scholarly journals Assessment of a novel mucolytic solution for dissolving mucus in pseudomyxoma peritonei: an ex vivo and in vitro study

2017 ◽  
Vol 2 (2) ◽  
pp. 111-117 ◽  
Author(s):  
Krishna Pillai ◽  
Javed Akhter ◽  
David L. Morris
Keyword(s):  
Pseudomyxoma Peritonei ◽  
Hyperthermic Intraperitoneal Chemotherapy ◽  
Ex Vivo ◽  
In Vitro Study ◽  
Ht29 Cell ◽  
Test Solution ◽  
Test Formulation ◽  
Peritoneal Washing ◽  
Ht29 Cell Line

AbstractBackgroundPseudomyxoma peritonei (PMP) is difficult to treat. Intraperitoneal delivery of mucolytic solutions might potentially improve therapy, in addition to surgical cytoreduction and hyperthermic intraperitoneal chemotherapy.MethodsComparison of mucolytic effect of two formulations (control: bromelain 300 µg/mL+N-Acetylcystein 250 mM; test: bromelain 200 µg/mL+200 mM cysteamine) in vitro on a mucin producing cell lines (HT29) and ex vivo on mucus obtained from 18 PMP patients. Mucin plugs were classified according to their density into three categories: hard, semi hard and soft. Simulation of peritoneal washing ex vivo using a closed heated circulating pump.ResultsSolubilisation was faster with the test vs. the control formulation (90 vs. 180 min) for dissolving the soft mucin plugs (p < 0.05). The test solution was also more effective in dissolving the hard mucus plugs compared to control (82.5±2.74 % vs. 36.33±3.27 %). All mucin types disintegrated in simulated peritoneal washing. Cytotoxicity of the test solution on HT29 cell line was time-dependent.ConclusionsThe test formulation is more effective and faster than the control formulation in dissolving mucus plugs of various densities. Mucus plugs were all solubilised after 40 min in simulated peritoneal washing. This novel mucolytic formulation might pave the way for an effective and less invasive therapy of PMP in the future.

Effects of Two Different Doses of Hirudin on APTT, Determined with Eight Different Reagents

1995 ◽  
Vol 73 (02) ◽  
pp. 219-222 ◽  
Author(s):  
Manuel Monreal ◽  
Luis Monreal ◽  
Rafael Ruiz de Gopegui ◽  
Yvonne Espada ◽  
Ana Maria Angles ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Anticoagulant Activity ◽  
Subcutaneous Administration ◽  
In Vitro Study ◽  
Ex Vivo Model ◽  
Suitable Candidate ◽  
Significant Prolongation ◽  
High Doses ◽  
Different Doses

SummaryThe APTT has been considered the most suitable candidate to monitor the anticoagulant activity of hirudin. However, its use is hampered by problems of standardization, which make the results heavily dependent on the responsiveness of the reagent used. Our aim was to investigate if this different responsiveness of different reagents when added in vitro is to be confirmed in an ex vivo study.Two different doses of r-hirudin (CGP 39393), 0.3 mg/kg and 1 mg/kg, were administered subcutaneously to 20 New Zealand male rabbits, and the differences in prolongation of APTT 2 and 12 h later were compared, using 8 widely used commercial reagents. All groups exhibited a significant prolongation of APTT 2 h after sc administration of hirudin, both at low and high doses. But this prolongation persisted 12 h later only when the PTTa reagent (Boehringer Mannheim) was used. In general, hirudin prolonged the APTT most with the silica- based reagents.In a further study, we compared the same APTT reagents in an in vitro study in which normal pooled plasma was mixed with increasing amount of hirudin. We failed to confirm a higher sensitivity for silica- containing reagents. Thus, we conclude that subcutaneous administration of hirudin prolongs the APTT most with the silica-based reagents, but this effect is exclusive for the ex vivo model.

scholarly journals 64Cu-ATSM/64Cu-Cl2 and their relationship to hypoxia in glioblastoma: a preclinical study

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Elodie A. Pérès ◽  
Jérôme Toutain ◽  
Louis-Paul Paty ◽  
Didier Divoux ◽  
Méziane Ibazizène ◽  
...  
Keyword(s):  
Ex Vivo ◽  
In Vitro Study ◽  
Tumor Location ◽  
Preclinical Study ◽  
Significant Rise ◽  
Lower Oxygen ◽  
Cu Complexes ◽  
Ex Vivo Study

Abstract Background Diacetyl-bis(N4-methylthiosemicarbazone), labeled with 64Cu (64Cu-ATSM) has been suggested as a promising tracer for imaging hypoxia. However, various controversial studies highlighted potential pitfalls that may disable its use as a selective hypoxic marker. They also highlighted that the results may be tumor location dependent. Here, we first analyzed uptake of Cu-ATSM and its less lipophilic counterpart Cu-Cl2 in the tumor over time in an orthotopic glioblastoma model. An in vitro study was also conducted to investigate the hypoxia-dependent copper uptake in tumor cells. We then further performed a comprehensive ex vivo study to compare 64Cu uptake to hypoxic markers, specific cellular reactions, and also transporter expression. Methods μPET was performed 14 days (18F-FMISO), 15 days (64Cu-ATSM and 64Cu-Cl2), and 16 days (64Cu-ATSM and 64Cu-Cl2) after C6 cell inoculation. Thereafter, the brains were withdrawn for further autoradiography and immunohistochemistry. C6 cells were also grown in hypoxic workstation to analyze cellular uptake of Cu complexes in different oxygen levels. Results In vivo results showed that Cu-ASTM and Cu-Cl2 accumulated in hypoxic areas of the tumors. Cu-ATSM also stained, to a lesser extent, non-hypoxic regions, such as regions of astrogliosis, with high expression of copper transporters and in particular DMT-1 and CTR1, and also characterized by the expression of elevated astrogliosis. In vitro results show that 64Cu-ATSM showed an increase in the uptake only in severe hypoxia at 0.5 and 0.2% of oxygen while for 64Cu-Cl2, the cell retention was significantly increased at 5% and 1% of oxygen with no significant rise at lower oxygen percentages. Conclusion In the present study, we show that Cu-complexes undoubtedly accumulate in hypoxic areas of the tumors. This uptake may be the reflection of a direct dependency to a redox metabolism and also a reflection of hypoxic-induced overexpression of transporters. We also show that Cu-ATSM also stained non-hypoxic regions such as astrogliosis.

scholarly journals Enhancing Anti-Tumoral Potential of CD-NHF by Modulating PI3K/Akt Axis in U87 Ex Vivo Glioma Model

2021 ◽  
Vol 22 (8) ◽  
pp. 3873
Author(s):  
Gabriel Luta ◽  
Mihail Butura ◽  
Adrian Tiron ◽  
Crina E. Tiron
Keyword(s):  
Cell Line ◽  
Ex Vivo ◽  
Glioma Cells ◽  
In Vitro Study ◽  
Immunofluorescent Staining ◽  
Expression Levels ◽  
Disease Diagnostics ◽  
Phosphorylated Akt ◽  
Significant Impairment

Background: In the latest years, there has been an increased interest in nanomaterials that may provide promising novel approaches to disease diagnostics and therapeutics. Our previous results demonstrated that Carbon-dots prepared from N-hydroxyphthalimide (CD-NHF) exhibited anti-tumoral activity on several cancer cell lines such as MDA-MB-231, A375, A549, and RPMI8226, while U87 glioma tumor cells were unaffected. Gliomas represent one of the most common types of human primary brain tumors and are responsible for the majority of deaths. In the present in vitro study, we expand our previous investigation on CD-NHF in the U87 cell line by adding different drug combinations. Methods: Cell viability, migration, invasion, and immunofluorescent staining of key molecular pathways have been assessed after various treatments with CD-NHF and/or K252A and AKTVIII inhibitors in the U87 cell line. Results: Association of an inhibitor strongly potentiates the anti-tumoral properties of CD-NHF identified by significant impairment of migration, invasion, and expression levels of phosphorylated Akt, p70S6Kinase, or by decreasing expression levels of Bcl-2, IL-6, STAT3, and Slug. Conclusions: Using simultaneously reduced doses of both CD-NHF and an inhibitor in order to reduce side effects, the viability and invasiveness of U87 glioma cells were significantly impaired.

scholarly journals Short-term oxaliplatin exposure according to established hyperthermic intraperitoneal chemotherapy (HIPEC) protocols lacks effectiveness in vitro and ex vivo

2019 ◽  
Author(s):  
Markus W. Löffler ◽  
Nick Seyfried ◽  
Markus Burkard ◽  
Benedikt Oswald ◽  
Alexander Tolios ◽  
...  
Keyword(s):  
Intraperitoneal Chemotherapy ◽  
Hyperthermic Intraperitoneal Chemotherapy ◽  
Drug Exposure ◽  
Ex Vivo ◽  
Medical Center ◽  
Drug Effects ◽  
Patient Treatment ◽  
Cell Shrinkage ◽  
Cell Assay

AbstractCytotoxicity of oxaliplatin-containing solutions (OCS), sampled during patient treatment with hyperthermic intraperitoneal chemotherapy (HIPEC), was assessed by well-established continuous impedance-based real-time cell analysis (RTCA) ex vivo. HIPEC treatment was replicated by exposing OAW-42 cancer cells to OCS for 30 or 60 minutes at 42 °C. In contrast to previous observations with continuous exposure, where cytotoxicity was proven, identical OCS obtained during HIPEC did not induce cell death reproducibly and showed strongly attenuated effects after only 30 minutes of application. Based on these unexpected findings, spike-ins of oxaliplatin (OX) into peritoneal dialysis solution (PDS) or dextrose 5 % in water (D5W) were used to replicate HIPEC conditions, as used in either our own protocols or the recently presented randomized controlled PRODIGE 7 trial, where OX HIPEC for 30 minutes failed to produce survival benefits in colorectal carcinoma patients. With OX-spiked into D5W or PDS at identical concentrations as used for PRODIGE 7 or conforming with own HIPEC protocols, we did not observe the expectable cytotoxic effects in RTCA, after replicating OX HIPEC for 30 minutes. These results were corroborated for both solvents at relevant drug concentrations by classical end-point assays for cytotoxicity in two cancer cell lines. Further results suggest that penetration depth, drug dosage, exposure time and drug solvents may constitute critical factors for HIPEC effectiveness. Accordingly, we witnessed substantial cell shrinkage with both PDS and D5W, potentially contributing to reduced drug effects. Based on these results, intensified pharmacological research seems warranted to establish effective HIPEC protocols.Key PointsOxaliplatin (OX)-containing solutions obtained during patient treatment with Hyperthermic intraperitoneal chemotherapy (HIPEC) unexpectedly showed low cytotoxicity in an impedance-based ex vivo cytotoxicity cell assay.OX cytotoxicity under HIPEC conditions could be enhanced by extending drug exposure to one hour by an impedance-based ex vivo cytotoxicity cell assay.HIPEC failed to show survival benefits in the randomized controlled PRODIGE 7 trial and was questioned in the aftermath.Clinically relevant OX concentrations applied in conjunction with hyperthermia (42 °C) for 30 minutes, as used either at our own medical center or according to the PRODIGE 7 trial, proved predominantly ineffective, when used according to HIPEC routines in an impedance-based in vitro cytotoxicity cell assay.Respective findings were corroborated in two different cell lines and by two established end-point assays, showing that 50 % cell death could not be reached by the same HIPEC treatment with OX, in contrast to continuous drug exposure.As potentially relevant factor, the thickness of the exposed cell layer was identified, requiring at least ~100 µm penetration depth for our model to indicate effectiveness.Additionally, we show relevant cell shrinkage by two drug diluents used either at our own medical center or according to the PRODIGE 7 trial, potentially associated with fluid shifts out of the cell and impaired drug effects.Our own as well as recent findings by Ubink et al. (Br J Surg. 2019. doi: 10.1002/bjs.11206) support the notion that lacking effectiveness of OX HIPEC may explain the negative PRODIGE 7 trial results.

scholarly journals Retinoblastoma protein induction by HIV viremia or CCR5 in monocytes exposed to HIV-1 mediates protection from activation-induced apoptosis: ex vivo and in vitro study

2012 ◽  
Vol 92 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Bethsebah Gekonge ◽  
Andrea D. Raymond ◽  
Xiangfan Yin ◽  
Jay Kostman ◽  
Karam Mounzer ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Retinoblastoma Protein ◽  
In Vitro Study ◽  
Vitro Study ◽  
Induced Apoptosis ◽  
Hiv 1

scholarly journals Biomechanical-Based Protocol for in vitro Study of Cartilage Response to Cyclic Loading: A Proof-of-Concept in Knee Osteoarthritis

2021 ◽  
Vol 9 ◽  
Author(s):  
Paolo Caravaggi ◽  
Elisa Assirelli ◽  
Andrea Ensini ◽  
Maurizio Ortolani ◽  
Erminia Mariani ◽  
...  
Keyword(s):  
Ex Vivo ◽  
In Vitro Study ◽  
Cartilage Explants ◽  
Joint Loading ◽  
Design And Optimization ◽  
Cartilage Metabolism ◽  
Beneficial Effects ◽  
Physiological Loading

Osteoarthritis (OA) is an evolving disease and a major cause of pain and impaired mobility. A deeper understanding of cartilage metabolism in response to loading is critical to achieve greater insight into OA mechanisms. While physiological joint loading helps maintain cartilage integrity, reduced or excessive loading have catabolic effects. The main scope of this study is to present an original methodology potentially capable to elucidate the effect of cyclic joint loading on cartilage metabolism, to identify mechanisms involved in preventing or slowing down OA progression, and to provide preliminary data on its application. In the proposed protocol, the combination of biomechanical data and medical imaging are integrated with molecular information about chondrocyte mechanotransduction and tissue homeostasis. The protocol appears to be flexible and suitable to analyze human OA knee cartilage explants, with different degrees of degeneration, undergoing ex vivo realistic cyclic joint loading estimated via gait analysis in patients simulating mild activities of daily living. The modulation of molecules involved in cartilage homeostasis, mechanotransduction, inflammation, pain and wound healing can be analyzed in chondrocytes and culture supernatants. A thorough analysis performed with the proposed methodology, combining in vivo functional biomechanical evaluations with ex vivo molecular assessments is expected to provide new insights on the beneficial effects of physiological loading and contribute to the design and optimization of non-pharmacological treatments limiting OA progression.

scholarly journals Expression/Activation of PAR-1 in Airway Epithelial Cells of COPD Patients: Ex Vivo/In Vitro Study

2021 ◽  
Vol 22 (19) ◽  
pp. 10703
Author(s):  
Angela Marina Montalbano ◽  
Giuseppina Chiappara ◽  
Giusy Daniela Albano ◽  
Maria Ferraro ◽  
Caterina Di Sano ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Ex Vivo ◽  
In Vitro Study ◽  
Bronchial Epithelial Cell ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Bronchial Epithelial ◽  
Copd Patients

The role of PAR-1 expression and activation was described in epithelial cells from the central and distal airways of COPD patients using an ex vivo/in vitro model. PAR-1 immunoreactivity was studied in epithelial cells from surgical specimens of the central and distal airways of COPD patients and healthy control (HC). Furthermore, PAR-1 expression and activation were measured in both the human bronchial epithelial cell line (16HBE) and normal human bronchial epithelial cells (NHBEs) exposed to cigarette smoke extract (CSE) (10%) or thrombin. Finally, cell proliferation, apoptosis, and IL-8 release were detected in stimulated NHBEs. We identified higher levels of PAR-1 expression/activation in epithelial cells from the central airways of COPD patients than in HC. Active PAR-1 increased in epithelial cells from central and distal airways of COPD, with higher levels in COPD smokers (correlated with pack-years) than in COPD ex-smokers. 16HBE and NHBEs exposed to CSE or thrombin showed increased levels of active PAR-1 (localized in the cytoplasm) than baseline conditions, while NHBEs treated with thrombin or CSE showed increased levels of IL-8 proteins, with an additional effect when used in combination. Smoking habits generate the upregulation of PAR-1 expression/activation in airway epithelial cells, and promoting IL-8 release might affect the recruitment of infiltrating cells in the airways of COPD patients.

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