (DALK). Within 2 hours after surgery, a confocal Brillouin microscope using a monochromatic laser at 532 nm was used to map the Brillouin frequency shifts of the corneas. The mean Brillouin shift in the anterior 200 μm of the keratoconic corneas at the cone was measured to be 7.99 ± 0.10 GHz, significantly lower than 8.17 ± 0.06 GHz of the healthy corneas (P < 0.001). The Brillouin shift in the keratoconic corneas decreased with depth from the anterior toward posterior regions with a steeper slope than in the healthy corneas (P < 0.001). Within keratoconic corneas, the Brillouin shift in regions away from the apex of the cone was significantly higher than within the cone region (P < 0.001). Brillouin measurements revealed notable differences between healthy and keratoconic corneas. Importantly, Brillouin imaging showed that the mechanical loss is primarily concentrated within the area of the keratoconic cone. Outside the cone, the Brillouin shift was comparable with that of healthy corneas. The results demonstrate the potential of Brillouin microscopy for diagnosis and treatment monitoring of keratoconus. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  8. An ex vivo culture system to study thyroid development.

    Science.gov (United States)

    Delmarcelle, Anne-Sophie; Villacorte, Mylah; Hick, Anne-Christine; Pierreux, Christophe E

    2014-06-06

    The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the "angio-follicular" units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood. This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo. Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR. In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

  9. Ex vivo pathomechanics of the canine Pond-Nuki model.

    Directory of Open Access Journals (Sweden)

    Antonio Pozzi

    Full Text Available BACKGROUND: Transection of the canine cranial cruciate ligament (CCL is a well-established osteoarthritis (OA model. The effect of CCL loss on contact pressure and joint alignment has not been quantified for stifle loading in standing. The purposes of the study were to measure femorotibial contact areas and stresses and joint alignment following transection of the CCL in an ex vivo model. We hypothesized that transection of the CCL would lead to abnormal kinematics, as well as alterations in contact mechanics of the femorotibial joint. METHODOLOGY/PRINCIPAL FINDINGS: Eight canine hindlimbs were tested in a servo-hydraulic materials testing machine using a custom made femoral jig. Contact area and pressure measurements, and femorotibial rotations and translations were measured in the normal and the CCL-deficient stifle in both standing and deep flexion angles. We found that at standing angle, transection of the CCL caused cranial translation and internal rotation of the tibia with a concurrent caudal shift of the contact area, an increase in peak pressure and a decrease in contact area. These changes were not noted in deep flexion. At standing, loss of CCL caused a redistribution of the joint pressure, with the caudal region of the compartment being overloaded and the rest of the joint being underloaded. CONCLUSION: In the Pond-Nuki model alterations in joint alignment are correlated with shifting of the contact points to infrequently loaded areas of the tibial plateau. The results of this study suggest that this cadaveric Pond-Nuki model simulates the biomechanical changes previously reported in the in-vivo Pond-Nuki model.

  10. Activity of taurolidine gels on ex vivo periodontal biofilm.

    Science.gov (United States)

    Pirracchio, Luca; Joos, Aline; Luder, Nina; Sculean, Anton; Eick, Sigrun

    2017-12-14

    The purpose of this study is to evaluate the activity of two different taurolidine (TAU) gels in comparison with a 0.2% chlorhexidine (CHX) gel on an ex vivo subgingival biofilm. Subgingival including supragingival biofilm samples from periodontitis patients were cultured for 10 days, before TAU 1% and TAU 3% gels and CHX gel were applied for 10 min and thereafter diluted with nutrient media to 10% for 50 min. One third of the samples were analyzed for bacterial counts, biofilm quantity, and biofilm metabolic activity. In the two other thirds, 90% of the nutrient media were replaced and biofilms were incubated for 23 h. The second third was analyzed in the same way as before. In the third part, patients' microorganisms were added again and incubated for additional 24 h to allow reformation of biofilm before proceeding to analysis. Decrease of bacterial counts in biofilms was highest following application of TAU 3% after 60 min (0.87 log10 cfu, corresponding 86.5%), 24 and 48 h (reformation of biofilms), respectively. All antimicrobials reduced biofilm quantity after 24 h (each p activity in biofilms was decreased at 60 min (each p gels, while the activity of the reformed biofilm was lower after application of all evaluated antimicrobials (each p activity of taurolidine gels clearly depends on its taurolidine concentration. A high concentrated taurolidine gel is equally active or even superior to 0.2% chlorhexidine gel. However, the activity of antimicrobials is limited in a complex established biofilm and underlines the pivotal role of mechanical biofilm disruption. Within their limits, the data suggest that TAU 3% gel might represent a potential alternative to 0.2% chlorhexidine gel.

  11. Cysteinyl leukotrienes mediate histamine hypersensitivity ex vivo by increasing histamine receptor numbers

    NARCIS (Netherlands)

    Pynaert, G.; Grooten, J.; van Deventer, S. J.; Peppelenbosch, M. P.

    1999-01-01

    Hyperresponsiveness to histamine is a key feature of a variety of pathological conditions, including bronchial asthma, food allergy, colitis ulcerosa, and topical allergic disorders. Cells isolated from hyperresponsive individuals do not display exaggerated histamine responses ex vivo and thus the

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  13. Possibility of ex vivo animal training model for colorectal endoscopic submucosal dissection.

    Science.gov (United States)

    Yoshida, Naohisa; Yagi, Nobuaki; Inada, Yutaka; Kugai, Munehiro; Kamada, Kazuhiro; Katada, Kazuhiro; Uchiyama, Kazuhiko; Ishikawa, Takeshi; Takagi, Tomohisa; Handa, Osamu; Konishi, Hideyuki; Kokura, Satoshi; Inoue, Ken; Wakabayashi, Naoki; Abe, Yasuhisa; Yanagisawa, Akio; Naito, Yuji

    2013-01-01

    Colorectal endoscopic submucosal dissection (ESD) has not been standardized due to technical difficulties and requires extensive training for reliability. Ex vivo animal model is convenient, but has no blood flow. The objective of this study is to evaluate the characteristics of various ex vivo animal models including a blood flow model for colorectal ESD training and the usefulness of practicing endoscopic hemostasis and closure using an animal model. Harvested porcine cecum, rectum, and stomach and bovine cecum and rectum were analyzed regarding ease of mucosal injection, degree of submucosal elevation, and status of the proper muscle layer. Ex vivo animal model with blood flow was made using the bovine cecum. The vessel around the cecum was detached, and red ink was injected. Endoscopic hemostasis for perioperative hemorrhage and endoscopic closure for perforation were performed in this model. Mucosal injection was easily performed in the bovine cecum and rectum. Submucosal elevation was low in the bovine cecum, while the proper muscle layer was not tight in the porcine rectum and bovine cecum. Endoscopic hemostasis were accomplished in six (60 %) out of ten procedures of the ex vivo blood flow model. In two non-experts, the completion rates of endoscopic closure were 40 and 60 % in the first five procedures. These rates became 100 % in the last five procedures. We have evaluated the characteristics of various ex vivo animal models and shown the possibility of training for endoscopic hemostasis and endoscopic closure in the ex vivo animal model.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-11-15

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

  15. Reproducibility and Variation of Diffusion Measures in the Squirrel Monkey Brain, In Vivo and Ex Vivo

    Science.gov (United States)

    Schilling, Kurt; Gao, Yurui; Stepniewska, Iwona; Choe, Ann S; Landman, Bennett A; Anderson, Adam W

    2016-01-01

    Purpose Animal models are needed to better understand the relationship between diffusion MRI (dMRI) and the underlying tissue microstructure. One promising model for validation studies is the common squirrel monkey, Saimiri sciureus. This study aims to determine (1) the reproducibility of in vivo diffusion measures both within and between subjects; (2) the agreement between in vivo and ex vivo data acquired from the same specimen and (3) normal diffusion values and their variation across brain regions. Methods Data were acquired from three healthy squirrel monkeys, each imaged twice in vivo and once ex vivo. Reproducibility of fractional anisotropy (FA), mean diffusivity (MD), and principal eigenvector (PEV) was assessed, and normal values were determined both in vivo and ex vivo. Results The calculated coefficients of variation (CVs) for both intra-subject and inter-subject MD were below 10% (low variability) while FA had a wider range of CVs, 2–14% intra-subject (moderate variability), and 3–31% inter-subject (high variability). MD in ex vivo tissue was lower than in vivo (30%–50% decrease), while FA values increased in all regions (30–39% increase). The mode of angular differences between in vivo and ex vivo PEVs was 12 degrees. Conclusion This study characterizes the diffusion properties of the squirrel monkey brain and serves as the groundwork for using the squirrel monkey, both in vivo and ex vivo, as a model for diffusion MRI studies. PMID:27587226

  16. Ex Vivo ERG analysis of photoreceptors using an In Vivo ERG system

    Science.gov (United States)

    Vinberg, Frans; Kolesnikov, Alexander V.; Kefalov, Vladimir J.

    2014-01-01

    The Function of the retina and effects of drugs on it can be assessed by recording transretinal voltage across isolated retina that is perfused with physiological medium. However, building ex vivo ERG apparatus requires substantial amount of time, resources and expertise. Here we adapted a commercial in vivo ERG system for transretinal ERG recordings from rod and cone photoreceptors and compared rod and cone signalling between ex vivo and in vivo environments. We found that the rod and cone a- and b-waves recorded with the transretinal ERG adapter and a standard in vivo ERG system are comparable to those obtained from live anesthetized animals. However, ex vivo responses are somewhat slower and their oscillatory potentials are suppressed as compared to those recorded in vivo. We found that rod amplification constant (A) was comparable between ex vivo and in vivo conditions, ∼10 - 30 s-2 depending on the choice of response normalization. We estimate that the A in cones is between 3 and 6 s-2 in ex vivo conditions and by assuming equal A in vivo we arrive to light funnelling factor of 3 for cones in the mouse retina. The ex vivo ERG adapter provides a simple and affordable alternative to designing a custom-built transretinal recordings setup for the study of photoreceptors. Our results provide a roadmap to the rigorous quantitative analysis of rod and cone responses made possible with such a system. PMID:24959652

  17. Experience with the first 50 ex vivo lung perfusions in clinical transplantation.

    Science.gov (United States)

    Cypel, Marcelo; Yeung, Jonathan C; Machuca, Tiago; Chen, Manyin; Singer, Lianne G; Yasufuku, Kazuhiro; de Perrot, Marc; Pierre, Andrew; Waddell, Thomas K; Keshavjee, Shaf

    2012-11-01

    Normothermic ex vivo lung perfusion is a novel method to evaluate and improve the function of injured donor lungs. We reviewed our experience with 50 consecutive transplants after ex vivo lung perfusion. A retrospective study using prospectively collected data was performed. High-risk brain death donor lungs (defined as Pao(2)/Fio(2) .05). Thirty-day mortality (4% in the ex vivo lung perfusion group and 3.5% in the control group, P = 1.00) and 1-year survival (87% in the ex vivo lung perfusion group and 86% in the control group, P = 1.00) were similar in both groups. Transplantation of high-risk donor lungs after 4 to 6 hours of ex vivo lung perfusion is safe, and outcomes are similar to those of conventional transplants. Ex vivo lung perfusion improved our center use of donor lungs, accounting for 20% of our current lung transplant activity. Copyright © 2012 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.

  18. The Human Intestinal Microbiome: A New Frontier of Human Biology

    OpenAIRE

    Hattori, Masahira; Taylor, Todd D.

    2009-01-01

    To analyze the vast number and variety of microorganisms inhabiting the human intestine, emerging metagenomic technologies are extremely powerful. The intestinal microbes are taxonomically complex and constitute an ecologically dynamic community (microbiota) that has long been believed to possess a strong impact on human physiology. Furthermore, they are heavily involved in the maturation and proliferation of human intestinal cells, helping to maintain their homeostasis and can be causative o...

  19. Ex Vivo Growth of Bioengineered Ligaments and Other Tissues

    Science.gov (United States)

    Altman, Gregory; Kaplan, David L.; Martin, Ivan; Vunjak-Novakovic, Gordana

    2005-01-01

    A method of growing bioengineered tissues for use in surgical replacement of damaged anterior cruciate ligaments has been invented. An anterior cruciate ligament is one of two ligaments (the other being the posterior cruciate ligament) that cross in the middle of a knee joint and act to prevent the bones in the knee from sliding forward and backward relative to each other. Anterior cruciate ligaments are frequently torn in sports injuries and traffic accidents, resulting in pain and severe limitations on mobility. By making it possible to grow replacement anterior cruciate ligaments that structurally and functionally resemble natural ones more closely than do totally synthetic replacements, the method could create new opportunities for full or nearly full restoration of functionality in injured knees. The method is also adaptable to the growth of bioengineered replacements for other ligaments (e.g., other knee ligaments as well as those in the hands, wrists, and elbows) and to the production of tissues other than ligaments, including cartilage, bones, muscles, and blood vessels. The method is based on the finding that the histomorphological properties of a bioengineered tissue grown in vitro from pluripotent cells within a matrix are affected by the direct application of mechanical force to the matrix during growth generation. This finding provides important new insights into the relationships among mechanical stress, biochemical and cell-immobilization methods, and cell differentiation, and is applicable to the production of the variety of tissues mentioned above. Moreover, this finding can be generalized to nonmechanical (e.g., chemical and electromagnetic) stimuli that are experienced in vivo by tissues of interest and, hence, the method can be modified to incorporate such stimuli in the ex vivo growth of replacements for the various tissues mentioned above. In this method, a three-dimensional matrix made of a suitable material is seeded with pluripotent stem

  20. Multispectral tissue characterization for intestinal anastomosis optimization

    Science.gov (United States)

    Cha, Jaepyeong; Shademan, Azad; Le, Hanh N. D.; Decker, Ryan; Kim, Peter C. W.; Kang, Jin U.; Krieger, Axel

    2015-10-01

    Intestinal anastomosis is a surgical procedure that restores bowel continuity after surgical resection to treat intestinal malignancy, inflammation, or obstruction. Despite the routine nature of intestinal anastomosis procedures, the rate of complications is high. Standard visual inspection cannot distinguish the tissue subsurface and small changes in spectral characteristics of the tissue, so existing tissue anastomosis techniques that rely on human vision to guide suturing could lead to problems such as bleeding and leakage from suturing sites. We present a proof-of-concept study using a portable multispectral imaging (MSI) platform for tissue characterization and preoperative surgical planning in intestinal anastomosis. The platform is composed of a fiber ring light-guided MSI system coupled with polarizers and image analysis software. The system is tested on ex vivo porcine intestine tissue, and we demonstrate the feasibility of identifying optimal regions for suture placement.

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

    Directory of Open Access Journals (Sweden)

    Michael D. Burkitt

    2017-02-01

    Full Text Available 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.

  3. Application of Ulex europaeus agglutinin I-modified liposomes for oral vaccine: Ex Vivo bioadhesion and in Vivo immunity.

    Science.gov (United States)

    Li, KeXin; Zhao, Xiuli; Xu, Shiyi; Pang, DaHai; Yang, ChunRong; Chen, DaWei

    2011-01-01

    The conjugation of Ulex europaeus agglutinin I (UEAI) onto surface of liposomes has been demonstrated to effectively improve the intestinal absorption of antigen, subsequently induced strong mucosal and systemic immune responses. In this context, we prepared bovine serum albumin (BSA)-encapsulating UEAI-modified liposomes (UEAI-LIP) and unmodified ones (LIP). The specific bioadhesion on mice gastro-intestinal mucosa was studied ex vivo. An important increase of interaction between UEAI-conjugated liposomes and the intestinal segments with Peyer's Patches (PPs) was observed compared with the unconjugated one (p<0.01). However, under the presence of α-L-fucose, which is the reported specific sugar for UEAI, specifically inhibited the activity of these conjugates. The immune-stimulating activity in vivo was studied by measuring immunoglobulin G (IgG) levels in serum and immunoglobulin A (IgA) levels in intestinal mucosal secretions following oral administration of BSA solution, LIP and UEAI-LIP in mice. Results indicate that antigen encapsulated in liposomes, especially the UEAI-modified ones, was favorable for inducing immune response. At 42 d after the first immunization, the highest IgG and IgA antibody levels produced by UEAI-LIP occurred, respectively showing 4.4-fold and 5-fold higher levels compared to those of the groups receiving BSA alone. This data demonstrated high potential of UEAI-modified liposomes for their use as carrier for oral vaccines.

  4. In vivo and ex vivo confocal endomicroscopy of pancreatic cystic lesions: A prospective study.

    Science.gov (United States)

    Krishna, Somashekar G; Modi, Rohan M; Kamboj, Amrit K; Swanson, Benjamin J; Hart, Phil A; Dillhoff, Mary E; Manilchuk, Andrei; Schmidt, Carl R; Conwell, Darwin L

    2017-05-14

    To investigate the reproducibility of the in vivo endoscopic ultrasound (EUS) - guided needle based confocal endomicroscopy (nCLE) image patterns in an ex vivo setting and compare these to surgical histopathology for characterizing pancreatic cystic lesions (PCLs). In a prospective study evaluating EUS-nCLE for evaluation of PCLs, 10 subjects underwent an in vivo nCLE (AQ-Flex nCLE miniprobe; Cellvizio, MaunaKea, Paris, France) during EUS and ex vivo probe based CLE (pCLE) of the PCL (Gastroflex ultrahigh definition probe, Cellvizio) after surgical resection. Biopsies were obtained from ex vivo CLE-imaged areas for comparative histopathology. All subjects received intravenous fluorescein prior to EUS and pancreatic surgery for in vivo and ex vivo CLE imaging respectively. A total of 10 subjects (mean age 53 ± 12 years; 5 female) with a mean PCL size of 34.8 ± 14.3 mm were enrolled. Surgical histopathology confirmed 2 intraductal papillary mucinous neoplasms (IPMNs), 3 mucinous cystic neoplasms (MCNs), 2 cystic neuroendocrine tumors (cystic-NETs), 1 serous cystadenoma (SCA), and 2 squamous lined PCLs. Characteristic in vivo nCLE image patterns included papillary projections for IPMNs, horizon-type epithelial bands for MCNs, nests and trabeculae of cells for cystic-NETs, and a "fern pattern" of vascularity for SCA. Identical image patterns were observed during ex vivo pCLE imaging of the surgically resected PCLs. Both in vivo and ex vivo CLE imaging findings correlated with surgical histopathology. In vivo nCLE patterns are reproducible in ex vivo pCLE for all major neoplastic PCLs. These findings add further support the application of EUS-nCLE as an imaging biomarker in the diagnosis of PCLs.

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

    Science.gov (United States)

    Nogales, Carlos Goes; Ferreira, Marina Beloti; Montemor, Antonio Fernando; Rodrigues, Maria Filomena de Andrade; Lage-Marques, José Luiz; Antoniazzi, João Humberto

    2016-01-01

    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 (pozone 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.

  6. Reovirus as a successful ex vivo purging modality for multiple myeloma.

    Science.gov (United States)

    Thirukkumaran, C M; Shi, Z Q; Luider, J; Kopciuk, K; Bahlis, N; Neri, P; Pho, M; Stewart, D; Mansoor, A; Morris, D G

    2014-01-01

    Autologous stem cell rescue (ASCT) following high-dose myeloablative chemotherapy is considered to be a therapeutic option for many multiple myeloma (MM) patients; however relapse post ASCT presents a major challenge. The oncolytic potential of reovirus has been previously demonstrated and is currently undergoing phase I monotherapy clinical trials for MM and phase II/III clinical trials for solid tumors. Here we tested the hypothesis that reovirus can successfully purge MM in a murine model that partially recapitulates human MM. RPMI 8226, MM1S, H929 and U266 human myeloma cell lines were exposed to reovirus and oncolysis was assessed. Apheresis product admixed with MM cells was purged with live reovirus (LV) or dead virus (DV) and purging efficacy was monitored via flow cytometry, reverse transcribed-PCR (RT-PCR) and disease relapse in non obese diabetic/severe combined immune deficient (NOD/SCID) mice. Significant LV purging was seen with MM1S, H929 and U266 and the complete ex vivo purging achieved with RPMI 8226 was confirmed by flow cytometry, RT-PCR and absence of disease relapse in vivo. Mice that received LV-purged autografts exhibited 100% survival in comparison to mice that received DV-purged controls. Reovirus's unique ability to kill MM while sparing hematopoietic stem cells places it as an attractive purging agent for MM during ASCT.

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

    Science.gov (United States)

    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 (pozone 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

  8. Establishment of an ex vivo pulpitis model by co-culturing immortalized dental pulp cells and macrophages.

    Science.gov (United States)

    Yonehiro, J; Yamashita, A; Yoshida, Y; Yoshizawa, S; Ohta, K; Kamata, N; Okihara, T; Nishimura, F

    2012-12-01

    To establish an ex vivo pulpitis model by co-culturing dental pulp cells with macrophages. As dental pulp cells, immortalized human dental pulp cells, named DP-1, were used, whilst as macrophage cell lines, the differentiated human monocytic cell line, THP-1, was used. In some experiments, primary dental pulp cells were isolated and used to confirm the results obtained in the experiments using immortalized cells. Co-culturing was performed using transwell systems. Inflammatory responses were evaluated by measuring cytokines produced by the cells. Co-culturing both cell types markedly up-regulated inflammatory cytokine production as compared with the cells cultured independently, suggesting that both cell types interact with each other to synergistically produce higher amounts of inflammatory cytokines. Interestingly, both DP-1 and primary dental pulp cells appeared to produce molecules stimulating macrophages to produce tumour necrosis factor-α-. Co-culturing immortalized dental pulp cells and macrophages may be a new ex vivo model for studying the pathophysiology of reversible pulpitis. © 2012 International Endodontic Journal.

  9. Rapid Stereomicroscopic Imaging of HER2 Overexpression in Ex Vivo Breast Tissue Using Topically Applied Silica-Based Gold Nanoshells

    Directory of Open Access Journals (Sweden)

    Lissett R. Bickford

    2012-01-01

    Full Text Available Tumor margin detection for patients undergoing breast conservation surgery primarily occurs postoperatively. Previously, we demonstrated that gold nanoshells rapidly enhance contrast of HER2 overexpression in ex vivo tissue sections. Our ultimate objective, however, is to discern HER2 overexpressing tissue from normal tissue in whole, nonsectioned, specimens to facilitate rapid diagnoses. Here, we use targeted nanoshells to quickly and effectively visualize HER2 receptor expression in intact ex vivo human breast tissue specimens. Punch biopsies of human breast tissue were analyzed after a brief 5-minute incubation with and without HER2-targeted silica-gold nanoshells using two-photon microscopy and stereomicroscopy. Labeling was subsequently verified using reflectance confocal microscopy, darkfield hyperspectral imaging, and immunohistochemistry to confirm levels of HER2 expression. Our results suggest that anti-HER2 nanoshells used in tandem with a near-infrared reflectance confocal microscope and a standard stereomicroscope may potentially be used to discern HER2-overexpressing cancerous tissue from normal tissue in near real time and offer a rapid supplement to current diagnostic techniques.

  10. Granulocyte-Colony Stimulating Factor related pathways tested on an endometrial ex-vivo model.

    Directory of Open Access Journals (Sweden)

    Mona Rahmati

    Full Text Available INTRODUCTION: Recombinant human Granulocyte-Colony Stimulating Factor (rhG-CSF supplementation seems to be a promising innovative therapy in reproductive medicine, used in case of recurrent miscarriage, embryo implantation failure or thin endometrium, although its mechanisms of action remain unknown. Our aim was to identify possible endometrial pathways influenced by rhG-CSF. MATERIALS AND METHODS: Hypothetical molecular interactions regulated by G-CSF were designed through a previous large scale endometrial microarray study. The variation of endometrial expression of selected target genes was confirmed in control and infertile patients. G-CSF supplementation influence on these targets was tested on an endometrial ex-vivo culture. Middle luteal phase endometrial biopsies were cultured on collagen sponge with or without rhG-CSF supplementation during 3 consecutive days. Variations of endometrial mRNA expression for the selected targets were studied by RT-PCR. RESULTS: At the highest dose of rhG-CSF stimulation, the mRNA expression of these selected target genes was significantly increased if compared with their expression without addition of rhG-CSF. The selected targets were G-CSF Receptor (G-CSFR, Integrin alpha-V/beta-3 (ITGB3 implicated in cell migration and embryo implantation, Plasminogen Activator Urokinase Receptor (PLAUR described as interacting with integrins and implicated in cell migration, Thymidine Phosphorylase (TYMP implicated in local angiogenesis, CD40 and its ligand CD40L involved in cell proliferation control. CONCLUSION: RhG-CSF seems able to influence endometrial expressions crucial for implantation process involving endometrial vascular remodelling, local immune modulation and cellular adhesion pathways. These variations observed in an ex-vivo model should be tested in-vivo. The strict indications or counter indication of rhG-CSF supplementation in reproductive field are not yet established, while the safety of its

  11. Evaluation of the Doxycycline Release from AH26 Sealer-Doxycycline Combination: An ex vivo Study.

    Science.gov (United States)

    Ashofteh Yazdi, Kazem; Shokouhinejad, Noushin; Moazeni, Esmaeil; Mirzayi Rad, Sina

    2011-01-01

    The purpose of this ex vivo study was to determine the releasing characteristics and doxycycline dentinal diffusion of AH26 sealer-doxycycline combination from apical 3mm of tooth root and apical foramen. One-hundred and two recently extracted single-rooted human teeth were decoronated and prepared with #3 and #4 Gates-Glidden drills and rotary Mtwo files. Smear layer was removed; all surfaces except for apical 3mm of each root were sealed with two coats of nail polish. To quantify the release and diffusion of the doxycycline at different time intervals (30 min, 48 and 72 h) after root canal obturation, the samples were randomly divided into three groups (n=30; 0.5 h, 48 h, 72 h). To evaluate the release of doxycycline from AH26 sealer-doxycycline combination at six concentrations of antibiotic including 0.5%, 1%, 2%, 5%, 10% and 20%; each experimental group was divided into six equal subgroups (n=5). Root canals were filled with gutta-percha and AH26-doxycycline combinations and then were placed in vials containing 1.25mL of phosphate buffer saline solution (PBS). After 30 min, 48 and 72 h, the amount of doxycycline released from specimens into PBS were determined by measuring the absorbance values using UV spectrophotometry at λ(max)=350 nm. Data were analyzed using two-way ANOVA. The findings of this study revealed that AH26 sealer-doxycycline combination released variable measures of antibiotic at each time interval and in the various concentrations. At 30 min, no statistically significant differences were obtained between the results of subgroups, but at 48 and 72 h these differences were significant (P<0.001). The results also showed that differences between 0.5 h, 48 h and 72 h were significant within subgroups (P<0.01). Under the conditions of this ex vivo study, doxycycline can be released from AH26 sealer-antibiotic combination through 3mm of apical root and apical foramen at 30 min, 48 and 72 h after mixing the sealer with doxycycline at concentrations

  12. The Human Milk Oligosaccharide 2′-Fucosyllactose Quenches Campylobacter jejuni–Induced Inflammation in Human Epithelial Cells HEp-2 and HT-29 and in Mouse Intestinal Mucosa123

    Science.gov (United States)

    Yu, Zhuo-Teng; Nanthakumar, N Nanda

    2016-01-01

    Background: Campylobacter jejuni causes diarrhea worldwide; young children are most susceptible. Binding of virulent C. jejuni to the intestinal mucosa is inhibited ex vivo by α1,2-fucosylated carbohydrate moieties, including human milk oligosaccharides (HMOSs). Objective: The simplest α1,2-fucosylated HMOS structure, 2′-fucosyllactose (2′-FL), can be predominant at ≤5 g/L milk. Although 2′-FL inhibits C. jejuni binding ex vivo and in vivo, the effects of 2′FL on the cell invasion central to C. jejuni pathogenesis have not been tested. Clinical isolates of C. jejuni infect humans, birds, and ferrets, limiting studies on its mammalian pathobiology. Methods: Human epithelial cells HEp-2 and HT-29 infected with the virulent C. jejuni strain 81-176 human isolate were treated with 5 g 2′-FL/L, and the degree of infection and inflammatory response was measured. Four-week-old male wild-type C57BL/6 mice were fed antibiotics to reduce their intestinal microbiota and were inoculated with C. jejuni strain 81-176. The sensitivity of the resulting acute transient enteric infection and immune response to inhibition by 2′-FL ingestion was tested. Results: In HEp-2 and HT-29 cells, 2′-FL attenuated 80% of C. jejuni invasion (P jejuni colonization by 80%, weight loss by 5%, histologic features of intestinal inflammation by 50–70%, and induction of inflammatory signaling molecules of the acute-phase mucosal immune response by 50–60% (P jejuni clinical disease, 2′-FL inhibited pathogenesis and its sequelae. These data strongly support the hypothesis that 2′-FL represents a new class of oral agent for prevention, and potentially for treatment, of specific enteric infectious diseases. PMID:27629573

  13. 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.

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

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    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.

  15. Senolytic drugs target alveolar epithelial cell function and attenuate experimental lung fibrosis ex vivo.

    Science.gov (United States)

    Lehmann, Mareike; Korfei, Martina; Mutze, Kathrin; Klee, Stephan; Skronska-Wasek, Wioletta; Alsafadi, Hani N; Ota, Chiharu; Costa, Rita; Schiller, Herbert B; Lindner, Michael; Wagner, Darcy E; Günther, Andreas; Königshoff, Melanie

    2017-08-01

    Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor prognosis and limited therapeutic options. The incidence of IPF increases with age, and ageing-related mechanisms such as cellular senescence have been proposed as pathogenic drivers. The lung alveolar epithelium represents a major site of tissue injury in IPF and senescence of this cell population is probably detrimental to lung repair. However, the potential pathomechanisms of alveolar epithelial cell senescence and the impact of senolytic drugs on senescent lung cells and fibrosis remain unknown. Here we demonstrate that lung epithelial cells exhibit increased P16 and P21 expression as well as senescence-associated β-galactosidase activity in experimental and human lung fibrosis tissue and primary cells.Primary fibrotic mouse alveolar epithelial type (AT)II cells secreted increased amounts of senescence-associated secretory phenotype (SASP) factors in vitro, as analysed using quantitative PCR, mass spectrometry and ELISA. Importantly, pharmacological clearance of senescent cells by induction of apoptosis in fibrotic ATII cells or ex vivo three-dimensional lung tissue cultures reduced SASP factors and extracellular matrix markers, while increasing alveolar epithelial markers.These data indicate that alveolar epithelial cell senescence contributes to lung fibrosis development and that senolytic drugs may be a viable therapeutic option for IPF. Copyright ©ERS 2017.

  16. Real-time motion compensation for EM bronchoscope tracking with smooth output - ex-vivo validation

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    Reichl, Tobias; Gergel, Ingmar; Menzel, Manuela; Hautmann, Hubert; Wegner, Ingmar; Meinzer, Hans-Peter; Navab, Nassir

    2012-02-01

    Navigated bronchoscopy provides benefits for endoscopists and patients, but accurate tracking information is needed. We present a novel real-time approach for bronchoscope tracking combining electromagnetic (EM) tracking, airway segmentation, and a continuous model of output. We augment a previously published approach by including segmentation information in the tracking optimization instead of image similarity. Thus, the new approach is feasible in real-time. Since the true bronchoscope trajectory is continuous, the output is modeled using splines and the control points are optimized with respect to displacement from EM tracking measurements and spatial relation to segmented airways. Accuracy of the proposed method and its components is evaluated on a ventilated porcine ex-vivo lung with respect to ground truth data acquired from a human expert. We demonstrate the robustness of the output of the proposed method against added artificial noise in the input data. Smoothness in terms of inter-frame distance is shown to remain below 2 mm, even when up to 5 mm of Gaussian noise are added to the input. The approach is shown to be easily extensible to include other measures like image similarity.

  17. Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples

    Science.gov (United States)

    Peters, Sven; Hammer, Martin; Schweitzer, Dietrich

    2011-07-01

    Two-photon excited fluorescence (TPEF) imaging of ocular tissue has recently become a promising tool in ophthalmology for diagnostic and research purposes. The feasibility and the advantages of TPEF imaging, namely deeper tissue penetration and improved high-resolution imaging of microstructures, have been demonstrated lately using human ocular samples. The autofluorescence properties of endogenous fluorophores in ocular fundus tissue are well known from spectrophotometric analysis. But fluorophores, especially when it comes to fluorescence lifetime, typically display a dependence of their fluorescence properties on local environmental parameters. Hence, a more detailed investigation of ocular fundus autofluorescence ideally in vivo is of utmost interest. The aim of this study is to determine space-resolved the stationary and time-resolved fluorescence properties of endogenous fluorophores in ex vivo porcine ocular fundus samples by means of two-photon excited fluorescence spectrum and lifetime imaging microscopy (FSIM/FLIM). By our first results, we characterized the autofluorescence of individual anatomical structures of porcine retina samples excited at 760 nm. The fluorescence properties of almost all investigated retinal layers are relatively homogenous. But as previously unknown, ganglion cell bodies show a significantly shorter fluorescence lifetime compared to the adjacent mueller cells. Since all retinal layers exhibit bi-exponential autofluorescence decays, we were able to achieve a more precise characterization of fluorescence properties of endogenous fluorophores compared to a present in vivo FLIM approach by confocal scanning laser ophthalmoscope (cSLO).

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

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    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.

  19. Ex vivo skin absorption of terpenes from Vicks VapoRub ointment.

    Science.gov (United States)

    Cal, Krzysztof; Sopala, Monika

    2008-08-01

    The pharmaceutical market offers a wide range of inhalant drug products applied on the skin that contain essential oils and/or their isolated compounds, i.e. terpenes. Because there are few data concerning the skin penetration of terpenes, especially from complex carriers, the goal of this study was to determine the ex vivo skin absorption kinetics of chosen terpenes, namely eucalyptol, menthol, camphor, alpha-pinene, and beta-pinene, from the product Vicks VapoRub. Human cadaver skin was placed in a flow-through diffusion chamber and the product was applied for 15, 30, and 60 min. After the application time the skin was separated into layers using a tape-stripping technique: three fractions of stratum corneum and epidermis with dermis, and terpenes amounts in the samples were determined by gas-chromatography. The investigated terpenes showed different absorption characteristics related to their physicochemical properties and did not permeate through the skin into the acceptor fluid. Eucalyptol had the largest total accumulation in the stratum corneum and in the epidermis with dermis, while alpha-pinene penetrated into the skin in the smallest amount. The short time in which saturation of the stratum corneum with the terpenes occurred and the high accumulation of most of the investigated terpenes in the skin layers proved that these compounds easily penetrate and permeate the stratum corneum and that in vivo they may easily penetrate into the blood circulation.

  20. 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.

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

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    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.

  2. 4D optical coherence tomography of aortic valve dynamics in a murine mouse model ex vivo

    Science.gov (United States)

    Schnabel, Christian; Jannasch, Anett; Faak, Saskia; Waldow, Thomas; Koch, Edmund

    2015-07-01

    The heart and its mechanical components, especially the heart valves and leaflets, are under enormous strain during lifetime. Like all highly stressed materials, also these biological components undergo fatigue and signs of wear, which impinge upon cardiac output and in the end on health and living comfort of affected patients. Thereby pathophysiological changes of the aortic valve leading to calcific aortic valve stenosis (AVS) as most frequent heart valve disease in humans are of particular interest. The knowledge about changes of the dynamic behavior during the course of this disease and the possibility of early stage diagnosis could lead to the development of new treatment strategies and drug-based options of prevention or therapy. ApoE-/- mice as established model of AVS versus wildtype mice were introduced in an ex vivo artificially stimulated heart model. 4D optical coherence tomography (OCT) in combination with high-speed video microscopy were applied to characterize dynamic behavior of the murine aortic valve and to characterize dynamic properties during artificial stimulation. OCT and high-speed video microscopy with high spatial and temporal resolution represent promising tools for the investigation of dynamic behavior and their changes in calcific aortic stenosis disease models in mice.

  3. In and ex-vivo Myocardial Tissue Temperature Monitoring by Combined Infrared and Ultrasonic Thermometries

    Science.gov (United States)

    Engrand, C.; Laux, D.; Ferrandis, J.-Y.; Sinquet, J.-C.; Demaria, R.; Le Clézio, E.

    The success of cardiac surgery essentially depends on tissue preservation during intervention. Consequently a hypothermic cardio-plegia is applied in order to avoid ischemia. However, myocardial temperature is not monitored during operation. The aim of this study is then to find a relevant and simple method for myocardial global temperature estimation in real time using both ultrasounds and infra-red thermography. In order to quantify the sensitivity of ultrasonic velocity to temperature, a 2.25 MHz ultrasonic probe was used for ex-vivo tests. Pig myocards (n=25) were placed in a thermostatically-controlled water bath and measurements of the ultrasound velocity were realized from 10 to 30 ˚C. The results of this study indicate that the specificity and sensitivity of the ultrasonic echo delay induced by the modification of temperature can be exploited for in-depth thermometry. In parallel, for TIR experiments, a bolometer was used to detect the myocardium surface thermal evolution during in-vivo pig heart experiments. Hypothermic cardioplegic solutions were injected and infra-red surface imaging was performed during one hour. In the near futur, the correlation of the ultrasound and the infrared measurements should allow the real time estimation of the global temperature of the heart. The final objective being to realize in vivo measurements on human hearts, this information may have a very high importance in terms of per-operation inspection as well as decision making process during medical interventions.

  4. Ex vivo Stimulation of Lymphocytes with IL-10 Mimics Sepsis-Induced Intrinsic T-Cell Alterations.

    Science.gov (United States)

    Poujol, Fanny; Monneret, Guillaume; Gallet-Gorius, Emmanuelle; Pachot, Alexandre; Textoris, Julien; Venet, Fabienne

    2018-02-01

    Profound T-cell alterations are observed in septic patients in association with increased risk of secondary infection and mortality. The pathophysiological mechanisms leading to such dysfunctions are not completely understood and direct and indirect mechanisms have been described. In this study we evaluated whether ex vivo stimulation of lymphocytes with IL-10, an immunosuppressive cytokine released at the systemic level during sepsis, could mimic sepsis-induced intrinsic T-cell alterations. We showed that recombinant human IL-10 priming of T cells altered their proliferative response to anti-CD2/CD3/CD28 antibody-coated beads and PHA stimulations, in a dose-dependent manner independently of accessory cells. This priming also significantly decreased T-cell secretion of IL-2 and IFNγ following stimulation. Furthermore, we demonstrated that IL-10 reduction of T-cell functionality was associated with increased FOXP3 expression in CD4+CD25+CD127- regulatory T cells as observed in sepsis. Finally, we found that blocking the increased IL-10 concentration in plasma from septic shock patients increased the proliferative response of responding T cells from healthy controls. We describe here an ex vivo model recapitulating features of sepsis-induced intrinsic T-cell alterations. This should help, in further studies, to decipher the pathophysiological mechanisms of T-cell alterations induced after septic shock.

  5. Effects of Maté Tea Intake on ex Vivo LDL Peroxidation Induced by Three Different Pathways

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    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

  6. Formulation, functional evaluation and ex vivo performance of thermoresponsive soluble gels - A platform for therapeutic delivery to mucosal sinus tissue.

    Science.gov (United States)

    Pandey, Preeti; Cabot, Peter J; Wallwork, Benjamin; Panizza, Benedict J; Parekh, Harendra S

    2017-01-01

    Mucoadhesive in situ gelling systems (soluble gels) have received considerable attention recently as effective stimuli-transforming vectors for a range of drug delivery applications. Considering this fact, the present work involves systematic formulation development, optimization, functional evaluation and ex vivo performance of thermosensitive soluble gels containing dexamethasone 21-phosphate disodium salt (DXN) as the model therapeutic. A series of in situ gel-forming systems comprising the thermoreversible polymer poloxamer-407 (P407), along with hydroxypropyl methyl cellulose (HPMC) and chitosan were first formulated. The optimized soluble gels were evaluated for their potential to promote greater retention at the mucosal surface, for improved therapeutic efficacy, compared to existing solution/suspension-based steroid formulations used clinically. Optimized soluble gels demonstrated a desirable gelation temperature with Newtonian fluid behaviour observed under storage conditions (4-8°C), and pseudoplastic fluid behaviour recorded at nasal cavity/sinus temperature (≈34°C). The in vitro characterization of formulations including rheological evaluation, textural analysis and mucoadhesion studies of the gel form were investigated. Considerable improvement in mechanical properties and mucoadhesion was observed with incorporation of HPMC and chitosan into the gelling systems. The lead poloxamer-based soluble gels, PGHC4 and PGHC7, which were carried through to ex vivo permeation studies displayed extended drug release profiles in conditions mimicking the human nasal cavity, which indicates their suitability for treating a range of conditions affecting the nasal cavity/sinuses. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Polymeric nanoparticles based topical gel of poorly soluble drug: Formulation, ex-vivo and in vivo evaluation

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    Mohammed Elmowafy

    2017-06-01

    Full Text Available The study was conducted to evaluate the potential application of nanocapsules and nanospheres as topical drug delivery systems for indomethacin (as model drug. Both were prepared by nanoprecipitation using poly (ɛ-caprolactone and hydroxypropyl β-cyclodextrin polymers and evaluated for morphology, particle size, zeta potential, EE% and in vitro drug release then incorporated in methylcellulose and Carbopol 940 gel bases and evaluated for in vitro release. The selected formulations and market product were evaluated for ex vivo human skin permeation and anti-inflammatory and analgesic activities by paw edema and hot plate methods respectively. Results showed that nanocapsules had slight higher EE% and larger particle sizes than nanospheres. In vitro release and zeta potential were nearly similar. Methylcellulose exhibited higher in vitro release than Carbopol 940 after 3 h (except NS2. Ex vivo skin permeation studies showed significant higher cumulative amount of IND (and flux from NC1/MC and NS1/MC (1573.06 ± 14.23 µg/cm2 and 1452.24 ± 23.18 µg/cm2 respectively than market product. They also showed enhancement ratio and permeability coefficient rate of ∼1.5 and ∼2 respectively. NC1/MC proved to be significantly higher pharmacodynamic effects than market product. All results showed that NC1/MC could provide a promising formula as a topical delivery of indomethacin.

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

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    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.

  9. Bioactive protein fraction DLBS1033 containing lumbrokinase isolated from Lumbricus rubellus: ex vivo, in vivo, and pharmaceutic studies.

    Science.gov (United States)

    Tjandrawinata, Raymond R; Trisina, Jessica; Rahayu, Puji; Prasetya, Lorentius Agung; Hanafiah, Aang; Rachmawati, Heni

    2014-01-01

    DLBS1033 is a bioactive protein fraction isolated from Lumbricus rubellus that tends to be unstable when exposed to the gastrointestinal environment. Accordingly, appropriate pharmaceutical development is needed to maximize absorption of the protein fraction in the gastrointestinal tract. In vitro, ex vivo, and in vivo stability assays were performed to study the stability of the bioactive protein fraction in gastric conditions. The bioactive protein fraction DLBS1033 was found to be unstable at low pH and in gastric fluid. The "enteric coating" formulation showed no leakage in gastric fluid-like medium and possessed a good release profile in simulated intestinal medium. DLBS1033 was absorbed through the small intestine in an intact protein form, confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis. This result confirmed that an enteric coating formula using methacrylic acid copolymer could protect DLBS1033 from the acidic condition of the stomach by preventing the release of DLBS1033 in the stomach, while promoting its release when reaching the intestine. From the blood concentration-versus-time curve, (99m)Tc-DLBS1033 showed a circulation half-life of 70 minutes. This relatively long biological half-life supports its function as a thrombolytic protein. Thus, an enteric delivery system is considered the best approach for DLBS1033 as an oral thrombolytic agent.

  10. Ex Vivo and In Vivo Mice Models to Study Blastocystis spp. Adhesion, Colonization and Pathology: Closer to Proving Koch's Postulates.

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    Sitara S R Ajjampur

    Full Text Available Blastocystis spp. are widely prevalent extra cellular, non-motile anerobic protists that inhabit the gastrointestinal tract. Although Blastocystis spp. have been associated with gastrointestinal symptoms, irritable bowel syndrome and urticaria, their clinical significance has remained controversial. We established an ex vivo mouse explant model to characterize adhesion in the context of tissue architecture and presence of the mucin layer. Using confocal microscopy with tissue whole mounts and two axenic isolates of Blastocystis spp., subtype 7 with notable differences in adhesion to intestinal epithelial cells (IEC, isolate B (ST7-B and isolate H (more adhesive, ST7-H, we showed that adhesion is both isolate dependent and tissue trophic. The more adhesive isolate, ST7-H was found to bind preferentially to the colon tissue than caecum and terminal ileum. Both isolates were also found to have mucinolytic effects. We then adapted a DSS colitis mouse model as a susceptible model to study colonization and acute infection by intra-caecal inoculation of trophic Blastocystis spp.cells. We found that the more adhesive isolate ST7-H was also a better colonizer with more mice shedding parasites and for a longer duration than ST7-B. Adhesion and colonization was also associated with increased virulence as ST7-H infected mice showed greater tissue damage than ST7-B. Both the ex vivo and in vivo models used in this study showed that Blastocystis spp. remain luminal and predominantly associated with mucin. This was further confirmed using colonic loop experiments. We were also successfully able to re-infect a second batch of mice with ST7-H isolates obtained from fecal cultures and demonstrated similar histopathological findings and tissue damage thereby coming closer to proving Koch's postulates for this parasite.

  11. Ex Vivo and In Vivo Mice Models to Study Blastocystis spp. Adhesion, Colonization and Pathology: Closer to Proving Koch's Postulates.

    Science.gov (United States)

    Ajjampur, Sitara S R; Png, Chin Wen; Chia, Wan Ni; Zhang, Yongliang; Tan, Kevin S W

    2016-01-01

    Blastocystis spp. are widely prevalent extra cellular, non-motile anerobic protists that inhabit the gastrointestinal tract. Although Blastocystis spp. have been associated with gastrointestinal symptoms, irritable bowel syndrome and urticaria, their clinical significance has remained controversial. We established an ex vivo mouse explant model to characterize adhesion in the context of tissue architecture and presence of the mucin layer. Using confocal microscopy with tissue whole mounts and two axenic isolates of Blastocystis spp., subtype 7 with notable differences in adhesion to intestinal epithelial cells (IEC), isolate B (ST7-B) and isolate H (more adhesive, ST7-H), we showed that adhesion is both isolate dependent and tissue trophic. The more adhesive isolate, ST7-H was found to bind preferentially to the colon tissue than caecum and terminal ileum. Both isolates were also found to have mucinolytic effects. We then adapted a DSS colitis mouse model as a susceptible model to study colonization and acute infection by intra-caecal inoculation of trophic Blastocystis spp.cells. We found that the more adhesive isolate ST7-H was also a better colonizer with more mice shedding parasites and for a longer duration than ST7-B. Adhesion and colonization was also associated with increased virulence as ST7-H infected mice showed greater tissue damage than ST7-B. Both the ex vivo and in vivo models used in this study showed that Blastocystis spp. remain luminal and predominantly associated with mucin. This was further confirmed using colonic loop experiments. We were also successfully able to re-infect a second batch of mice with ST7-H isolates obtained from fecal cultures and demonstrated similar histopathological findings and tissue damage thereby coming closer to proving Koch's postulates for this parasite.

  12. Effect of flunixin meglumine and firocoxib on ex vivo cyclooxygenase activity in horses undergoing elective surgery.

    Science.gov (United States)

    Duz, Marco; Parkin, Tim D; Cullander, Rose M; Marshall, John F

    2015-03-01

    To evaluate ex vivo cyclooxygenase (COX) inhibition and compare in vitro and ex vivo COX-1 inhibition by flunixin meglumine and firocoxib in horses. 4 healthy horses for in vitro experiments and 12 healthy horses (6 males and 6 females; 5 Thoroughbreds, 5 Warmbloods, and 2 ponies) undergoing elective surgery for ex vivo experiments. 12 horses received flunixin meglumine (1.1 mg/kg, IV, q 12 h) or firocoxib (0.09 mg/kg, IV, q 24 h). Blood samples were collected before (baseline) and 2 and 24 hours after NSAID administration. Prostanoids (thromboxane B2, prostaglandin E2, and prostaglandin E metabolites) served as indicators of COX activity, and serum drug concentrations were measured by use of high-performance liquid chromatography. An in vitro coagulation-induced thromboxane B2 assay was used to calculate drug concentration-COX-1 inhibition curves. Effect of time and treatment on COX activity was determined. Agreement between in vitro and ex vivo measurement of COX activity was assessed with Bland-Altman analysis. At 2 and 24 hours after NSAID administration, COX-1 activity was reduced, compared with baseline activity, for the flunixin meglumine group only and relative COX-1 activity was significantly greater for the firocoxib group, compared with that for the flunixin meglumine group. There was no significant change in COX-2 activity after surgery for either group. Bland-Altman analysis revealed poor agreement between in vitro and ex vivo measurement of COX-1 activity. Compared with flunixin meglumine, firocoxib had COX-1-sparing effects ex vivo in equine patients that underwent elective surgery.

  13. Ex-vivo imaging of blood and lymphatic vessels in conjunctiva using optical coherence tomography

    Science.gov (United States)

    Gong, Peijun; Karnowski, Karol; Yu, Paula; An, Dong; Yu, Dao-Yi; Sampson, David D.

    2017-04-01

    Label-free imaging of the blood and lymphatic vessel networks of the conjunctiva of the eye is important in assessing the drainage pathways affected by glaucoma. We utilize the characteristically low signal in optical coherence tomography (OCT) provided by such vessels in ex vivo tissue to characterize their morphology in two and three dimensions. We demonstrate this method on conjunctiva from six porcine eyes, showing the ready visualization of both vessel networks. Such ex vivo characterization is a necessary precursor for future in vivo studies directed towards improving glaucoma surgery.

  14. Comparison of In-Vivo and Ex-Vivo Viscoelastic Behavior of the Spinal Cord.

    Science.gov (United States)

    Ramo, Nicole L; Shetye, Snehal S; Streijger, Femke; Lee, Jae H T; Troyer, Kevin L; Kwon, Brian K; Cripton, Peter; Puttlitz, Christian M

    2017-12-26

    Despite efforts to simulate the in-vivo environment, post-mortem degradation and lack of blood perfusion complicate the use of ex-vivo derived material models in computational studies of spinal cord injury. In order to quantify the mechanical changes that manifest ex-vivo, the viscoelastic behavior of in-vivo and ex-vivo porcine spinal cord samples were compared. Stress-relaxation data from each condition were fit to a non-linear viscoelastic model using a novel characterization technique called the direct fit method. To validate the presented material models, the parameters obtained for each condition were used to predict the respective dynamic cyclic response. Both ex-vivo and in-vivo samples displayed non-linear viscoelastic behavior with a significant increase in relaxation with applied strain. However, at all three strain magnitudes compared, ex-vivo samples experienced a higher stress and greater relaxation than in-vivo samples. Significant differences between model parameters also showed distinct relaxation behaviors, especially in non-linear relaxation modulus components associated with the short-term response (0.1 to 1 second). The results of this study underscore the necessity of utilizing material models developed from in-vivo experimental data for studies of spinal cord injury, where the time-dependent properties are critical. The ability of each material model to accurately predict the dynamic cyclic response validates the presented methodology and supports the use of the in-vivo model in future high-resolution finite element modeling efforts. Neural tissues (such as the brain and spinal cord) display time-dependent, or viscoelastic, mechanical behavior making it difficult to model how they respond to various loading conditions, including injury. Methods that aim to characterize the behavior of the spinal cord almost exclusively use ex-vivo cadaveric or animal samples, despite evidence that time after death affects the behavior compared to that in a

  15. Effects of Digested Onion Extracts on Intestinal Gene Expression: An Interspecies Comparison Using Different Intestine Models.

    Science.gov (United States)

    de Wit, Nicole J W; Hulst, Marcel; Govers, Coen; van der Meulen, Jan; van Hoef, Angeline; Stoopen, Geert; Hamers, Astrid; Hoekman, Arjan; de Vos, Ric; Bovee, Toine F H; Smits, Mari; Mes, Jurriaan J; Hendriksen, Peter J M

    2016-01-01

    Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, we explored the applicability of in vitro (human Caco-2 cells) and ex vivo intestine models (rat precision cut intestine slices and the pig in-situ small intestinal segment perfusion (SISP) technique) to study the effect of food compounds. In vitro digested yellow (YOd) and white onion extracts (WOd) were used as model food compounds and transcriptomics was applied to obtain more insight into which extent mode of actions depend on the model. The three intestine models shared 9,140 genes which were used to compare the responses to digested onions between the models. Unsupervised clustering analysis showed that genes up- or down-regulated by WOd in human Caco-2 cells and rat intestine slices were similarly regulated by YOd, indicating comparable modes of action for the two onion species. Highly variable responses to onion were found in the pig SISP model. By focussing only on genes with significant differential expression, in combination with a fold change > 1.5, 15 genes showed similar onion-induced expression in human Caco-2 cells and rat intestine slices and 2 overlapping genes were found between the human Caco-2 and pig SISP model. Pathway analyses revealed that mainly processes related to oxidative stress, and especially the Keap1-Nrf2 pathway, were affected by onions in all three models. Our data fit with previous in vivo studies showing that the beneficial effects of onions are mostly linked to their antioxidant properties. Taken together, our data indicate that each of the in vitro and ex vivo intestine models used in this study, taking into account their limitations, can be used to determine modes of action of nutritional compounds and can thereby reduce the number of animals used in conventional nutritional intervention studies.

  16. Characterization of ex vivo cultured limbal, conjunctival, and oral mucosal cells: A comparative study with implications in transplantation medicine

    Science.gov (United States)

    Dhamodaran, Kamesh; Subramani, Murali; Jeyabalan, Nallathambi; Ponnalagu, Murugeswari; Chevour, Priyanka; Shetty, Reshma; Matalia, Himanshu; Shetty, Rohit; Prince, Sabina Evan

    2015-01-01

    Purpose Limbal epithelial stem cell deficiency is caused by exposure of the cornea to thermal, chemical, or radiation burns or by diseases (aniridia and Stevens-Johnson syndrome). Autologous cell transplantation is a widely used therapeutic modality for restoring the corneal surface in such pathological conditions. Ex vivo cultured limbal, conjunctival, and oral biopsies have been widely used to reconstruct the corneal surface with variable outcomes. Culture characterization of the ex vivo cultured cells would provide insight and clues into the underlying signaling mechanisms that would aid in determining the probable transplantation outcome. Comparison of the vital proteins and genes among the three ex vivo cultured tissues has implications in clinical practice. To address this issue, we characterized and compared the proliferative and differentiated properties of ex vivo cultured limbal, conjunctival, and oral biopsies used for cell-based therapy for corneal surface restoration. Methods Limbal, conjunctival, and oral biopsies were collected with informed patient consent. Explant cultures were established on the denuded human amniotic membrane with corneal lineage differentiation medium. The day 14 cultures were characterized for epithelial and corneal lineage-specific markers using reverse transcription (RT)–PCR for cytokeratin 3, 4, 12, 13, 15, connexin 43, vimentin, p63α, and ABCG2 markers. mRNA expression was estimated in day 14 cultures with real-time quantitative real time (qRT)-PCR for pluripotency markers (OCT4, SOX2, NANOG), putative corneal stem cell markers (ABCG2 and p63α), proliferation markers (cyclin d1, Ki-67, PCNA, and CDC20), apoptotic markers (BCL2, BAX, caspase 3, and caspase 9), Notch signaling pathway markers (Notch1, Jagged1, Hes1, Hes3, Hes5, and Hey1), and autophagic markers (LC3A, LC3B, ATG7, RAB7, LAMP1, and LAMP2). Fluorescence-activated cell sorter profiling was performed for pluripotent markers and putative corneal stem cell

  17. 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.

  18. In vitro and ex vivo infection models help assess the molecular aspects of the interaction of Trichophyton rubrum with the host milieu.

    Science.gov (United States)

    Peres, Nalu Teixeira de Aguiar; Silva, Larissa Gomes da; Santos, Rodrigo da Silva; Jacob, Tiago Rinaldi; Persinoti, Gabriela Felix; Rocha, Lenaldo Branco; Falcão, Juliana Pfrimer; Rossi, Antonio; Martinez-Rossi, Nilce Maria

    2016-05-01

    Dermatophytes are fungal pathogens that cause cutaneous infections such as onychomycosis and athlete's foot in both healthy and immunocompromised patients.Trichophyton rubrum is the most prevalent dermatophyte causing human nail and skin infections worldwide, and because of its anthropophilic nature, animal infection models are limited. The purpose of this work was to compare the expression profile of T. rubrum genes encoding putative virulence factors during growth in ex vivo and in vitro infection models. The efficiency of the ex vivo skin infection model was confirmed by scanning electron microscopy (SEM), which showed that the conidia had produced hyphae that penetrated into the epidermis. Quantitative RT-PCR (qRT-PCR) analysis showed that the expression of some genes is modulated in response to the infection model used, as compared to that observed in cells grown in glucose-containing media. We concluded that ex vivo infection models help assess the molecular aspects of the interaction of T. rubrum with the host milieu, and thus provide insights into the modulation of genes during infection. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Faecalibacterium prausnitzii and human intestinal health

    NARCIS (Netherlands)

    Miquel, S.; Martin, R.; Rossi, O.; Bermudez-Humaran, L.G.; Chatel, J.M.; Sokol, H.; Thomas, M.; Wells, J.M.; Langella, P.

    2013-01-01

    Faecalibacterium prausnitzii is the most abundant bacterium in the human intestinal microbiota of healthy adults, representing more than 5% of the total bacterial population. Over the past five years, an increasing number of studies have clearly described the importance of this highly metabolically

  20. Ex vivo changes in blood glucose levels seldom change blood glucose control algorithm recommendations

    NARCIS (Netherlands)

    de Groene, L.; Harmsen, R. E.; Binnekade, J. M.; Spronk, P. E.; Schultz, M. J.

    2010-01-01

    Background. Hyperglycemia and glycemic variabilities are associated with adverse outcomes in critically ill patients. Blood glucose control with insulin mandates an adequate and precise assessment of blood glucose levels. Blood glucose levels, however, can change ex vivo after sampling. The aim of

  1. A rapid ex vivo tissue model for optimising drug detection and ionisation in MALDI imaging studies.

    Science.gov (United States)

    Huber, K; Aichler, M; Sun, N; Buck, A; Li, Z; Fernandez, I E; Hauck, S M; Zitzelsberger, H; Eickelberg, O; Janssen, K P; Keller, U; Walch, A

    2014-10-01

    The aim of this study was to establish an ex vivo model for a faster optimisation of sample preparation procedures, for example matrix choice, in matrix-assisted laser desorption/ionisation (MALDI) drug imaging studies. The ionisation properties of four drugs, afatinib, erlotinib, irinotecan and pirfenidone, were determined in an ex vivo tissue experiment by spotting decreasing dilution series onto liver sections. Hereby, the drug signals were distinctly detectable using different matrix compounds, which allowed the selection of the optimal matrix for each drug. The analysis of afatinib and erlotinib yielded high drug signals with α-cyano-4-hydroxycinnamic acid matrix, whereas 2,3-dihydroxybenzoic acid was identified as optimal matrix for irinotecan and pirfenidone detection. Our method was validated by a MALDI drug imaging approach of in vivo treated mouse tissue resulting in corresponding findings, indicating the spotting method as an appropriate approach to determine the matrix of choice. The present study shows the accordance between the detection of ex vivo spotted drugs and in vivo administered drugs by MALDI-TOF and MALDI-FT-ICR imaging, which has not been demonstrated so far. Our data suggest the ex vivo tissue spotting method as an easy and reliable model to optimise MALDI imaging measurements and to predict drug detection in tissue sections derived from treated mice prior to the recruitment of laboratory animals, which helps to save animals, time and costs.

  2. Ex vivo trypanostatic effect of stem-bark extracts of Securidaca ...

    African Journals Online (AJOL)

    User

    2016-12-21

    Dec 21, 2016 ... Current treatment of trypanosomosis in sub-Saharan Africa is associated with widespread inefficiency. There is therefore the need to find more effective drugs against the disease from promising traditional medicinal herbs. This work is aimed at evaluating ex vivo anti-trypanosomal effect of stem-bark ...

  3. The performance of a hydrogel nucleus pulposus prosthesis in an ex vivo canine model

    NARCIS (Netherlands)

    Bergknut, N.|info:eu-repo/dai/nl/314418059; Smolders, L.A.; Koole, L.H.; Voorhout, G.|info:eu-repo/dai/nl/073903329; Hagman, R.E.; Lagerstedt, A.S.; Saralidze, K.; Hazewinkel, H.A.W.|info:eu-repo/dai/nl/070975760

    2010-01-01

    Biomaterials. 2010 Sep;31(26):6782-8. Epub 2010 Jun 12. The performance of a hydrogel nucleus pulposus prosthesis in an ex vivo canine model. Bergknut N, Smolders LA, Koole LH, Voorhout G, Hagman RE, Lagerstedt AS, Saralidze K, Hazewinkel HA, van der Veen AJ, Meij BP. Department of Clinical Sciences

  4. 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

    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

  5. Ex-vivo evaluation of crab shell chitosan as absorption enhancer in ...

    African Journals Online (AJOL)

    This study was aimed at evaluating crab shell chitosan as absorption enhancer in ciprofloxacin tablet formulation using the ex-vivo model. Six batches of ciprofloxacin tablets containing varying concentrations of crab shell-derived chitosan ranging from 0 to 5% w/w at 1% w/w intervals were produced. Batch CTS-0 ...

  6. Preparation, characterization and ex vivo evaluation of an orally disintegrating film formulation containing pyrazinamide

    CSIR Research Space (South Africa)

    Adeleke, Oluwatoyin A

    2015-10-01

    Full Text Available The purpose of this research is to prepare, characterize as well as assess the ex vivo buccal drug delivery and cytotoxicity of an orally disintegrating film (ODF) loaded with pyrazinamide. Pyrazinamide (solubility = 15 mg/mL at 25°C; log P = -1...

  7. Validation of myocardial perfusion quantification by dynamic CT in an ex-vivo porcine heart model

    NARCIS (Netherlands)

    Pelgrim, Gert Jan; Das, Marco; van Tuijl, Sjoerd; van Assen, Marly; Prinzen, Frits W; Stijnen, Marco; Oudkerk, Matthijs; Wildberger, Joachim E; Vliegenthart, Rozemarijn

    2017-01-01

    To test the accuracy of quantification of myocardial perfusion imaging (MPI) using computed tomography (CT) in ex-vivo porcine models. Five isolated porcine hearts were perfused according to Langendorff. Hearts were perfused using retrograde flow through the aorta and blood flow, blood pressure and

  8. 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

  9. Ex vivo humidifying capacity and patient acceptability of stoma cloths in laryngectomized individuals

    NARCIS (Netherlands)

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

    Background Heat and moisture exchangers (HMEs) improve respiratory function after laryngectomy, but there is virtually no information on the benefit of traditional stoma cloths or other covers. Methods Two sequential studies were performed: (1) an ex vivo test was used to compare the humidifying

  10. Ex vivo humidifying capacity and patient acceptability of stoma cloths in laryngectomized individuals

    NARCIS (Netherlands)

    Lansaat, Liset; van den Boer, Cindy; Muller, Sara H.; van der Noort, Vincent; van den Brekel, Michiel W. M.; Hilgers, Frans J. M.

    2017-01-01

    BackgroundHeat and moisture exchangers (HMEs) improve respiratory function after laryngectomy, but there is virtually no information on the benefit of traditional stoma cloths or other covers. MethodsTwo sequential studies were performed: (1) an ex vivo test was used to compare the humidifying

  11. 3D morphological analysis of the mouse cerebral vasculature: Comparison of in vivo and ex vivo methods.

    Science.gov (United States)

    Steinman, Joe; Koletar, Margaret M; Stefanovic, Bojana; Sled, John G

    2017-01-01

    Ex vivo 2-photon fluorescence microscopy (2PFM) with optical clearing enables vascular imaging deep into tissue. However, optical clearing may also produce spherical aberrations if the objective lens is not index-matched to the clearing material, while the perfusion, clearing, and fixation procedure may alter vascular morphology. We compared in vivo and ex vivo 2PFM in mice, focusing on apparent differences in microvascular signal and morphology. Following in vivo imaging, the mice (four total) were perfused with a fluorescent gel and their brains fructose-cleared. The brain regions imaged in vivo were imaged ex vivo. Vessels were segmented in both images using an automated tracing algorithm that accounts for the spatially varying PSF in the ex vivo images. This spatial variance is induced by spherical aberrations caused by imaging fructose-cleared tissue with a water-immersion objective. Alignment of the ex vivo image to the in vivo image through a non-linear warping algorithm enabled comparison of apparent vessel diameter, as well as differences in signal. Shrinkage varied as a function of diameter, with capillaries rendered smaller ex vivo by 13%, while penetrating vessels shrunk by 34%. The pial vasculature attenuated in vivo microvascular signal by 40% 300 μm below the tissue surface, but this effect was absent ex vivo. On the whole, ex vivo imaging was found to be valuable for studying deep cortical vasculature.

  12. Preserving and evaluating hearts with ex vivo machine perfusion: an avenue to improve early graft performance and expand the donor pool.

    Science.gov (United States)

    Collins, Michael J; Moainie, Sina L; Griffith, Bartley P; Poston, Robert S

    2008-08-01

    Cardiac transplantation remains the first choice for the surgical treatment of end stage heart failure. An inadequate supply of donor grafts that meet existing criteria has limited the application of this therapy to suitable candidates and increased interest in extended criteria donors. Although cold storage (CS) is a time-tested method for the preservation of hearts during the ex vivo transport interval, its disadvantages are highlighted in hearts from the extended criteria donor. In contrast, transport of high-risk hearts using hypothermic machine perfusion (MP) provides continuous support of aerobic metabolism and ongoing washout of metabolic byproducts. Perhaps more importantly, monitoring the organ's response to this intervention provides insight into the viability of a heart initially deemed as extended criteria. Obviously, ex vivo MP introduces challenges, such as ensuring homogeneous tissue perfusion and avoiding myocardial edema. Though numerous groups have experimented with this technology, the best perfusate and perfusion parameters needed to achieve optimal results remain unclear. In the present review, we outline the benefits of ex vivo MP with particular attention to how the challenges can be addressed in order to achieve the most consistent results in a large animal model of the ideal heart donor. We provide evidence that MP can be used to resuscitate and evaluate hearts from animal and human extended criteria donors, including the non-heart beating donor, which we feel is the most compelling argument for why this technology is likely to impact the donor pool.

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

    Directory of Open Access Journals (Sweden)

    Carlos Goes NOGALES

    Full Text Available 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.

  14. Ex vivo tracheomalacia model with 3D-printed external tracheal splint.

    Science.gov (United States)

    Kaye, Rachel; Goldstein, Todd; Aronowitz, Danielle; Grande, Daniel A; Zeltsman, David; Smith, Lee P

    2017-04-01

    To design and evaluate an ex vivo model of tracheomalacia with and without a three-dimensional (3D)-printed external tracheal splint. Prospective, ex vivo animal trial. Three groups of ex vivo porcine tracheas were used: 1) control (unmanipulated trachea), 2) tracheomalacia (tracheal rings partially incised and crushed), and 3) splinted tracheomalacia (external custom tracheal splint fitted onto group 2 trachea). Each end of an ex vivo trachea was sealed with a custom-designed and 3D-printed cap; a transducer was placed through one end to measure the pressure inside the trachea. Although the negative pressure was applied to the tracheal lumen, the tracheal wall collapse was measured externally and internally using a bronchoscope. Each group had at least three recorded trials. Tracheal diameter was evaluated using ImageJ software (National Institutes of Health, Bethesda, MD) and was averaged between two raters. Average tracheal occlusion percentage was compared using Student t test. The average occlusion was 31% for group 1, 87.4% for group 2, and 20% for group 3. Significant differences were found between the control and tracheomalacia groups (P splinted tracheomalacia groups (P splinted tracheomalacia groups (P = 0.13). Applied pressure was plotted against occlusion and regression line slope differed between the tracheomalacia (0.91) and control (0.12) or splinted tracheomalacia (0.39) groups. We demonstrate the potential for an ex vivo tracheomalacia model to reproduce airway collapse and show that this collapse can be treated successfully with a 3D-printed external splint. These results are promising and justify further studies. N/A. Laryngoscope, 127:950-955, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  15. Microwave ablation of primary and secondary liver tumours: ex vivo, in vivo, and clinical characterisation.

    Science.gov (United States)

    Amabile, Claudio; Ahmed, Muneeb; Solbiati, Luigi; Meloni, Maria Franca; Solbiati, Marco; Cassarino, Simone; Tosoratti, Nevio; Nissenbaum, Yitzhak; Ierace, Tiziana; Goldberg, S Nahum

    2017-02-01

    The aim of this study was to compare the performance of a microwave ablation (MWA) apparatus in preclinical and clinical settings. The same commercial 2.45 GHz MWA apparatus was used throughout this study. In total 108 ablations at powers ranging from 20 to 130 W and lasting from 3 to 30 min were obtained on ex vivo bovine liver; 28 ablations at 60 W, 80 W and 100 W lasting 5 and 10 min were then obtained in an in vivo swine model. Finally, 32 hepatocellular carcinomas (HCCs) and 19 liver metastases in 46 patients were treated percutaneously by administering 60 W for either 5 or 10 min. The treatment outcome was characterised in terms of maximum longitudinal and transversal axis of the induced ablation zone. Ex vivo ablation volumes increased linearly with deposited energy (r2 = 0.97), with higher sphericity obtained at lower power for longer ablation times. Larger ablations were obtained on liver metastases compared to HCCs treated with 60 W for 10 min (p vivo swine model at 60 W were substantially smaller than the ex vivo and clinical results (either populations). No statistically significant difference was observed between ex vivo results at 60 W and HCC results (p > 0.08). For the selected MW ablation device, ex vivo data on bovine liver was more predictive of the actual clinical performance on liver malignancies than an in vivo porcine model. Equivalent MW treatments yielded a significantly different response for HCC and metastases at higher deposited energy, suggesting that outcomes are not only device-specific but must also be characterised on a tissue-by-tissue basis.

  16. Prevention of coronal discoloration induced by regenerative endodontic treatment in an ex vivo model.

    Science.gov (United States)

    Shokouhinejad, Noushin; Khoshkhounejad, Mehrfam; Alikhasi, Marzieh; Bagheri, Parisa; Camilleri, Josette

    2017-10-31

    The aim of this study was to assess the effect of sealing the pulp chamber walls with a dentin-bonding agent (DBA) on prevention of discoloration induced by regenerative endodontic procedures (REPs) in an ex vivo model. Ninety-six bovine incisors were prepared and randomly divided into two groups. In one group, the pulp chamber walls were sealed with DBA before placement of triple antibiotic paste (TAP) containing minocycline inside the root canals, but in the other group, DBA was not applied. After 4 weeks, the root canals were filled with human blood and each group was then randomly divided into four subgroups (n = 12) according to the endodontic cements placed over the blood clot (ProRoot MTA, OrthoMTA, RetroMTA, or Biodentine). The color changes (∆E) were measured at different steps. The data were analyzed using t test and two-way ANOVA. The specimens in which dentinal walls of pulp chamber were sealed with DBA showed significantly less coronal discoloration at each step of regenerative treatment (p endodontic cements did not result in significant difference in coronal discoloration (p > 0.05). Sealing the pulp chamber walls before insertion of TAP decreased coronal discoloration following REP using different endodontic cements but did not prevent it. Discoloration of teeth undergoing REPs is an unfavorable outcome. Considering the significant contribution of TAP containing minocycline to the coronal tooth discoloration even after sealing the pulp chamber walls, the revision of current guidelines in relation to the use of TAP with minocycline might need to be revised.

  17. Effects of peptide therapy on ex vivo T-cell responses.

    Science.gov (United States)

    Marcotte, G V; Braun, C M; Norman, P S; Nicodemus, C F; Kagey-Sobotka, A; Lichtenstein, L M; Essayan, D M

    1998-04-01

    Peptide therapy targets T cells directly with short peptides containing multiple T-cell receptor epitopes. Murine studies suggest T-cell anergy as the mechanism of action; however, changes in T-cell cytokine profiles may be more relevant in human beings. We sought to study the effects of peptide therapy on ex vivo antigen-specific T-cell responses. Antigen-specific T-cell lines were generated from subjects enrolled in a double-blind, placebo controlled, two-dose study of the ALLERVAX CAT therapeutic, containing Fel d 1 peptides (ImmuLogic Pharmaceutical Corp., Waltham, Mass.) (n = 7, 8, and 7, respectively, for groups receiving placebo, 75 microg, or 750 microg). Each subject had three lines propagated before and after receiving peptide therapy; antigens used were cat hair extract, Fel d 1 peptides, and tetanus toxoid (negative control). Proliferative responses and cytokine generation from each line were assessed after two restimulations with antigen and autologous antigen-presenting cells. The Fel d 1 peptide lines showed a dose-dependent decrease of IL-4 production (p = 0.02 and 0.025, respectively, for the 750 microg group vs both the 75 microg and placebo groups). IL-4 production from the cat hair allergen extract lines and interferon-gamma production from both the Fel d 1 peptide lines and cat hair allergen extract lines showed no statistically significant changes. The control tetanus toxoid lines showed no changes in cytokine production; there were no significant changes in proliferation with any of the antigens in any of the treatment groups. In the clinical arm of the trial, only the 750 microg dose of peptides produced a significant response. Peptide therapy induces a significant, dose-dependent decrease in peptide-stimulated IL-4 production, consistent with either a shift in T-cell phenotype or peptide-specific T-cell tolerance.

  18. Ex vivo evaluation of new 2D and 3D dental radiographic technology for detecting caries

    Science.gov (United States)

    Tyndall, Donald; Mol, André; Everett, Eric T; Bangdiwala, Ananta

    2016-01-01

    Objectives: Proximal dental caries remains a prevalent disease with only modest detection rates by current diagnostic systems. Many new systems are available without controlled validation of diagnostic efficacy. The objective of this study was to evaluate the diagnostic efficacy of three potentially promising new imaging systems. Methods: This study evaluated the caries detection efficacy of Schick 33 (Sirona Dental, Salzburg, Austria) intraoral digital detector images employing an advanced sharpening filter, Planmeca ProMax® (Planmeca Inc., Helsinki, Finland) extraoral “panoramic bitewing” images and Sirona Orthophos XG3D (Sirona Dental) CBCT images with advanced artefact reduction. Conventional photostimulable phosphor images served as the control modality. An ex vivo study design using extracted human teeth, ten expert observers and micro-CT ground truth was employed. Results: Receiver operating characteristic analysis indicated similar diagnostic efficacy of all systems (ANOVA p > 0.05). The sensitivity of the Schick 33 images (0.48) was significantly lower than the other modalities (0.53–0.62). The specificity of the Planmeca images (0.86) was significantly lower than Schick 33 (0.96) and XG3D (0.97). The XG3D showed significantly better cavitation detection sensitivity (0.62) than the other modalities (0.48–0.57). Conclusions: The Schick 33 images demonstrated reduced caries sensitivity, whereas the Planmeca panoramic bitewing images demonstrated reduced specificity. XG3D with artefact reduction demonstrated elevated sensitivity and specificity for caries detection, improved depth accuracy and substantially improved cavitation detection. Care must be taken to recognize potential false-positive caries lesions with Planmeca panoramic bitewing images. Use of CBCT for caries detection must be carefully balanced with the presence of metal artefacts, time commitment, financial cost and radiation dose. PMID:26670605

  19. Aloin delivery on buccal mucosa: ex vivo studies and design of a new locoregional dosing system.

    Science.gov (United States)

    De Caro, Viviana; Scaturro, Anna Lisa; Di Prima, Giulia; Avellone, Giuseppe; Sutera, Flavia Maria; Di Fede, Olga; Campisi, Giuseppina; Giannola, Libero Italo

    2015-01-01

    Chemoprevention of potential malignant disorders or cancerous lesions that affect oral mucosae requires extended duration of treatment. Locoregional delivery of natural products could represent a promising strategy for this purpose. To investigate the aptitude of aloin to permeate through, or accumulate in, the buccal mucosa and to develop a new prolonged oro-mucosal drug delivery system. Permeation/accumulation of aloin from Curacao Aloe (containing 50% barbaloin) was evaluated ex vivo, using porcine buccal mucosa as the most useful model to simulate human epithelium. Oro-mucosal matrix tablets were prepared by dispersing aloin (10% w/w) in Eudragit® RS 100 as, biocompatible, low permeable, pH-independent, and non-swelling polymer. The prepared tablets were evaluated for drug-polymer compatibility, weight variation, drug uniformity content, diameter, thickness, hardness, friability, swelling, mucoadhesive strength, and drug release. Aloin has low tendency to cross buccal mucosa, permeation is marginal, and high drug amounts remain entrapped into the epithelium. Matrix tablets characteristics were in agreement with pharmacopoeial requirements. Drug release showed highly reproducible Higuchian profile. Delivery through matrix tablets promoted drug accumulation in the mucosal tissue. Following application of matrix tablets on porcine buccal mucosa, the amount of discharged drug recovered in the tissue should be sufficient to produce the desired effects, providing therapeutic drug levels directly at the site of action. Aloin-loaded tablets are valid candidates for prevention/treatment of potentially malignant disorders and oral cancer and could potentially lead to clinically relevant drug delivery system as coadjuvant of conventional chemotherapy/radiation therapy.

  20. HPMA copolymer conjugate with pirarubicin: In vitro and ex vivo stability and drug release study.

    Science.gov (United States)

    Islam, Waliul; Fang, Jun; Etrych, Tomas; Chytil, Petr; Ulbrich, Karel; Sakoguchi, Akihiro; Kusakabe, Katsuki; Maeda, Hiroshi

    2017-11-10

    We have developed a tumor environment-responsive polymeric anticancer prodrug containing pirarubicin (THP) conjugated to N-(2-hydroxypropyl) methacrylamide copolymer (PHPMA), [P-THP], through a spacer containing pH-sensitive hydrazone bond, that showed remarkable therapeutic effect against various tumor models and in a human pilot study. Toward clinical development, here we report THP release profile from its HPMA copolymer conjugate, the conjugate stability, protein and cell-binding and solubility of P-THP. Size exclusion chromatography of P-THP (molecular weight 38 kDa) showed similar hydrodynamic volume as bovine serum albumin (BSA) in aqueous solution, with no apparent interactions with BSA, nor aggregation by itself. pH-responsive release of free THP was reconfirmed at pHs 6.5 and lower. The drug release was significantly affected by a type of used buffer. Phosphate buffer seems to facilitate faster hydrazone bond cleavage at pH 7.4 whereas higher stability was achieved in L-arginine solution which yielded only little cleavage and THP release, approx. 15% within 2 weeks at the same pH at 25 °C. Furthermore, ex vivo study using sera of different animal species showed very high stability of P-THP. Incubation with blood showed high stability of P-THP during circulation, without binding to blood cells. These findings revealed that L-arginine solution provides appropriate media for formulation of P-THP infusion solution as tumor-targeted polymeric anticancer drug based on EPR effect. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Comparison of culture media for ex vivo cultivation of limbal epithelial progenitor cells.

    Science.gov (United States)

    Loureiro, Renata Ruoco; Cristovam, Priscila Cardoso; Martins, Caio Marques; Covre, Joyce Luciana; Sobrinho, Juliana Aparecida; Ricardo, José Reinaldo da Silva; Hazarbassanov, Rossen Myhailov; Höfling-Lima, Ana Luisa; Belfort, Rubens; Nishi, Mauro; Gomes, José Álvaro Pereira

    2013-01-01

    To compare the effectiveness of three culture media for growth, proliferation, differentiation, and viability of ex vivo cultured limbal epithelial progenitor cells. Limbal epithelial progenitor cell cultures were established from ten human corneal rims and grew on plastic wells in three culture media: supplemental hormonal epithelial medium (SHEM), keratinocyte serum-free medium (KSFM), and Epilife. The performance of culturing limbal epithelial progenitor cells in each medium was evaluated according to the following parameters: growth area of epithelial migration; immunocytochemistry for adenosine 5'-triphosphate-binding cassette member 2 (ABCG2), p63, Ki67, cytokeratin 3 (CK3), and vimentin (VMT) and real-time reverse transcription polymerase chain reaction (RT-PCR) for CK3, ABCG2, and p63, and cell viability using Hoechst staining. Limbal epithelial progenitor cells cultivated in SHEM showed a tendency to faster migration, compared to KSFM and Epilife. Immunocytochemical analysis showed that proliferated cells in the SHEM had lower expression for markers related to progenitor epithelial cells (ABCG2) and putative progenitor cells (p63), and a higher percentage of positive cells for differentiated epithelium (CK3) when compared to KSFM and Epilife. In PCR analysis, ABCG2 expression was statistically higher for Epilife compared to SHEM. Expression of p63 was statistically higher for Epilife compared to SHEM and KSFM. However, CK3 expression was statistically lower for KSFM compared to SHEM. Based on our findings, we concluded that cells cultured in KSFM and Epilife media presented a higher percentage of limbal epithelial progenitor cells, compared to SHEM.

  2. 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.

  3. 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.

  4. Ex vivo evaluation of new 2D and 3D dental radiographic technology for detecting caries.

    Science.gov (United States)

    Gaalaas, Laurence; Tyndall, Donald; Mol, André; Everett, Eric T; Bangdiwala, Ananta

    2016-01-01

    Proximal dental caries remains a prevalent disease with only modest detection rates by current diagnostic systems. Many new systems are available without controlled validation of diagnostic efficacy. The objective of this study was to evaluate the diagnostic efficacy of three potentially promising new imaging systems. This study evaluated the caries detection efficacy of Schick 33 (Sirona Dental, Salzburg, Austria) intraoral digital detector images employing an advanced sharpening filter, Planmeca ProMax(®) (Planmeca Inc., Helsinki, Finland) extraoral "panoramic bitewing" images and Sirona Orthophos XG3D (Sirona Dental) CBCT images with advanced artefact reduction. Conventional photostimulable phosphor images served as the control modality. An ex vivo study design using extracted human teeth, ten expert observers and micro-CT ground truth was employed. Receiver operating characteristic analysis indicated similar diagnostic efficacy of all systems (ANOVA p > 0.05). The sensitivity of the Schick 33 images (0.48) was significantly lower than the other modalities (0.53-0.62). The specificity of the Planmeca images (0.86) was significantly lower than Schick 33 (0.96) and XG3D (0.97). The XG3D showed significantly better cavitation detection sensitivity (0.62) than the other modalities (0.48-0.57). The Schick 33 images demonstrated reduced caries sensitivity, whereas the Planmeca panoramic bitewing images demonstrated reduced specificity. XG3D with artefact reduction demonstrated elevated sensitivity and specificity for caries detection, improved depth accuracy and substantially improved cavitation detection. Care must be taken to recognize potential false-positive caries lesions with Planmeca panoramic bitewing images. Use of CBCT for caries detection must be carefully balanced with the presence of metal artefacts, time commitment, financial cost and radiation dose.

  5. Intraoral versus extraoral bitewing radiography in detection of enamel proximal caries: an ex vivo study.

    Science.gov (United States)

    Abu El-Ela, Walaa Hussein; Farid, Mary Medhat; Mostafa, Mostafa Saad El-Din

    2016-01-01

    To compare the diagnostic accuracy of digital intraoral and extraoral bitewing (EO BW) radiography in the detection of enamel proximal caries regardless of their ability to separate contacts. Artificial caries with different degrees of decalcification was induced in 80 human sound premolars and molars using formic acid. Intraoral radiographs were taken with photostimulable phosphor plate (PSP) and complementary metal oxide semiconductor (CMOS), using the paralleling bitewing technique. Extraoral bitewing radiographs were obtained using Sirona digital panoramic X-ray unit (Sirona Siemens, Bensheim, Germany). In total, 160 proximal surfaces were assessed by 2 observers twice. Area under the receiver operating characteristic curve (Az) values for each image type, observer and reading were compared using z-tests, with a significance level of p ≤ 0.05. Sensitivity, specificity, positive-predictive value and negative-predictive value for each observer and reading were calculated. Spearman's test showed a strong positive correlation between the duration of demineralization and histological grading of carious teeth surfaces. For the three radiographic techniques, intraobserver reliability was strong to excellent. Moreover, interobserver agreement was strong. The differences between all detection methods were not statistically significant (p > 0.05). Intraoral bitewing using CMOS sensor had the highest sensitivity while EO BW had the highest specificity in the detection of incipient proximal caries. Within the limits of the ex vivo design, the difference in diagnostic accuracy between the three radiographic techniques was not significant. EO BW could be used in the detection of enamel proximal caries with results comparable with intraoral bitewing with PSP plate and CMOS sensor.

  6. In vitro and ex vivo antimicrobial efficacy of nano-MgO in the elimination of endodontic pathogens.

    Science.gov (United States)

    Monzavi, Abbas; Eshraghi, Saeed; Hashemian, Roxana; Momen-Heravi, Fatemeh

    2015-03-01

    The use of metal oxide nanoparticles has attracted lots of attention, mostly because of their promising antimicrobial activity along with their biocompatibility with mammalian cells. This study aims to investigate the in vitro and ex vivo antimicrobial efficiency of nano-magnesium oxide (MgO) aqueous solution against endodontic pathogens. The cytotoxicity of different concentrations of nano-MgO was assessed using lactate dehydrogenase cytotoxicity assay (LDH assay). A comparison of the antimicrobial efficiency of several concentrations of nano-MgO solution, sodium hypochlorite (NaOCl), and chlorhexidine (CHX) gluconate against Staphylococcus aureus, Enterococcus faecalis, and Candida albicans was made using the direct contact method. An ex vivo model of decoronated and experimentally infected human teeth was employed to compare the efficiency of nano-MgO (5 mg/L) solution with NaOCl (5.25 %) in the elimination of E. faecalis. There was no statistically significant difference between nano-MgO solutions (10 and 5 mg/L), 5.25 % NaOCl, and 2 % CHX gluconate in terms of the required time to inhibit the growth of the tested pathogens (p > 0.05). The LDH assay showed no cytotoxicity of different concentrations of nano-MgO used in this study (p MgO (5 mg/L) and NaOCl (5.25 %)-treated teeth (5-6 log scale reduction). However, the nano-MgO group showed a significant decrease in colony-forming units per milliliter (7 log scale), 24 h post-irrigation (p MgO group (2-3 log scale difference) compared to the NaOCl group, indicating long-term antibacterial activity of nano-MgO (p MgO group. The detection limit was 10 CFU/mL. Nano-MgO aqueous solutions represent promising antimicrobial activities, both in vitro and ex vivo with minimal toxicity. Compared to NaOCl (5.25 %), nano-MgO (5 mg/L) exhibits statistically significant long-term efficiency in the elimination of E. faecalis in the root canal system. After further investigations, nano-MgO could be considered as a new

  7. SR 11302, an AP-1 Inhibitor, Reduces Metastatic Lesion Formation in Ex Vivo 4D Lung Cancer Model.

    Science.gov (United States)

    Mishra, Dhruva Kumar; Kim, Min P

    2017-12-01

    Activator protein (AP) -1 is a transcription factor, plays important role in cell differentiation, proliferation and apoptosis. Analysis of tumor cells grown on ex vivo 4D lung cancer model shows increase in components of AP-1, c-Fos and c-Jun in circulating tumor cells (CTC) compared to primary tumor. Our aim was to determine whether the AP-1 inhibitor SR11302 reduces metastatic lesion formation in the 4D model. Human lung cancer cell lines A549, H1299, and H460 were grown in the 4D model and treated with SR11302 (1 μM). We compared the number of cells in the metastatic site upon SR11302 treatment and number of viable CTCs isolated from the 4D model with parental cells treated/untreated with SR11302 on a petri dish. There were significantly fewer tumor cells per high-power field on metastatic site in 4D model seeded with H460 (p = 0.009), A549 (p = 0.01), or H1299 (p = 0.02) cells treated with SR11302. Furthermore, the CTCs from SR11302 treated 4D models, seeded with H460 (p = 0.04), A549 (p = 0.008), or H1299 (p = 0.01) cells had significantly fewer viable tumor cells after 4 days in culture than the respective untreated control. However, the SR11302 had no impact on the viability of parental H460 (p = 0.87), A549 (p = 0.93), or H1299 (p = 0.25) cells grown on a petri dish (2D). SR11302 reduces metastatic lesion formation in the ex vivo 4D lung cancer model due to the presence of an independent yet common pathway among three cell lines. The ex vivo 4D model may provide a tool to better understand the complex process of metastasis.

  8. 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.

    Directory of Open Access Journals (Sweden)

    Christopher Ueck

    Full Text Available 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

  9. Human intestinal spirochetosis – a review

    Directory of Open Access Journals (Sweden)

    Gebbers, Jan-Olaf

    2010-01-01

    Full Text Available Human intestinal spirochetosis (IS is a condition defined histologically by the presence of spirochetal microorganisms attached to the apical cell membrane of the colorectal epithelium. Intestinal spirochetes comprise a heterogeneous group of bacteria. In humans, Brachyspira aalborgi and Brachyspira pilosicoli predominate. Prevalence rates of IS are low where living standards are high, in contrast to poorly developed areas where IS is common. Homosexuals and HIV-infected individuals are at high risk of being colonized. Clinical significance in individual cases has remained unclear up to now. A review of the literature assumes that invasion of spirochetes beyond the surface epithelium may be associated with gastrointestinal symptoms which respond to antibiotic treatment (metronidazole, whereas individuals lacking this feature may be mostly asymptomatic. Of unknown reason, homosexual and HIV-positive men as well as children are more likely to be symptomatic irrespective of invasion. Rare cases of spirochetemia and multiple organ failure have been reported in critically ill patients with IS.

  10. [Ex Vivo Testing of Mechanical Properties of Canine Metacarpal/Metatarsal Bones after Simulated Implant Removal].

    Science.gov (United States)

    Srnec, R; Fedorová, P; Pěnčík, J; Vojtová, L; Sedlinská, M; Nečas, A

    2016-01-01

    EXP, were compared and the difference was found to be statistically significant (p ≤ 0.05). CONCLUSIONS The recently developed biodegradable polymer-composite gel is easy and quick to apply to any defect, regardless of its shape, in bone tissue. The ex vivo mechanical tests on canine short bones showed that the composite applied to defects, which simulated holes left after screw removal, provided sufficient mechanical support to the bone architecture. The results of measuring maximum loading forces were statistically significant. However, before the composite could be recommended for use in veterinary or human medical practice, thorough pre-clinical studies will be required. fracture fixation, mechanical testing, bone plate, cortical screw, refracture.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. The human intestinal microbiome: a new frontier of human biology.

    Science.gov (United States)

    Hattori, Masahira; Taylor, Todd D

    2009-02-01

    To analyze the vast number and variety of microorganisms inhabiting the human intestine, emerging metagenomic technologies are extremely powerful. The intestinal microbes are taxonomically complex and constitute an ecologically dynamic community (microbiota) that has long been believed to possess a strong impact on human physiology. Furthermore, they are heavily involved in the maturation and proliferation of human intestinal cells, helping to maintain their homeostasis and can be causative of various diseases, such as inflammatory bowel disease and obesity. A simplified animal model system has provided the mechanistic basis for the molecular interactions that occur at the interface between such microbes and host intestinal epithelia. Through metagenomic analysis, it is now possible to comprehensively explore the genetic nature of the intestinal microbiome, the mutually interacting system comprising the host cells and the residing microbial community. The human microbiome project was recently launched as an international collaborative research effort to further promote this newly developing field and to pave the way to a new frontier of human biology, which will provide new strategies for the maintenance of human health.

  13. Diversity of human small intestinal Streptococcus and Veillonella populations

    NARCIS (Netherlands)

    Bogert, B. van den; Erkus, O.; Boekhorst, J.; Goffau, M. de; Smid, E.J.; Zoetendal, E.G.; Kleerebezem, M.

    2013-01-01

    Molecular and cultivation approaches were employed to study the phylogenetic richness and temporal dynamics of Streptococcus and Veillonella populations in the small intestine. Microbial profiling of human small intestinal samples collected from four ileostomy subjects at four time points displayed

  14. Diversity of human small intestinal Streptococcus and Veillonella populations

    NARCIS (Netherlands)

    van den Bogert, Bartholomeus; Erkus, Oylum; Boekhorst, Jos; de Goffau, Marcus; Smid, Eddy J.; Zoetendal, Erwin G.; Kleerebezem, Michiel

    Molecular and cultivation approaches were employed to study the phylogenetic richness and temporal dynamics of Streptococcus and Veillonella populations in the small intestine. Microbial profiling of human small intestinal samples collected from four ileostomy subjects at four time points displayed

  15. Noncontact optical measurement of lens capsule thickness ex vivo

    Science.gov (United States)

    Ziebarth, Noel M.; Manns, Fabrice; Uhlhorn, Stephen; Parel, Jean-Marie

    2004-07-01

    Purpose: To design a non-contact optical system to measure lens capsule thickness in cadaver eyes. Methods: The optical system uses a 670nm laser beam delivered to a single-mode fiber coupler. The output of the fiber coupler is focused onto the tissue using an aspheric lens (NA=0.68) mounted on a motorized translation stage. Light reflected from the sample is collected by the fiber coupler and sent to a silicon photodiode connected to a power meter. Peaks in the power signal are detected when the focal point of the aspheric lens coincides with the capsule boundaries. The capsule thickness is proportional to the distance between successive peaks. Anterior and posterior lens capsule thickness measurements were performed on 13 human, 10 monkey, and 34 New Zealand white rabbit lenses. The cadaver eyes were prepared for optical measurements by bonding a PMMA ring on the sclera. The posterior pole was sectioned, excess vitreous was removed, and the eye was placed on a Teflon slide. The cornea and iris were then sectioned. After the experiments, the lenses were excised, placed in 10% buffered formalin, and prepared for histology. Results: Central anterior lens capsule thickness was 9.4+/-2.9μm (human), 11.2+/-6.6μm (monkey), and 10.3+/-3.6μm (rabbit) optically and 14.9+/-1.6μm (human), 17.7+/-4.9μm (monkey), and 12.6+/-2.3μm (rabbit) histologically. The values for the central posterior capsule were 9.4+/-2.9μm (human), 6.6+/-2.5μm (monkey), and 7.9+/-2.3μm (rabbit) optically and 4.6+/-1.4μm (human), 4.5+/-1.2μm (monkey), and 5.7+/-1.7μm (rabbit) histologically. Conclusions: This study demonstrates that a non-contact optical system can successfully measure lens capsule thickness in cadaver eyes.

  16. Bioactive protein fraction DLBS1033 containing lumbrokinase isolated from Lumbricus rubellus: ex vivo, in vivo, and pharmaceutic studies

    Directory of Open Access Journals (Sweden)

    Tjandrawinata RR

    2014-09-01

    Full Text Available Raymond R Tjandrawinata,1 Jessica Trisina,1 Puji Rahayu,1 Lorentius Agung Prasetya,1 Aang Hanafiah,2 Heni Rachmawati3 1Dexa Laboratories of Biomolecular Sciences, Dexa Medica, Cikarang, Indonesia; 2National Nuclear Energy Agency, Bandung, Indonesia; 3School of Pharmacy, Bandung Institute of Technology, Bandung, Indonesia Abstract: DLBS1033 is a bioactive protein fraction isolated from Lumbricus rubellus that tends to be unstable when exposed to the gastrointestinal environment. Accordingly, appropriate pharmaceutical development is needed to maximize absorption of the protein fraction in the gastrointestinal tract. In vitro, ex vivo, and in vivo stability assays were performed to study the stability of the bioactive protein fraction in gastric conditions. The bioactive protein fraction DLBS1033 was found to be unstable at low pH and in gastric fluid. The “enteric coating” formulation showed no leakage in gastric fluid–like medium and possessed a good release profile in simulated intestinal medium. DLBS1033 was absorbed through the small intestine in an intact protein form, confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE analysis. This result confirmed that an enteric coating formula using methacrylic acid copolymer could protect DLBS1033 from the acidic condition of the stomach by preventing the release of DLBS1033 in the stomach, while promoting its release when reaching the intestine. From the blood concentration–versus-time curve, 99mTc-DLBS1033 showed a circulation half-life of 70 minutes. This relatively long biological half-life supports its function as a thrombolytic protein. Thus, an enteric delivery system is considered the best approach for DLBS1033 as an oral thrombolytic agent. Keywords: bioactive protein fraction, enteric coated tablet, pharmacodynamic

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

    Science.gov (United States)

    Fan, Xiaobing; Macleod, Kay; Mustafi, Devkumar; Conzen, Suzanne D.; Markiewicz, Erica; Zamora, Marta; Vosicky, James; Mueller, Jeffrey; Karczmar, Gregory S.

    2015-01-01

    Ex vivo MRI may aid in the evaluation of surgical specimens, and provide valuable information regarding the micro-anatomy of mammary/breast cancer. The use of ex vivo MRI to study mouse mammary cancer would be enhanced if there is a strong correlation between parameters derived from in vivo and ex vivo scans. Here, we report the correlation between apparent diffusion coefficient (ADC) and T2 values measured in vivo and ex vivo in mouse mammary glands with in situ cancers (mammary intraepithelial neoplasia (MIN)) and invasive cancers (those which spread outside the ducts into surrounding tissue). MRI experiments were performed on the Polyoma middle T oncoprotein breast cancer mouse model (n = 15) in a 9.4T scanner. For in vivo experiments, T2-weighted (T2W) images were acquired to identify abnormal regions, then ADC and T2 values were measured for nine selected slices. For ex vivo experiments, a midline incision was made along the spine, and then skin, glands, and tumors were gently peeled from the body. Tissue was fixed in formalin, placed around a mouse-sized sponge, and sutured together mimicking the geometry of the gland when attached to the mouse. The same pulse sequences used for in vivo experiments were repeated for ex vivo scans at room temperature. Regions of interest were manually traced on T2W images defining features that could be identified on in vivo and ex vivo images. The results demonstrate a strong positive correlations between in vivo and ex vivo invasive cancers for ADC (r = 0.89, p vivo and ex vivo in situ cancers for ADC (r = 0.61, p vivo ADC value was about 54% of the in vivo value; and the average ex vivo T2 was similar to the in vivo value for cancers. Although motion, fixation, and temperature differences affect ADC and T2, these results show a reliable relationship between ADC and T2 in vivo and ex vivo. As a result ex vivo images can provide valuable information with clinical and research applications. PMID:26208092

  18. Mucoadhesive buccal tablets containing silymarin Eudragit-loaded nanoparticles: formulation, characterisation and ex vivo permeation.

    Science.gov (United States)

    El-Nahas, Amira E; Allam, Ahmed N; El-Kamel, Amal H

    2017-08-01

    Eudragit-loaded silymarin nanoparticles (SNPs) and their formulation into buccal mucoadhesive tablets were investigated to improve the low bioavailability of silymarin through buccal delivery. Characterisation of SNPs and silymarin buccal tablets (SBTs) containing the optimised NPs were performed. Ex vivo permeability of nominated SBTs were assessed using chicken pouch mucosa compared to SNPs and drug suspension followed by histopathological examination. Selected SNPs had a small size (77%) with drug release of about 90% after 6 h. For STBs, all physicochemical parameters were satisfactory for different polymers used. DSC and FT-IR studies suggested the presence of silymarin in an amorphous state. Ex vivo permeation significantly emphasised the great enhancement of silymarin permeation after NPs formation and much more increase after formulating into BTs relative to the corresponding drug dispersion with confirmed membrane integrity. Incorporation of SNPs into BTs could be an efficient vehicle for delivery of silymarin.

  19. 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).

  20. Shaping of Natural Killer Cell Antitumor Activity by Ex Vivo Cultivation

    Science.gov (United States)

    Granzin, Markus; Wagner, Juliane; Köhl, Ulrike; Cerwenka, Adelheid; Huppert, Volker; Ullrich, Evelyn

    2017-01-01

    Natural killer (NK) cells are a promising tool for the use in adoptive immunotherapy, since they efficiently recognize and kill tumor cells. In this context, ex vivo cultivation is an attractive option to increase NK cells in numbers and to improve their antitumor potential prior to clinical applications. Consequently, various strategies to generate NK cells for adoptive immunotherapy have been developed. Here, we give an overview of different NK cell cultivation approaches and their impact on shaping the NK cell antitumor activity. So far, the cytokines interleukin (IL)-2, IL-12, IL-15, IL-18, and IL-21 are used to culture and expand NK cells. The selection of the respective cytokine combination is an important factor that directly affects NK cell maturation, proliferation, survival, distribution of NK cell subpopulations, activation, and function in terms of cytokine production and cytotoxic potential. Importantly, cytokines can upregulate the expression of certain activating receptors on NK cells, thereby increasing their responsiveness against tumor cells that express the corresponding ligands. Apart from using cytokines, cocultivation with autologous accessory non-NK cells or addition of growth-inactivated feeder cells are approaches for NK cell cultivation with pronounced effects on NK cell activation and expansion. Furthermore, ex vivo cultivation was reported to prime NK cells for the killing of tumor cells that were previously resistant to NK cell attack. In general, NK cells become frequently dysfunctional in cancer patients, for instance, by downregulation of NK cell activating receptors, disabling them in their antitumor response. In such scenario, ex vivo cultivation can be helpful to arm NK cells with enhanced antitumor properties to overcome immunosuppression. In this review, we summarize the current knowledge on NK cell modulation by different ex vivo cultivation strategies focused on increasing NK cytotoxicity for clinical application in malignant

  1. Multimodality scoring of chondral injuries in the equine fetlock joint ex vivo

    OpenAIRE

    Sarin Jaakko Kalevi; Brommer Harold; Argüelles David; Puhakka Pia Henriikka; Inkinen Satu Irene; Afara Isaac Oluwaseun; Saarakkala Simo; Töyräs Juha

    2017-01-01

    Objective We investigate the potential of a prototype multimodality arthroscope, combining ultrasound, optical coherence tomography (OCT) and arthroscopic indentation device, for assessing cartilage lesions, and compare the reliability of this approach with conventional arthroscopic scoring ex vivo. Design Areas of interest (AIs, N = 43) were selected from equine fetlock joints (N = 5). Blind-coded AIs were independently scored by two equine surgeons employing International Cartilag...

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

    Science.gov (United States)

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

    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. PMID:22147969

  3. Ex vivo comparative study on three sinus lift tools for transcrestal detaching maxillary sinus mucosa.

    Science.gov (United States)

    Li, Yanfeng; Hu, Pin; Han, Yishi; Fan, Jiadong; Dong, Xinming; Ren, Huan; Yang, Chunhao; Shi, Tingting; Xia, Dong

    2017-07-04

    The objective of this study was to comparatively evaluate 3 different sinus lift tools, namely umbrella-shaped sinus lift curette YSL-04, our recently designed probe-improved sinus lift curettes, and our newly invented elevator 014, using our previous developed goat ex vivo models for direct visualizing the effectiveness of detaching sinus mucosa in real time. Goat ex vivo models for direct visualizing the effectiveness of detaching sinus mucosa in real time were generated according to our previously developed protocol. The effectiveness for each tool was evaluated through the length of sinus mucosa detached in mesial and distal directions or buccal and palatal directions, and the space volume created by detaching maxillary sinus mucosa in mesial, distal, buccal and palatal directions. The results showed that all 3 sinus lift tools could transcrestally detach the maxillary sinus mucosa and create extra space under the elevated sinus floor on the goat ex vivo sinus models. Moreover, our newly invented elevator 014 had advantages over the other 2 in term of the capability to detach the sinus mucosa. Our newly invented elevator 014 might be a promising tool for detaching maxillary sinus mucosa in transcrestal maxillary sinus floor elevation.

  4. Evaluation of the effect of cryopreservation on ex vivo expansion of hematopoietic progenitors from cord blood.

    Science.gov (United States)

    Lazzari, L; Lucchi, S; Montemurro, T; Porretti, L; Lopa, R; Rebulla, P; Sirchia, G

    2001-10-01

    In previous studies, we identified a cytokine cocktail including thrombopoietin, Flt-3 ligand, interleukin (IL)-6 and IL-11 in serum-free medium, suitable to induce significant and sustained ex vivo expansion of primitive hematopoietic stem cells (HSCs) from cord blood (CB) for up to 10 weeks. The aim of the present study was to evaluate the effects of cryopreservation on ex vivo expansion of HSCs and their committed progenitors. CD34+ cells were purified from CB units, each of which was processed in part as such and in part as cryopreserved and thawed, then expanded for 5 weeks in serum-free medium with the cytokine cocktail described above. We determined the number of nucleated cells (NC), CD34+, CD34+/38(-)/33(-), CD34+/61+, CD61+ cells and the clonogenic potential. After 2 weeks the median fold expansion of NC, CD34+ and CD34+/38(-)/33(-) cells was around two log both with fresh and cryopreserved CB and the expansion continued similarly until week 5. Our data suggest that this serum free protocol induces similar ex vivo expansion of HSCs and their committed progenitors from both fresh and cryopreserved CB. Our findings can be useful in view of clinical applications, since CB used for transplantation is stored in the cryopreserved state.

  5. Ex vivo efficacy of gemifloxacin in experimental keratitis induced by methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Marino, Andreana; Blanco, Anna Rita; Ginestra, Giovanna; Nostro, Antonia; Bisignano, Giuseppe

    2016-10-01

    In recent years, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) strains has been observed in ocular infections. Resistance of MRSA to second- and third-generation fluoroquinolones has increased interest in the fourth-generation fluoroquinolones. In this study, the antibacterial activity of gemifloxacin against MRSA ocular isolates in vitro and in a modified ex vivo rabbit keratitis model was investigated. In vitro susceptibility test results indicated that the minimum inhibitory concentrations (MICs) of gemifloxacin were lower than the MICs of other fluoroquinolones, including moxifloxacin (MIC50 range, 0.016-0.032 µg/mL; MIC90 range, 0.047-0.094 µg/mL). Results from the ex vivo keratitis model showed a statistically significant decrease in MRSA counts (0.5-2 log10 CFU/g; P keratitis. In addition, this reproducible, ethical and economic ex vivo infection model can be used as a mechanistically-based alternative to in vivo animal testing, bridging the gap between in vitro and in vivo results. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  6. 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.

  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. Ex vivo expanded autologous polyclonal regulatory T cells suppress inhibitor formation in hemophilia

    Directory of Open Access Journals (Sweden)

    Debalina Sarkar

    2014-01-01

    Full Text Available Adoptive cell therapy utilizing ex vivo expanded polyclonal CD4+CD25+FOXP3+ regulatory T cells (Treg is in use in clinical trials for the treatment of type 1 diabetes and prevention of graft versus host disease in bone marrow transplantation. Here, we seek to evaluate this approach in the treatment of inherited protein deficiencies, i.e., hemophilia, which is often complicated by antibody formation against the therapeutic protein. Treg from mice that express green fluorescent protein–marked FoxP3 were highly purified by two-step magnetic/flow sorting and ex vivo expanded 50- to 100-fold over a 2-week culture period upon stimulation with antibody-coated microbeads. FoxP3 expression was maintained in >80% of expanded Treg, which also expressed high levels of CD62L and CTLA-4. Transplanted Treg suppressed inhibitory antibody formation against coagulation factors VIII and IX in protein and gene therapies in strain-matched hemophilia A and B mice, including in mice with pre-existing antibodies. Although transplanted Treg became undetectable within 2 weeks, suppression persisted for >2 months. Additional studies suggested that antigen-specific suppression emerged due to induction of endogenous Treg. The outcomes of these studies support the concept that cell therapy with ex vivo expanded autologous Treg can be used successfully to minimize immune responses in gene and protein replacement therapies.

  9. Ex Vivo and in Vivo Administration of Fluorescent CNA35 Specifically Marks Cardiac Fibrosis

    Directory of Open Access Journals (Sweden)

    Sanne de Jong

    2014-12-01

    Full Text Available Cardiac fibrosis is a major hallmark of cardiac diseases. For evaluation of cardiac fibrosis, the development of highly specific and preferably noninvasive methods is desired. Our aim was to evaluate CNA35, a protein known to specifically bind to collagen, as a specific marker of cardiac fibrosis. Fluorescently labeled CNA35 was applied ex vivo on tissue sections of fibrotic rat, mouse, and canine myocardium. After quantification of CNA35, sections were examined with picrosirius red (PSR and compared to CNA35. Furthermore, fluorescently labeled CNA35 was administered in vivo in mice. Hearts were isolated, and CNA35 labeling was examined in tissue sections. Serial sections were histologically examined with PSR. Ex vivo application of CNA35 showed specific binding to collagen and a high correlation with PSR (Pearson r = .86 for mice/rats and r = .98 for canine; both p < .001. After in vivo administration, CNA35 labeling was observed around individual cardiomyocytes, indicating its ability to penetrate cardiac endothelium. High correlation was observed between CNA35 and PSR (r = .91, p < .001. CNA35 specifically binds to cardiac collagen and can cross the endothelial barrier. Therefore, labeled CNA35 is useful to specifically detect collagen both ex vivo and in vivo and potentially can be converted to a noninvasive method to detect cardiac fibrosis.

  10. 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. Copyright © 2014. Published by Elsevier Ltd.

  11. Perfusion device for liver preservation ex vivo before transplantation: first experimental study

    Directory of Open Access Journals (Sweden)

    O. N. Reznik

    2017-01-01

    Full Text Available Introduction. Successful liver transplantation including from donors with a sudden irreversible cardiac arrest requires the use of modern hardware and technical support to maintain, select and sustain organ viability for the period from harvesting to transplantation to the recipient.Materials and methods. Hardware-software system (HSS developed by the Russian State Scientific Center for Robotics and Technical Cybernetics (RTC was used for testing of normothermic perfusion of donor’s liver ex vivo. The experiment was conducted on the isolated pig liver (Duroc breed in accordance with the ethical principles.Result. During perfusion spontaneous recovery of bile outflow through the cannula installed in the common bile duct (volume of bile released – 240 ml was observed, and the color and uniformity of the perfused liver did not differ from the normal parameters. Biochemical indicators were stabilized at the physiological values after 40 minutes of perfusion procedure.Conclusion. Isolated liver transplant was completely restored after 30 minutes of warm ischemia and was functioning well due to ex vivo perfusion procedure on the new perfusion device. The first case of the new device usage for normothermic liver ex vivo demonstrated hopeful results to be further investigated.

  12. Ex vivo culture platform for assessment of cartilage repair treatment strategies.

    Science.gov (United States)

    Schwab, Andrea; Meeuwsen, Annick; Ehlicke, Franziska; Hansmann, Jan; Mulder, Lars; Smits, Anthal; Walles, Heike; Kock, Linda

    2017-01-01

    There is a great need for valuable ex vivo models that allow for assessment of cartilage repair strategies to reduce the high number of animal experiments. In this paper we present three studies with our novel ex vivo osteochondral culture platform. It consists of two separated media compartments for cartilage and bone, which better represents the in vivo situation and enables supply of factors specific to the different needs of bone and cartilage. We investigated whether separation of the cartilage and bone compartments and/or culture media results in the maintenance of viability, structural and functional properties of cartilage tissue. Next, we evaluated for how long we can preserve cartilage matrix stability of osteochondral explants during long-term culture over 84 days. Finally, we determined the optimal defect size that does not show spontaneous self-healing in this culture system. It was demonstrated that separated compartments for cartilage and bone in combination with tissue-specific medium allow for long-term culture of osteochondral explants while maintaining cartilage viability, matrix tissue content, structure and mechanical properties for at least 56 days. Furthermore, we could create critical size cartilage defects of different sizes in the model. The osteochondral model represents a valuable preclinical ex vivo tool for studying clinically relevant cartilage therapies, such as cartilage biomaterials, for their regenerative potential, for evaluation of drug and cell therapies, or to study mechanisms of cartilage regeneration. It will undoubtedly reduce the number of animals needed for in vivo testing.

  13. Arterial Retention of Remnant Lipoproteins Ex Vivo Is Increased in Insulin Resistance Because of Increased Arterial Biglycan and Production of Cholesterol-Rich Atherogenic Particles That Can Be Improved by Ezetimibe in the JCR:LA-cp Rat

    Science.gov (United States)

    Mangat, Rabban; Warnakula, Samantha; Borthwick, Faye; Hassanali, Zahra; Uwiera, Richard R.E.; Russell, James C.; Cheeseman, Christopher I.; Vine, Donna F.; Proctor, Spencer D

    2012-01-01

    Background Literature supports the “response-to-retention” hypothesis—that during insulin resistance, impaired metabolism of remnant lipoproteins can contribute to accelerated cardiovascular disease progression. We used the JCR:LA-cp rat model of metabolic syndrome (MetS) to determine the extent of arterial accumulation of intestinal-derived remnants ex vivo and potential mechanisms that contribute to exacerbated cholesterol deposition in insulin resistance. Methods and Results Arteries from control and MetS (insulin-resistant) JCR:LA-cp rats were perfused ex vivo with Cy5-labeled remnant lipoproteins, and their arterial retention was quantified by confocal microscopy. Arterial proteoglycans were isolated from control and MetS rats at 6, 12, and 32 weeks of age. There was a significant increase in the arterial retention of remnants and in associated cholesterol accumulation in MetS rats as compared to control rats. Mechanistic studies reveal that increased cholesterol deposition is a result of greater arterial biglycan content; longer glycosaminoglycans and increased production of cholesterol-rich intestinal-derived remnants, as compared to controls. Additionally, perfusion of vessels treated with ezetimibe, alone or in combination with simvastatin, with remnants isolated from the respective treatment group reduced ex vivo arterial retention of remnant-derived cholesterol ex vivo as compared to untreated controls. Conclusions Increased progression of atherosclerotic cardiovascular disease in MetS and type 2 diabetes mellitus might be explained in part by an increase in the arterial retention of cholesterol-rich remnants. Furthermore, ezetimibe alone or in combination treatment with simvastatin could be beneficial in ameliorating atherosclerotic cardiovascular disease in insulin resistance and MetS. PMID:23316299

  14. Cardiac phenotyping in ex vivo murine embryos using microMRI.

    Science.gov (United States)

    Cleary, Jon O; Price, Anthony N; Thomas, David L; Scambler, Peter J; Kyriakopoulou, Vanessa; McCue, Karen; Schneider, Jürgen E; Ordidge, Roger J; Lythgoe, Mark F

    2009-10-01

    Microscopic MRI (microMRI) is an emerging technique for high-throughput phenotyping of transgenic mouse embryos, and is capable of visualising abnormalities in cardiac development. To identify cardiac defects in embryos, we have optimised embryo preparation and MR acquisition parameters to maximise image quality and assess the phenotypic changes in chromodomain helicase DNA-binding protein 7 (Chd7) transgenic mice. microMRI methods rely on tissue penetration with a gadolinium chelate contrast agent to reduce tissue T(1), thus improving signal-to-noise ratio (SNR) in rapid gradient echo sequences. We investigated 15.5 days post coitum (dpc) wild-type CD-1 embryos fixed in gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) solutions for either 3 days (2 and 4 mM) or 2 weeks (2, 4, 8 and 16 mM). To assess penetration of the contrast agent into heart tissue and enable image contrast simulations, T(1) and T(*) (2) were measured in heart and background agarose. Compared to 3-day, 2-week fixation showed reduced mean T(1) in the heart at both 2 and 4 mM concentrations (p < 0.0001), resulting in calculated signal gains of 23% (2 mM) and 29% (4 mM). Using T(1) and T(*) (2) values from 2-week concentrations, computer simulation of heart and background signal, and ex vivo 3D gradient echo imaging, we demonstrated that 2-week fixed embryos in 8 mM Gd-DTPA in combination with optimised parameters (TE/TR/alpha/number of averages: 9 ms/20 ms/60 degrees /7) produced the largest SNR in the heart (23.2 +/- 1.0) and heart chamber contrast-to-noise ratio (CNR) (27.1 +/- 1.6). These optimised parameters were then applied to an MRI screen of embryos heterozygous for the gene Chd7, implicated in coloboma of the eye, heart defects, atresia of the choanae, retardation of growth, genital/urinary abnormalities, ear abnormalities and deafness (CHARGE) syndrome (a condition partly characterised by cardiovascular birth defects in humans). A ventricular septal defect was readily identified

  15. Alternative Functional In Vitro Models of Human Intestinal Epithelia

    Directory of Open Access Journals (Sweden)

    Amanda L Kauffman

    2013-07-01

    Full Text Available Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We sought to evaluate and compare two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs and induced pluripotent stem cell (iPSC-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, our previously described 3-dimensional intestinal organogenesis method was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.

  16. Evaluacion ex vivo de la retencion de estructuras en zirconia--ytria de procera[R] con diferentes cementos

    National Research Council Canada - National Science Library

    Mejia Bravo, Richard Milton; Catarroso Perez, Carlos Bernardo; Ruiz Restrepo, Xiomara Cristina; Espitia Mesa, Jose Fernando; Moreno Castillo, Jenny Alexandra; Montoya Sepulveda, Andres Felipe

    2014-01-01

    Introduccion: en tanto no se encuentra en la literatura un protocolo fundamentado de cementacion para restauraciones elaboradas en zirconia-ytria, el proposito de este estudio fue evaluar ex vivo la retencion...

  17. In vivo and ex vivo sentinel node mapping does not identify the same lymph nodes in colon cancer

    DEFF Research Database (Denmark)

    Andersen, Helene Schou; Bennedsen, Astrid Louise Bjørn; Burgdorf, Stefan Kobbelgaard

    2017-01-01

    sentinel node mapping in vivo with indocyanine green and ex vivo with methylene blue in order to evaluate if the sentinel lymph nodes can be identified by both techniques. METHODS: Patients with colon cancer UICC stage I-III were included from two institutions in Denmark from February 2015 to January 2016....... In vivo sentinel node mapping with indocyanine green during laparoscopy and ex vivo sentinel node mapping with methylene blue were performed in all patients. RESULTS: Twenty-nine patients were included. The in vivo sentinel node mapping was successful in 19 cases, and ex vivo sentinel node mapping...... mapping. Lymph node metastases were found in 10 patients, and only two had metastases in a sentinel node. CONCLUSION: Placing a deposit in relation to the tumor by indocyanine green in vivo or of methylene blue ex vivo could only identify sentinel lymph nodes in a small group of patients....

  18. Ex vivo paracrine properties of cardiac tissue: Effects of chronic heart failure.

    Science.gov (United States)

    Boucek, Robert J; Steele, Jasmine; Jacobs, Jeffery P; Steele, Peter; Asante-Korang, Alfred; Quintessenza, James; Steele, Ann

    2015-06-01

    Cardiac regenerative responses are responsive to paracrine factors. We hypothesize that chronic heart failure (HF) in pediatric patients affects cardiac paracrine signaling relevant to resident c-kit(+)cluster of differentiation (CD)34- cardiac stem cells (CSCs). Discarded atrial septum (huAS) and atrial appendages (huAA) from pediatric patients with HF (huAA-HF; n = 10) or without HF (n = 3) were explanted and suspension explant cultured in media. Conditioned media were screened for 120 human factors using unedited monoclonal antibody-based arrays. Significantly expressed (relative chemiluminescence >30 of 100) factors are reported (secretome). Emigrated cells were immunoselected for c-kit and enumerated as CSCs. After culture Day 7, CSCs emigrate from huAA but not huAS. The huAA secretome during CSC emigration included hepatocyte growth factor (HGF), epithelial cell-derived neutrophil attractant-78 (ENA-78)/chemokine (C-X-C motif) ligand (CXCL) 5, growth-regulated oncogene-α (GRO-α)/CXCL1, and macrophage migration inhibitory factor (MIF), candidate pro-migratory factors not present in the huAS secretome. Survival/proliferation of emigrated CSCs required coculture with cardiac tissue or tissue-conditioned media. Removal of huAA (Day 14) resulted in the loss of all emigrated CSCs (Day 28) and in decreased expression of 13 factors, including HGF, ENA-78/CXCL5, urokinase-type plasminogen activator receptor (uPAR)/CD87, and neutrophil-activating protein-2 (NAP-2)/CXCL7 candidate pro-survival factors. Secretomes of atrial appendages from HF patients have lower expression of 14 factors, including HGF, ENA-78/CXCL5, GRO-α/CXCL1, MIF, NAP-2/CXCL7, uPAR/CD87, and macrophage inflammatory protein-1α compared with AA from patients without HF. Suspension explant culturing models paracrine and innate CSC interactions in the heart. In pediatric patients, heart failure has an enduring effect on the ex vivo cardiac-derived secretome, with lower expression of candidate pro

  19. Suppression by thimerosal of ex-vivo CD4+ T cell response to influenza vaccine and induction of apoptosis in primary memory T cells.

    Directory of Open Access Journals (Sweden)

    Emily Loison

    Full Text Available Thimerosal is a preservative used widely in vaccine formulations to prevent bacterial and fungal contamination in multidose vials of vaccine. Thimerosal was included in the multidose non-adjuvanted pandemic 2009 H1N1 vaccine Panenza. In the context of the analysis of the ex-vivo T cell responses directed against influenza vaccine, we discovered the in vitro toxicity Panenza, due to its content in thimerosal. Because thimerosal may skew the immune response to vaccines, we investigated in detail the ex-vivo effects of thimerosal on the fate and functions of T cells in response to TCR ligation. We report that ex-vivo exposure of quiescent or TCR-activated primary human T cells to thimerosal induced a dose-dependent apoptotic cell death associated with depolarization of mitochondrial membrane, generation of reactive oxygen species, cytochrome c release from the mitochondria and caspase-3 activation. Moreover, exposure to non-toxic concentrations of thimerosal induced cell cycle arrest in G0/G1 phase of TCR-activated T cells, and inhibition of the release of proinflammatory cytokines such as IFN gamma, IL-1 beta, TNF alpha, IL-2, as well as the chemokine MCP1. No shift towards Th2 or Th17 cells was detected. Overall these results underline the proapoptotic effect of thimerosal on primary human lymphocytes at concentrations 100 times less to those contained in the multidose vaccine, and they reveal the inhibitory effect of this preservative on T-cell proliferation and functions at nanomolar concentrations.

  20. Dynamic efficiency of the human intestinal microbiota.

    Science.gov (United States)

    Candela, Marco; Biagi, Elena; Turroni, Silvia; Maccaferri, Simone; Figini, Paolo; Brigidi, Patrizia

    2015-06-01

    The emerging dynamic dimensions of the human intestinal microbiota (IM) are challenging the traditional definition of healthy gut microbiota, principally based on the static concepts of phylogenetic and functional core. On the other hand, recent researches are revealing that the microbiota plasticity is strategic for several aspects of our biology, addressing the different immunological and metabolic needs at various ages, and adjusting the ecosystem services in response to different lifestyle, physiological states or diets. In light of these studies, we propose to revise the traditional concept of healthy human IM, including its degree of plasticity among the fundamental requisites for providing host health. In order to make a model taking into account the relative importance of IM core functions and plasticity for the maintenance of host health, we address to Economics, where the efficiency of a productive system is measured by computing static and dynamic parameters.

  1. Minimal vascular flows cause strong heat sink effects in hepatic radiofrequency ablation ex vivo.

    Science.gov (United States)

    Lehmann, Kai S; Poch, Franz G M; Rieder, Christian; Schenk, Andrea; Stroux, Andrea; Frericks, Bernd B; Gemeinhardt, Ole; Holmer, Christoph; Kreis, Martin E; Ritz, Jörg P; Zurbuchen, Urte

    2016-08-01

    The present paper aims to assess the lower threshold of vascular flow rate on the heat sink effect in bipolar radiofrequency ablation (RFA) ex vivo. Glass tubes (vessels) of 3.4 mm inner diameter were introduced in parallel to bipolar RFA applicators into porcine liver ex vivo. Vessels were perfused with flow rates of 0 to 1,500 ml/min. RFA (30 W power, 15 kJ energy input) was carried out at room temperature and 37°C. Heat sink effects were assessed in RFA cross sections by the decrease in ablation radius, area and by a high-resolution sector planimetry. Flow rates of 1 ml/min already caused a significant cooling effect (P ≤ 0.001). The heat sink effect reached a maximum at 10 ml/min (18.4 mm/s) and remained stable for flow rates up to 1,500 ml/min. Minimal vascular flows of ≥1 ml/min cause a significant heat sink effect in hepatic RFA ex vivo. A lower limit for volumetric flow rate was not found. The maximum of the heat sink effect was reached at a flow rate of 10 ml/min and remained stable for flow rates up to 1,500 ml/min. Hepatic inflow occlusion should be considered in RFA close to hepatic vessels. © 2016 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

  2. Differences in transmural pressure and axial loading ex vivo affect arterial remodeling and material properties.

    Science.gov (United States)

    Lawrence, Amanda R; Gooch, Keith J

    2009-10-01

    Arterial axial strains, present in the in vivo environment, often become reduced due to either bypass grafting or the normal aging process. Since the prevalence of hypertension increases with aging, arteries are often exposed to both decreased axial stretch and increased transmural pressure. The combined effects of these mechanical stimuli on the mechanical properties of vessels have not previously been determined. Porcine carotid arteries were cultured for 9 days at normal and reduced axial stretch ratios in the presence of normotensive and hypertensive transmural pressures using ex viv