5-chloromethylfluorescein Diacetate Research Articles

Fluorescent non transgenic schistosoma to decipher host-parasite phenotype compatibility.

Schistosomiasis is considered as a significant public health problem, imposing a deeper understanding of the intricate interplay between parasites and their hosts. Unfortunately, current invasive methodologies employed to study the compatibility and the parasite development impose limitations on exploring diverse strains under various environmental conditions, thereby impeding progress in the field. In this study, we demonstrate the usefulness for the trematode parasite Schistosma mansoni, leveranging a fluorescence-imaging-based approach that employs fluorescein 5-chloromethylfluorescein diacetate (CMFDA) and 5-chloromethylfluorescein diacetate (CMAC) as organism tracker for intramolluscan studies involving the host snail Biomphalaria glabrata. These probes represent key tools for qualitatively assessing snail infections with unmatched accuracy and precision. By monitoring the fluorescence of parasites within the snail vector, our method exposes an unprecedented glimpse into the host-parasite compatibility landscape. The simplicity and sensitivity of our approach render it an ideal choice for evolutionary studies, as it sheds light on the intricate mechanisms governing host-parasite interactions. Fluorescent probe-based methods play a pivotal role in characterizing factors influencing parasite development and phenotype of compatibility, paving the way for innovative, effective, and sustainable solutions to enhance our understanding host-parasite immunobiological interaction and compatibility.

CHANGES TO THE F-ACTIN DISTRIBUTION OF THE CHONDROCYTE CYTOSKELETON IN EARLY OSTEOARTHRITIS

Cartilage degeneration and loss are key events in the initiation and progression of osteoarthritis (OA). Changes to chondrocyte volume and morphology (in the form of cytoplasmic processes) and thus cell phenotype are implicated, as they lead to the production of a mechanically-weakened extracellular matrix. The chondrocyte cytoskeleton is intimately linked to cell volume and morphology and hence we have investigated alterations to levels and distribution of chondrocyte F-actin that occur during early OA.The femoral heads (FH) from hip joints (N=16) were obtained with ethical permission and patient consent following femoral neck fracture. Cartilage was assessed as grade 0 (non-degenerate) and grade 1 (superficial fibrillation) using OARSI criteria. In situ chondrocyte volume and F-actin distribution were assessed using the fluorescent indicators (5-chloromethyl fluorescein diacetate (CMFDA)) and phalloidin, and imaged and quantified by confocal microscopy, ImarisTM and ImageJ software.There were no differences between the volume or total F-actin levels of in situ chondrocytes within the superficial zone of grade 0 (n=164 cells) compared to grade 1 (n=145) cartilage (P>0.05). However, a more detailed analysis of phalloidin labelling was performed, which demonstrated significant increases in both intense punctuate (IP) or intense areas (IA) (P<0.0001; P=0.0175 respectively). A preliminary analysis of IP and IA F-actin labelling suggested that while the former did not appear to be associated with changes to chondrocyte morphology, most of the cytoplasmic processes were associated with the presence of IA at the starting point of the protrusion.These results demonstrate marked changes to F-actin distribution in chondrocytes in the very early stages of cartilage degeneration as occurs in OA. These subtle changes are probably an early indication of a change to the chondrocyte phenotype and thus worthy of further study as they may lead to deleterious alterations to matrix metabolism and ultimately cartilage weakening.

Establishment of a Patient-Derived Organoid Culture Platform for Mantle Cell Lymphoma Drug Sensitivity and CAR T Cell Anti-Tumor Activity Assay

Background: With BTK inhibitors and brexucabtagene autoleucel CAR-T cell therapy approved by FDA to treat B-cell malignancies, survival of patients with mantle cell lymphoma (MCL) has been greatly improved. However, frequent therapeutic relapses in patients remains as an unmet medical challenge. Preclinical tumor mouse models such as patient-derived xenografts (PDXs) are often used for drug sensitivity and treatment prediction. However, these conventional cancer models are time-consuming and expensive. In recent years, development and application of patient-derived organoid (PDO) models have led to many discoveries in various types of solid tumors. PDO models have demonstrated many advantages over PDX models. For example, PDOs can be established using biopsies as small as needle biopsies yet with much higher success rates. However, a PDO model has not been reported yet in MCL or in a few other hematologic malignancies. Therefore, in this study, with successful establishment of MCL PDO models, we optimized our protocols and applied this model for drug sensitivity screens and rapid evaluation of immunotherapies. Methods: Patient samples were lysed with RBC lysis buffer and washed with PBS, then cell pellets were resuspended in culture medium containing cytokine cocktails. The cell suspension was aliquoted into V-bottom 96-well plates and spun. The cell aggregates within 50% Matrigel were gently transferred into multiple-well plates as needed. After solidification of the cell-Matrigel mixture, pre-warmed culture medium was added to cover them. The PDOs in 24 or 96-well plates were treated with drug or co-cultured with CAR T cells for 3-5 days. The cell viability and T cell killing activity were determined using CellTiter-Glo 3D assay kits. Results: We successfully established and optimized an organoid culture procedure and culture medium for diverse types of biopsy from patients with MCL. The success rate of MCL organoid development from diverse MCL biopsies was greater than 90%, much higher than for PDX. H&E staining of both tumors and their derived organoids revealed similar histological features (Figure 1A). To examine the drug sensitivity of primary MCL cells, organoid cultures derived from the patient apheresis biopsies that were either BTK inhibitor-sensitive (naive) or multiple drug resistant (to acalabrutinib, rituximab, and venetoclax) were treated with the BTK inhibitors ibrutinib or pirtobrutinib or else doxorubicin (as a control) for 48 h. Cell viability assays indicated that MCL cells in organoid cultures maintained the responses of the original tumor cells to drugs used in clinical treatment. We also demonstrated that the MCL organoids had the same major cell lineage populations as the original biopsy. These results support the notion that the PDO in vitro assay has great potential to evaluate and predict patient responses. In addition, we applied this PDO platform to evaluate CAR T cell anti-tumor activity by co-culturing the primary MCL cells with CD19-targeting CAR T cells in organoids for 3 days. To visualize and quantitate tumor killing potential, the MCL cells were labelled with the green dye 5-chloromethyl fluorescein diacetate. The cocultures were examined by imaging and FACS analysis. Compared to the same batch of peripheral blood monocytes used to generating CAR T cells, the CD19-CAR T cells very effectively eradicated CD19+ MCL cells from the co-cultures (Figure 1B), indicating that the PDO platform is a promising in vitro assay for rapid examining CAR T cell activity. Importantly, we validated drug responses in parallel in PDO and PDX models derived from the same tumors. Our preliminary results displayed high consistency between these assay systems, suggesting that PDO-based assay provides an encouraging platform for predicting clinical treatment response. Conclusion: We have successfully established and optimized an MCL PDO platform that can be used to perform reliable and fast assays for drug sensitivity and CAR T cell anti-tumor activity in vitro. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phospholipase A2 increases the sensitivity of doxorubicin induced cell death in 3D breast cancer cell models

Doxorubicin is a common drug used for various cancer treatments including breast, ovarian, bladder, and lung cancer. However, inefficient penetration of cancer drugs into the interior of the three-dimensional (3D) tumor tissue limits the drug delivery. Increasing the dosage of drugs is an unacceptable solution, as patients may have a toxic reaction. Therefore, efficient drug delivery is crucial. We hypothesized that the addition of phospholipase A2 (PLA2), an enzyme found in bee venom that hydrolyzes the bond connecting fatty acid and glycerol, would increase the permeability of the drug for efficient drug penetration. We designed a 3D spheroid tumor model because it mimics an actual tumor environment better than the monolayer two-dimensional (2D) cell culture. We performed dye permeability assay using 5-chloromethylfluorescein diacetate (CMFDA), a green fluorescent probe, on 3D spheroid cells with concentration-dependent PLA2 treatment. We found that 1 mM PLA2 had the highest percentage of green fluorescence cells, indicating the highest permeability. Subsequently, we hypothesized that cotreatment of PLA2 and doxorubicin may increase drug sensitivity of doxorubicin. After analyzing the half-maximal inhibitory concentration, IC50 (639.8 nM) of doxorubicin on the MDA-MB-231 3D spheroid, the cotreatment of PLA2 and doxorubicin exhibited lower 3D spheroid cell viability compared to doxorubicin treatment alone. This indicates that PLA2 may be a useful drug to assist in more effective doxorubicin treatment for breast cancer. Increased efficiency in drug delivery would allow lower concentrations of drugs to be used, minimizing damage to normal cells.

The hepatocyte export carrier inhibition assay improves the separation of hepatotoxic from non-hepatotoxic compounds

An in vitro/in silico method that determines the risk of human drug induced liver injury in relation to oral doses and blood concentrations of drugs was recently introduced. This method utilizes information on the maximal blood concentration (Cmax) for a specific dose of a test compound, which can be estimated using physiologically-based pharmacokinetic modelling, and a cytotoxicity test in cultured human hepatocytes. In the present study, we analyzed if the addition of an assay that measures the inhibition of bile acid export carriers, like BSEP and/or MRP2, to the existing method improves the differentiation of hepatotoxic and non-hepatotoxic compounds. Therefore, an export assay for 5-chloromethylfluorescein diacetate (CMFDA) was established. We tested 36 compounds in a concentration-dependent manner for which the risk of hepatotoxicity for specific oral doses and the capacity to inhibit hepatocyte export carriers are known. Compared to the CTB cytotoxicity test, substantially lower EC10 values were obtained using the CMFDA assay for several known BSEP and/or MRP2 inhibitors. To quantify if the addition of the CMFDA assay to our test system improves the overall separation of hepatotoxic from non-hepatotoxic compounds, the toxicity separation index (TSI) was calculated. We obtained a better TSI using the lower alert concentration from either the CMFDA or the CTB test (TSI: 0.886) compared to considering the CTB test alone (TSI: 0.775). In conclusion, the data show that integration of the CMFDA assay with an in vitro test battery improves the differentiation of hepatotoxic and non-hepatotoxic compounds in a set of compounds that includes bile acid export carrier inhibitors.

Preliminary verification studies of the motility and fluorescence assay (MFA) for ballast water quality monitoring

The International Maritime Organization (IMO) has adopted a ballast water convention to prevent spread of invasive species with ballast water and with it comes the need for fast and reliable tests to estimate the number of viable organisms found in ballast water.During 2017 and 2018, computer-assisted automatic quantification of plankton was compared with direct microscopic counts. Analysed samples represented simulations of ballast water samples from Denmark. Samples consisted of live plankton monocultures, mixed cultures, or ambient organisms from local seawater. The aim was to validate the performance and efficiency of the automated Motility and Fluorescence Assay (MFA) system BallastWISE, which automatically counts motile and fluorescent organisms in the fractions ≥10 - <50 μm and ≥ 50 μm, according to the ballast water discharge standard.Standardized counting methods for comparison were completed under microscope. Organisms in the ≥10 - <50 μm size range were analysed using an FDA (Fluorescein diacetate) and CMFDA (5-chloromethylfluorescein diacetate) staining method, and a standard movement and response to stimuli technique was used for the ≥50 μm size range.BallastWISE results correlated well with direct microscopic counts, showing promising counting performance and efficiency when it comes to rapid analysis of ballast water plankton samples. Quantification of monoculture and mixed cultures using BallastWISE correlated well with manual viable counts (Tetraselmis suecica in size range ≥ 10 - <50 μm: Pearson correlation (r) = 1.0; Tigriopus californicus and Brachionus plicatilis in size range ≥ 50 μm: r = 0.99). BallastWISE counts also correlated well with viable microscopic counts of ambient organisms in natural seawater (size range ≥ 10 - <50 μm: r = 0.97 and size range ≥ 50 μm: r = 0.89). We propose that computer-assisted quantification of plankton provides a rapid and precise alternative to traditional counting and identification of organisms in water samples such as ballast water.

Triphenyltin disrupts intracellular Zn2+ homeostasis in rat thymic lymphocytes

Triphenyltin (TPT), previously used as an agricultural fungicide and industrial antifoulant, is now considered an environmental pollutant. The effect of TPT on human health is concerning due to its presence as a contaminant in seafood. In this study, the changes in intracellular Zn2+ concentration ([Zn2+]i) and cellular content of nonprotein thiols ([NPT]i) induced by triphenyltin chloride (TPTCH), were measured in rat thymic lymphocytes. This was studied by flow-cytometry using the fluorescent probes FluoZin-3-AM and 5-chloromethylfluorescein diacetate (5-CMF-DA). Incubation with TPTCH, at 0.1 μM or more (up to 3 μM), increased [Zn2+]i in a concentration-dependent manner. The TPTCH-induced elevation in [Zn2+]i was due to the increase in membrane Zn2+ permeability and intracellular Zn2+ release. Incubation with TPTCH at 0.3 μM significantly increased [NPT]i levels, whereas the addition of an intracellular Zn2+ chelator had no effect on the same. TPT at higher concentrations (1 or 3 μM) reduced [NPT]i. TPT may disturb intracellular Zn2+ signaling in lymphocytes that disturbs cellular functions.

Dragotcytosis: Elucidation of the Mechanism for Cryptococcus neoformans Macrophage-to-Macrophage Transfer.

Cryptococcus neoformans is a pathogenic yeast capable of a unique and intriguing form of cell-to-cell transfer between macrophage cells. The mechanism for cell-to-cell transfer is not understood. In this study, we imaged mouse macrophages with CellTracker Green 5-chloromethylfluorescein diacetate-labeled cytosol to ascertain whether cytosol was shared between donor and acceptor macrophages. Analysis of several transfer events detected no transfer of cytosol from donor-to-acceptor mouse macrophages. However, blocking Fc and complement receptors resulted in a major diminution of cell-to-cell transfer events. The timing of cell-to-cell transfer (11.17 min) closely approximated the sum of phagocytosis (4.18 min) and exocytosis (6.71 min) times. We propose that macrophage cell-to-cell transfer represents a nonlytic exocytosis event, followed by phagocytosis into a macrophage that is in close proximity, and name this process Dragotcytosis ("Dragot" is a Greek surname meaning "sentinel"), as it represents sharing of a microbe between two sentinel cells of the innate immune system.

The Effect of Rhodamine-Derived Superparamagnetic Maghemite Nanoparticles on the Motility of Human Mesenchymal Stem Cells and Mouse Embryonic Fibroblast Cells.

Nanoparticles have become popular in life sciences in the last few years. They have been produced in many variants and have recently been used in both biological experiments and in clinical applications. Due to concerns over nanomaterial risks, there has been a dramatic increase in investigations focused on safety research. The aim of this paper is to present the advanced testing of rhodamine-derived superparamagnetic maghemite nanoparticles (SAMN-R), which are used for their nontoxicity, biocompatibility, biodegradability, and magnetic properties. Recent results were expanded upon from the basic cytotoxic tests to evaluate cell proliferation and migration potential. Two cell types were used for the cell proliferation and tracking study: mouse embryonic fibroblast cells (3T3) and human mesenchymal stem cells (hMSCs). Advanced microscopic methods allowed for the precise quantification of the function of both cell types. This study has demonstrated that a dose of nanoparticles lower than 20 µg·cm−2 per area of the dish does not negatively affect the cells’ morphology, migration, cytoskeletal function, proliferation, potential for wound healing, and single-cell migration in comparison to standard CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate). A higher dose of nanoparticles could be a potential risk for cytoskeletal folding and detachment of the cells from the solid extracellular matrix.

In Vitro Monocyte/Macrophage Phagocytosis Assay for the Prediction of Drug-Induced Thrombocytopenia.

Phagocytosis of platelets by monocytes and macrophages is a primary mechanism of platelet clearance in vivo and has been increasingly implicated in playing an important role in thrombocytopenia mediated by monoclonal antibodies intended for therapeutic purposes. In the present article, we describe an in vitro flow cytometry assay to assess the effect of antibody-mediated platelet phagocytosis by monocytes. Freshly isolated platelets were labeled with a fluorescent probe, 5-chloromethylfluorescein diacetate (CMFDA) and then co-cultured with isolated peripheral blood mononuclear cells (PBMCs) from the same donor in the presence of increasing concentrations of a monoclonal antibody drug. After incubation, an increase in CMFDA fluorescence intensity of CD14 positive monocytes was evaluated by flow cytometry as an assessment for drug-mediated platelet phagocytosis by monocytes. The assay has been evaluated using both human and cynomolgus monkey cells for the prediction of drug-induced thrombocytopenia. © 2019 by John Wiley & Sons, Inc.

Characterisation of a functional rat hepatocyte spheroid model

Many in vitro liver cell models, such as 2D systems, that are used to assess the hepatotoxic potential of xenobiotics suffer major limitations arising from a lack of preservation of physiological phenotype and metabolic competence. To circumvent some of these limitations there has been increased focus on producing more representative 3D models. Here we have used a novel approach to construct a size-controllable 3D hepatic spheroid model using freshly isolated primary rat hepatocytes (PRH) utilising the liquid-overlay technique whereby PRH spontaneously self-assemble in to 3D microtissues. This system produces viable spheroids with a compact in vivo-like structure for up to 21 days with sustained albumin production for the duration of the culture period. F-actin was seen throughout the spheroid body and P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) transporters had polarised expression on the canalicular membrane of hepatocytes within the spheroids upon formation (day 3). The MRP2 transporter was able to functionally transport 5 μM 5-chloromethylfluorescein diacetate (CMFDA) substrates into these canalicular structures. These PRH spheroids display in vivo characteristics including direct cell-cell contacts, cellular polarisation, 3D cellular morphology, and formation of functional secondary structures throughout the spheroid. Such a well-characterised system could be readily exploited for pre-clinical and non-clinical repeat-dose investigations and could make a significant contribution to replace, reduce and refine the use of animals for applied research.

Cytotoxicity of benzophenone-3, an organic ultraviolet filter, caused by increased intracellular Zn2+ levels in rat thymocytes

Benzophenone-3 (BP-3) is often used as an ultraviolet (UV) light filter in sunscreen products. Although BP-3 protects the human skin and hair from damage caused by excessive UV radiation, it is reported to exhibit toxic effects in human. However, the cytotoxicity of BP-3 on various cells is still not well understood. In the present study, the cytotoxicity of BP-3 against rat thymocytes was evaluated using a flow cytometric technique with fluorescent probes. Cell mortality increased significantly after 3 h of exposure to 300 μM BP-3, whereas the mean intensity of 5-chloromethylfluorescein diacetate (5-CMF) fluorescence and cellular content of non-protein thiols decreased significantly. However, the membrane potential of thymocytes was not change by BP-3 treatment. Moreover, intracellular Zn2+ levels increased significantly in a concentration-dependent manner in response to 30 μM BP-3 or higher. The BP-3-induced changes in intracellular Zn2+ levels and non-protein thiol content increased the vulnerability of thymocytes to oxidative stress. We concluded that BP-3-induced cytotoxicity may be caused by oxidative stress associated with an increase in intracellular Zn2+ levels.

A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis.

ObjectivesStaphylococcus aureus (S. aureus) is the most commonly implicated organism in septic arthritis, a condition that may be highly destructive to articular cartilage. Previous studies investigating laboratory and clinical strains of S. aureus have demonstrated that potent toxins induced significant chondrocyte death, although the precise toxin or toxins that were involved was unknown. In this study, we used isogenic S. aureus mutants to assess the influence of alpha (Hla)-, beta (Hlb)-, and gamma (Hlg)-haemolysins, toxins considered important for the destruction of host tissue, on in situ bovine chondrocyte viability.MethodsBovine cartilage explants were cultured with isogenic S. aureus mutants and/or their culture supernatants. Chondrocyte viability was then assessed within defined regions of interest in the axial and coronal plane following live- and dead-cell imaging using the fluorescent probes 5-chloromethylfluorescein diacetate and propidium iodide, respectively, and confocal laser-scanning microscopy.ResultsHla-producing mutants caused substantial chondrocyte death compared with the toxin-deficient control (Hla-Hlb-Hlg-), whilst mutants producing Hlb and Hlg in the absence of Hla induced minimal chondrocyte death. Coronal studies established that Hla-induced chondrocyte death started in the superficial zone of cartilage and spread to deeper layers, whereas Hlb and Hlg toxins were without significant effect.ConclusionThis study identified Hla as a highly potent S. aureus toxin that caused rapid chondrocyte death in bovine cartilage, with other toxins or metabolic products produced by the bacteria playing a minor role. The identification of Hla in mediating chondrocyte death may assist in the development of therapeutic strategies aimed at reducing the extent of cartilage damage during and after an episode of septic arthritis.Cite this article: I. D. M. Smith, K. M. Milto, C. J. Doherty, S. G. B. Amyes, A. H. R. W. Simpson, A. C. Hall. A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis. Bone Joint Res 2018;7:457–467. DOI: 10.1302/2046-3758.77.BJR-2017-0165.R1.

The Biological Basis for Ballast Water Performance Standards: “Viable/Non-Viable” or “Live/Dead”?

The shipping industry is critical to international commerce; however, contemporary shipping practices involve uptake and discharge of ballast water, which introduces the potential for transfer of nonindigenous, invasive species among geographically distinct habitats. To counteract this hazard, regulations for ballast water management have been implemented by the International Maritime Organization (IMO) and by regulatory agencies such as the United States Coast Guard (USCG). IMO and USCG discharge standards are numerically identical, but involve different definitions of treatment end points, which are based on fundamentally different biological assays for quantification of ballast water treatment effectiveness. Available assays for quantification of the responses of organisms in the 10-50 μm size range include vital stains based on fluorescein diacetate (FDA), sometimes used in combination with 5-chloromethylfluorescein diacetate (CMFDA), observations of motility, and the most probable number dilution culture method (MPN). The mechanisms and implications of these assays are discussed relative to the Type Approval process, which quantitatively evaluates compliance with ballast water discharge standards (BWDSs) under controlled shipboard and land-based tests. For antimicrobial processes that accomplish treatment by preventing subsequent replication of the target species, the FDA/CMFDA and MPN methods can yield dramatically different results. An important example of a treatment process that is affected by the choice of assay is ultraviolet (UV) irradiation. Results of laboratory and field experiments have demonstrated UV-based technologies to be effective for accomplishing the objectives of ballast water treatment (inactivation of cellular reproduction), when the MPN assay is used as the basis for evaluation. The FDA, CMFDA, motility, and MPN methods are subject to well recognized sources of error; however, the MPN method is based on a response that is consistent with the objectives of ballast water management as well as the mechanism of action of UV-based inactivation. Complementary assays are available for use in compliance testing; however, the development of relevant indicative tests remains as a research priority. Historical lessons learned from applications of vital stains (and other indirect methods) for quantification of microbial responses to UV irradiation in other settings also support the use of assays that provide a direct measure of growth and reproduction, such as MPN. Collectively, these observations point to the use of MPN assays as the standard for type testing, especially when UV-based treatment is employed.

Characterization of the glutathione S-transferases that belong to the GSTFuA class in Ceriporiopsis subvermispora: Implications in intracellular detoxification and metabolism of wood-derived compounds

Glutathione S-transferases (GSTs) of wood-degrading fungi play essential roles in cellular detoxification processes and endogenous metabolism. Fungal GSTs of GSTFuA class are suggested to be involved in lignin degradation. Ceriporiopsis subvermispora is one of the important model fungi of the selective lignin degraders, we found it interesting to study its GSTs. Here, we characterized the activities of two GSTs of the GSTFuA class of C. subvermispora (CsGST63524 and CsGST83044). A high-yield expression systems involving Escherichia coli was developed for each of these enzymes. Both enzymes were found to exhibit GSH-conjugation activity toward 1-chloro-2,4-dinitrobenzene, and GSH-peroxidase activity toward cumene hydroperoxide. Both enzymes showed high GSH-conjugation activity under basic conditions (pH8.0 to 9.0), and the optimum temperature for their activity was 40°C. In addition, three fluorescent compounds were used i.e., methylumbelliferyl acetovanillone was used to monitor etherase activity, and 5-chloromethylfluorescein diacetate and 4-methylumbelliferyl acetate to monitor esterase activity. CsGST83044 exhibited both etherase and esterase activities, while CsGST63524 displayed only esterase activity, which was much higher than that of CsGST83044. These findings imply the functional diversity of the GSTFuA class GSTs of C. subvermispora, suggesting that each protein plays distinctive roles in both the fungal detoxification system and wood compound metabolism.

A novel organ culture model of a joint for the evaluation of static and dynamic load on articular cartilage.

ObjectivesThe purpose of this study was to create a novel ex vivo organ culture model for evaluating the effects of static and dynamic load on cartilage.MethodsThe metatarsophalangeal joints of 12 fresh cadaveric bovine feet were skinned and dissected aseptically, and cultured for up to four weeks. Dynamic movement was applied using a custom-made machine on six joints, with the others cultured under static conditions. Chondrocyte viability and matrix glycosaminoglycan (GAG) content were evaluated by the cell viability probes, 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), and dimethylmethylene blue (DMMB) assay, respectively.ResultsChondrocyte viability in the static model decreased significantly from 89.9% (sd 2.5%) (Day 0) to 66.5% (sd 13.1%) (Day 28), 94.7% (sd 1.1%) to 80. 9% (sd 5.8%) and 80.1% (sd 3.0%) to 46.9% (sd 8.5%) in the superficial quarter, central half and deep quarter of cartilage, respectively (p < 0.001 in each zone; one-way analysis of variance). The GAG content decreased significantly from 6.01 μg/mg (sd 0.06) (Day 0) to 4.71 μg/mg (sd 0.06) (Day 28) (p < 0.001; one-way analysis of variance). However, with dynamic movement, chondrocyte viability and GAG content were maintained at the Day 0 level over the four-week period without a significant change (chondrocyte viability: 92.0% (sd 4.0%) (Day 0) to 89.9% (sd 0.2%) (Day 28), 93.1% (sd 1.5%) to 93.8% (sd 0.9%) and 85.6% (sd 0.8%) to 84.0% (sd 2.9%) in the three corresponding zones; GAG content: 6.18 μg/mg (sd 0.15) (Day 0) to 6.06 μg/mg (sd 0.09) (Day 28)).ConclusionDynamic joint movement maintained chondrocyte viability and cartilage GAG content. This long-term whole joint culture model could be of value in providing a more natural and controlled platform for investigating the influence of joint movement on articular cartilage, and for evaluating novel therapies for cartilage repair.Cite this article: Y-C. Lin, A. C. Hall, A. H. R. W. Simpson. A novel organ culture model of a joint for the evaluation of static and dynamic load on articular cartilage. Bone Joint Res 2018;7:205–212. DOI: 10.1302/2046-3758.73.BJR-2017-0320.

Zinc-dependent and independent actions of hydroxyhydroquinone on rat thymic lymphocytes

Coffee contains hydroxyhydroquinone (HHQ). HHQ is one of the by-products released during bean roasting. Therefore, it is important to elucidate the bioactivity of HHQ to predict its beneficial or adverse effects on humans. We studied zinc-dependent and independent actions of commercially procured synthetic HHQ in rat thymocytes using flow cytometric techniques with propidium iodide, FluoZin-3-AM, 5-chloromethylfluorescein diacetate, and annexin V-FITC. HHQ at 1050 µM elevated intracellular Zn2+ levels by releasing intracellular Zn2+. HHQ at 10 µM increased cellular thiol content in a zinc-dependent manner. However, HHQ at 30–50 µM reduced cellular thiol content. Although the latter actions of HHQ (30–50 µM) were suggested to increase cell vulnerability to oxidative stress, HHQ at 0.3–100 µM significantly protected cells against oxidative stress induced by H2O2. The process of cell death induced by H2O2 was delayed by HHQ, although both H2O2 and HHQ increased the population of annexin V-positive living cells. However, HHQ at 10–30 µM promoted cell death induced by A23187, a calcium ionophore. HHQ at 10–30 µM exerted contrasting effects on cell death caused by oxidative stress and Ca2+ overload. Because HHQ is considered to possess diverse cellular actions, coffee with reduced amount of HHQ may be preferable to avoid potential adverse effects.

The clustering and morphology of chondrocytes in normal and mildly degenerate human femoral head cartilage studied by confocal laser scanning microscopy.

Chondrocytes are the major cell type present in hyaline cartilage and they play a crucial role in maintaining the mechanical resilience of the tissue through a balance of the synthesis and breakdown of extracellular matrix macromolecules. Histological assessment of cartilage suggests that articular chondrocytes insitu typically occur singly and demonstrate a rounded/elliptical morphology. However, there are suggestions that their grouping and fine shape is more complex and that these change with cartilage degeneration as occurs in osteoarthritis. In the present study we have used confocal laser scanning microscopy and fluorescently labelled insitu human chondrocytes and advanced imaging software to visualise chondrocyte clustering and detailed morphology within grade-0 (non-degenerate) and grade-1 (mildly degenerate) cartilage from human femoral heads. Graded human cartilage explants were incubated with 5-chloromethylfluorescein diacetate and propidium iodide to identify the morphology and viability, respectively, of insitu chondrocytes within superficial, mid- and deep zones. In grade-0 cartilage, the analysis of confocal microscope images showed that although the majority of chondrocytes were single and morphologically normal, clusters (i.e. three or more chondrocytes within the enclosed lacunar space) were occasionally observed in the superficial zone, and 15-25% of the cell population exhibited at least one cytoplasmic process of ~ 5μm in length. With degeneration, cluster number increased (~ 50%) but not significantly; however, the number of cells/cluster (P<0.001) and the percentage of cells forming clusters increased (P=0.0013). In the superficial zone but not the mid- or deep zones, the volume of clusters and average volume of chondrocytes in clusters increased (P<0.001 and P<0.05, respectively). The percentage of chondrocytes with processes, the number of processes/cell and the length of processes/cell increased in the superficial zone of grade-1 cartilage (P=0.0098, P=0.02 and P<0.001, respectively). Processes were categorised based on length (L0 - no cytoplasmic processes; L1<5μm; 5<L2≤10μm; 10<L3≤15μm; L4>15μm). With cartilage degeneration, for chondrocytes in all zones, there was a significant decrease (P=0.015) in the percentage of chondrocytes with 'normal' morphology (i.e. L0), with no change in the percentage of cells with L1 processes; however, there were significant increases in the other categories. In grade-0 cartilage, chondrocyte clustering and morphological abnormalities occurred and with degeneration these were exacerbated, particularly in the superficial zone. Chondrocyte clustering and abnormal morphology are associated with aberrant matrix metabolism, suggesting that these early changes to chondrocyte properties may be associated with cartilage degeneration.

Elevation of sensitivity to anticancer agents of human lung adenocarcinoma A549 cells by knockdown of claudin-2 expression in monolayer and spheroid culture models

Claudins, tight junctional proteins, regulate the paracellular permeability of ions and small molecules. Claudin-2 is highly expressed in human lung adenocarcinoma cells and is involved in the up-regulation of cell proliferation. However, the effect of claudin-2 on cellular sensitivity to anticancer agents has not been clarified. The cytotoxicity of anticancer agents such as cisplatin, gefitinib and doxorubicin (DXR) was increased by claudin-2 knockdown in A549 cells. Claudin-2 knockdown also significantly decreased the expression level of multidrug resistance-associated protein/ABCC2. The expression levels of other drug efflux transporters were unchanged. The intracellular accumulation of 5-chloromethylfluorescein diacetate (CMFDA) and DXR, substrates of ABCC2, was increased by claudin-2 knockdown, whereas the efflux was decreased. MK-571, an inhibitor of ABCC2, enhanced the cytotoxicity of anticancer agents. Claudin-2 knockdown decreased the levels of p-c-Jun and nuclear Sp1. SP600125, an inhibitor of c-Jun, and mithramycin, an inhibitor of Sp1, decreased the level of ABCC2. The promoter activity of ABCC2 was decreased by claudin-2 knockdown, SP600125 and mithramycin treatments, suggesting that claudin-2 is involved in the up-regulation of ABCC2 expression at the transcriptional level. Claudin-2 knockdown increased the paracellular permeability of DXR in a 2D monolayer culture model. In addition, the accumulation of DXR into spheroids was enhanced by claudin-2 knockdown, resulting in a reduction in cell viability. We suggest that claudin-2 may be a novel therapeutic target in lung adenocarcinoma, because claudin-2 knockdown increased the accumulation of anticancer agents in cancer cells and spheroids.

Minocycline Loaded Hybrid Composites Nanoparticles for Mesenchymal Stem Cells Differentiation into Osteogenesis.

Bone transplants are used to treat fractures and increase new tissue development in bone tissue engineering. Grafting of massive implantations showing slow curing rate and results in cell death for poor vascularization. The potentials of biocomposite scaffolds to mimic extracellular matrix (ECM) and including new biomaterials could produce a better substitute for new bone tissue formation. A purpose of this study is to analyze polycaprolactone/silk fibroin/hyaluronic acid/minocycline hydrochloride (PCL/SF/HA/MH) nanoparticles initiate human mesenchymal stem cells (MSCs) proliferation and differentiation into osteogenesis. Electrospraying technique was used to develop PCL, PCL/SF, PCL/SF/HA and PCL/SF/HA/MH hybrid biocomposite nanoparticles and characterization was analyzed by field emission scanning electron microscope (FESEM), contact angle and Fourier transform infrared spectroscopy (FT-IR). The obtained results proved that the particle diameter and water contact angle obtained around 0.54 ± 0.12 to 3.2 ± 0.18 µm and 43.93 ± 10.8° to 133.1 ± 12.4° respectively. The cell proliferation and cell-nanoparticle interactions analyzed using (3-(4,5-dimethyl thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) MTS assay (Promega, Madison, WI, USA), FESEM for cell morphology and 5-Chloromethylfluorescein diacetate (CMFDA) dye for imaging live cells. Osteogenic differentiation was proved by expression of osteocalcin, alkaline phosphatase activity (ALP) and mineralization was confirmed by using alizarin red (ARS). The quantity of cells was considerably increased in PCL/SF/HA/MH nanoparticles when compare to all other biocomposite nanoparticles and the cell interaction was observed more on PCL/SF/HA/MH nanoparticles. The electrosprayed PCL/SF/HA/MH biocomposite nanoparticle significantly initiated increased cell proliferation, osteogenic differentiation and mineralization, which provide huge potential for bone tissue engineering.