Number Of Cells In Culture Research Articles

HPV16 increases the number of migratory cancer stem cells and modulates their miRNA expression profile in oropharyngeal cancer.

Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous-cell-carcinoma (OPSCC). Although HPV+ OPSCC metastasize faster than HPV- tumors, they have a better prognosis. The molecular and cellular alterations underlying this pathobiology of HPV+ OPSCC remain elusive. In this study, we examined whether expression of HPV16-E6E7 targets the number of migratory and stationary cancer stem cells (CSC). Furthermore, we wanted to elucidate if aberrantly expressed miRNAs in migratory CSC may be responsible for progression of OPSCCs and whether they may serve as potential novel biomarkers for increased potential of metastasis. Our studies revealed that HPV16-E6E7 expression leads to an increase in the number of stationary (CD44high /EpCAMhigh ) stem cells in primary keratinocyte cultures. Most importantly, expression of E6E7 in the cell line H357 increased the migratory (CD44high /EpCAMlow ) CSC pool. This increase in migratory CSCs could also be confirmed in HPV+ OPSCC. Differentially expressed miRNAs from HPV16-E6E7 positive CD44high /EpCAMlow CSCs were validated by RT-qPCR and in situ hybridization on HPV16+ OPSCCs. These experiments led to the identification of miR-3194-5p, which is upregulated in primary HPV16+ OPSCC and matched metastasis. MiR-1281 was also found to be highly expressed in HPV+ and HPV- metastasis. As inhibition of this miRNA led to a markedly reduction of CD44high /EpCAMlow cells, it may prove to be a promising drug target. Taken together, our findings highlight the capability of HPV16 to modify the phenotype of infected stem cells and that miR-1281 and miR3194-5p may represent promising targets to block metastatic spread of OPSCC.

Normoxic to hypoxic switch of pre-activated NK cells leads to robust proliferation and enhanced effector function via stabilization of HIF-1α and inhibition of apoptosis

Abstract Although mature NK cells can function within hypoxic lymphatics and tissues, they show impaired anti-tumor functions in ex-vivo hypoxic environments. This raises a question regarding additional cues that may exist to allow full NK cell activation within the hypoxic tumor microenvironment. Here, we provide evidence that pre-activation of NK cells prior to hypoxic exposure is key to overcoming hypoxia-mediated impairment and inducing greater NK cell proliferation with enhanced cytolytic activity. Specifically, exposing pre-activated NK cells to 1.5% of pO2 hypoxia following 7-9 days of normoxic culture allowed HIF-1α stabilization and its target gene expression, leading to specific upregulation of NKp44 activating receptor and increased STAT3 phosphorylation. These hypoxia-exposed NK cells demonstrate concomitant reduction of apoptotic and p21/p53 senescence pathways, resulting in greater cell numbers in culture. Therefore, pre-existing proliferative signals allows HIF-1α-mediated adaptation of NK cells toward highly proliferative and cytolytic phenotypes, which may occur in vivo throughout tumor immunosurveillance.

196 - Development of a closed bag type humidifying container using a polyethersulfone nanofiber sheet membranes

We newly developed the nano fiber sheet (NF) which exhibit superior functions of both moisture permeability and waterproofing performance. Base on the urgent need to prevent cross contamination, especially in the management of CO2 cell culture incubators in aseptically controlled cell processing facility, we designed closed bag type humidification container comprised with this NF (Aquavator), and evaluated practical usefulness using cell culture assay. The nano fiber was made of polyethersulfone as φ350 nm in diameter, and constructed into a nonwoven fabric sheet with an average pore diameter of 450 nm by electrospinning method. The thickness of nano fiber sheets are adjustable between 20 to 90 µm. Aquavator was configured as a bag type container having an NF membrane on its one side; 27 × 31 cm square. Performance comparison with the other moisture-permeable waterproofing materials (Tyvek®, Beauty-ace®) are conducted. By the comparison with water bat (Conventional), a practical usability equipped in CO2 incubator and biological influence using cell culture assay are examined. Assessed parameters are %Room humidity, temperature, cultured cell number and viability, apoptosis. The results are the follows; 1) Aquavator exhibited superior performance both in moisture permeability and waterproofing by approximately 10 times higher than conventional products. (2) The adjustment ability of humidity by Aquavator showed equivalent to Conventional apparatus. (3) Cell culture analysis revealed no significant differences in cultured cell number growth, cell viability and apoptosis (3.29 ± 0.32, 3.04 ± 0.33) after 6 days culture between Aquavator and Conventional apparatus. We conclude that Aquavator produce equivalent performance of humidification as conventional water bat and contribute remarkable benefit to prevent cross contamination in CO2 cell culture incubator management.

Resveratrol Suppresses the Growth and Enhances Retinoic Acid Sensitivity of Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid cancer (ATC) is a highly lethal undifferentiated malignancy without reliable therapies. Retinoic acid (RA) has been employed to promote redifferentiation of thyroid cancers by increasing their I131 uptake and radio-sensitivity, but its effect(s) on ATCs has not yet been ascertained. Likewise, resveratrol induces cancer redifferentiation but, also in this case, its effects on ATCs remain unknown. These issues have been addresses in the current study using three human ATC cell lines (THJ-11T, THJ-16T, and THJ-21T) through multiple experimental approaches. The results reveal that RA exerts a small inhibitory effect on these cell lines. In comparison with normally cultured cells, the total cell number in resveratrol-treated THJ-16T and THJ-21T cultures significantly decreased (p < 0.05), and this effect was accompanied by reduced Cyclin D1 immuno-labeling, increased apoptotic fractions, and distinct caspase-3 activation. Resveratrol failed to inhibit growth but enhanced RA sensitivity of THJ-11T cells, suppressed peroxisome proliferator-activated receptor-β/δ (PPAR-β/δ), and upregulated cellular retinoic acid-binding protein 2 (CRABP2) and retinoic acid receptor beta (RAR-β) expression. Increased thyroglobulin (Tg) and E-cadherin levels and appearance of membranous E-cadherin were evidenced in resveratrol-treated THJ-11T cells. Our results demonstrate for the first time: (1) the therapeutic value of resveratrol by itself or in combination with RA in the management of ATCs, (2) the capacity of resveratrol to overcome RA resistance in ATC cells by reprogramming CRABP2/RAR- and fatty acid-binding protein 5 (FABP5)/PPAR-β/δ-mediated RA signaling, and (3) the redifferentiating potential of resveratrol in ATC cells.

Virulence Factors in Candida albicans and Streptococcus mutans Biofilms Mediated by Farnesol.

The aim of this study was to evaluate the effect of farnesol on the production of acids and hydrolytic enzymes by biofilms of Streptococcus mutans and Candida albicans. The present study also evaluated the time-kill curve and the effect of farnesol on matrix composition and structure of single-species and dual-species biofilms. Farnesol, at subinhibitory concentrations, showed a significant reduction in S. mutans biofilm acid production, but did not alter C. albicans hydrolytic enzyme production. The number of cultivable cells of both microorganisms was significantly reduced after 8h of contact with farnesol. Extracellular matrix protein content was reduced for biofilms formed in the presence of farnesol. In addition, confocal laser scanning and scanning electron microscopy displayed structural alterations in all biofilms treated with farnesol, which included reduction in viable cells and extracellular matrix. In conclusion, farnesol showed favorable properties controlling some virulence factors of S. mutans and C. albicans biofilms. These findings should stimulate further studies using this quorum-sensing molecule, combined with other drugs, to prevent or treat biofilm-associated oral diseases.

Increasing the autotrophic growth of Chlorella USTB-01 via the control of bacterial contamination by Bdellovibrio USTB-06.

(i) To obtain and identify the predatory bacteria for the control of contaminated bacteria and to promote the autotrophic growth of Chlorella USTB-01. (ii) To identify and measure the different cell numbers in microalgal culture using flow cytometer. A predatory bacterial strain was isolated using Escherichia coli BL21 as a sole prey host, which was identified as Bdellovibrio USTB-06 by the analysis of 16S rDNA sequence. A flow cytometer was successfully used to identify and measure the cell numbers of Chlorella USTB-01, the contaminated bacteria and Bdellovibrio USTB-06 simultaneously in the autotrophic culture of Chlorella USTB-01 according to the identification of the different cell sizes. With the addition of Bdellovibrio USTB-06 at initial 104 plaque-forming units per ml, the contaminated bacteria severely decreased by about five counts (in log10 CFU per ml) and the growth of Chlorella USTB-01 was greatly increased by 37·0% compared with those of control respectively. Bdellovibrio USTB-06 could effectively promote the growth of Chlorella USTB-01 via the killing of the contaminated bacteria. Our study reveals a good biotechnology method to increase the growth of Chlorella USTB-01 which is very important in the industry of microalgal culture.

Ex vivo expansion of human peripheral blood natural killer cells and cytotoxic T lymphocytes from lung cancer patients.

Lung cancer is the most common type of cancer with the highest cancer-associated mortality rates worldwide, as well as in Vietnam. Numerous studies have demonstrated that higher numbers and higher rate of activity of infiltrating natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) in the tumor are closely correlated with positive prognosis, tumor size decrease and longer survival of lung cancer patients. In the present study, the effectiveness of BINKIT® kit in the ex vivo expansion of NK cells and CTLs in the peripheral blood of 7 patients aged between 30 and 84 years with metastatic lung cancer was evaluated. After 21 days of culture, the average number of CTLs (CD3+CD8+) increased by 742.3-fold in the CTL culture, accounting for 72.2% of the cultured cell population, and the mean cell viability was 95.7%. In the NK cell culture, the average number of NK cells (CD3−CD56+) increased by 637.5-fold, accounting for 84.3% of the cultured cell population, with an average viability of 94.7%. The percentage of active NK cells (CD3−CD56+ bright) was 82.1%, which increased by 408.9-fold. Notably, a close correlation was identified between the numbers of cytokine-induced killer (CD3+CD56+) and NK (CD3−CD56+) cells in the NK cell culture (P<0.05). In the two culture conditions (namely NK cell and CTL cultures), no clear correlation was identified between the rate of initial immune cells in the peripheral blood and the corresponding number following ex vivo expansion (P>0.05). These results revealed that the method of expansion and activation of NK cells and CTLs from peripheral blood was successfully applied using BINKIT, and reached the requirements for clinical applications in cancer treatment in Vietnam.

Differential Neuroprotective Effects of Interleukin-1 Receptor Antagonist on Spinal Cord Neurons after Excitotoxic Injury

Secondary damage following spinal cord injury (SCI) induces neuronal damage through inflammatory and excitotoxic pathways. We hypothesized that the interleukin-1 receptor antagonist (IL1RA) protects neuronal populations and suppresses apoptosis and gliosis after injury. Spinal cord slice cultures (SCSCs) were subjected to excitotoxic injury with N-methyl-<smlcap>D</smlcap>-aspartate (NMDA) and treated with IL1RA. Immunohistochemistry for neuronal nuclei (NeuN), MacII, glial fibrillary acidic protein, and TdT-mediated dUTP nick end labelling stains were used to evaluate neuronal survival, glial activation, and apoptosis. Treatment with IL1RA significantly reduced the number of apoptotic cells in both NMDA-lesioned and unlesioned cultures. Experimental injury with NMDA reduced the number of NeuN-positive ventral horn neurons, and IL1RA treatment counteracted this loss 1 day after injury. However, IL1RA had no effect on the number of presumable Renshaw cells, identified by their selective expression of the cholinergic nicotinic α₂-receptor subunit (Chrna2). Activated microglial cells were more numerous in NMDA-lesioned cultures 1 day after injury, and IL1RA significantly reduced their numbers. We conclude that IL1RA modulates neuronal apoptosis and microglial activation in excitotoxically injured SCSCs. Renshaw cells were more susceptible to excitotoxic injury than other neurons and were not rescued by IL1RA treatment. Modulation of IL-1-mediated pathways may thus be effective in reducing excitotoxically induced neuronal damage after SCI, however only in specific neuronal populations, such as ventral horn neurons. These findings motivate further investigations of the possibility to antagonize inflammatory pathways after SCI.

Vinblastine and antihelmintic mebendazole potentiate temozolomide in resistant gliomas.

Glioblastoma (GBM) is a very aggressive tumor that has not had substantial therapeutic improvement since the introduction of temozolomide (TMZ) in combination with radiotherapy. Combining TMZ with other chemotherapeutic agents is a strategy that could be further explored for GBM. To search for molecular predictors of TMZ resistance, the TCGA (The Cancer Genome Atlas) database was utilized to assess the impact of specific genes on TMZ response. Patients whose tumors expressed low levels of FGFR3 and AKT2 responded poorly to TMZ. Combination treatment of vinblastine (VBL) plus mebendazole (MBZ) with TMZ was more effective in reducing cell number in most cultures when compared to TMZ alone, especially in cells with low expression levels of FGFR3 and AKT2. Cell cycle distribution and nuclear morphometric analysis indicated that the triple combination of TMZ, VBL and MBZ (TVM) was able to induce polyploidy and senescence, in addition to increasing the Notch3 RNA level in patient-derived gliomas. Thus, this set of data suggests that the triple combination of TMZ, VBL and MBZ may be a considerable therapeutic alternative for the TMZ-tolerant gliomas that harbor low expression of FGFR3/AKT2.

Cell Counting and Viability Assessment of 2D and 3D Cell Cultures: Expected Reliability of the Trypan Blue Assay

BackgroundWhatever the target of an experiment in cell biology, cell counting and viability assessment are always computed. The Trypan Blue (TB) assay was proposed about a century ago and is still the most widely used method to perform cell viability analysis. Furthermore, the combined use of TB with a haemocytometer is also considered the standard approach to estimate cell population density. There are numerous research articles reporting the use of TB assays to compute cell number and viability of 2D and 3D cultures. However, the literature still lacks studies regarding the reliability of the TB assay in terms of assessment of its repeatability and reproducibility.MethodsWe compared the TB assay's measurements obtained by two biologists who analysed 105 different samples in double-blind for a total of 210 counts performed. We measured: (a) the repeatability of the count performed by the same operator; (b) the reproducibility of counts performed by the two operators.ResultsThere were no significant differences in the results obtained with 2D and 3D cell cultures: we estimated an approximate variability of 5% when the TB assay was used to assess the viability of the culture, and a variability of around 20% when it was used to determine the cell population density.ConclusionsThe main aim of this study was to make researchers aware of potential measurement errors when TB is used with a haemocytometer for counting and viability measurements in 2D and 3D cultures. We believe that these results can help researchers to determine whether the expected reliability of the TB assay is compliant with their applications.

Antifungal activity of tyrosol and farnesol used in combination against Candida species in the planktonic state or forming biofilms.

This study examined the antifungal activity of the combination of tyrosol and farnesol against Candida albicans and Candida glabrata in the planktonic state or forming biofilms. The effect of drug association against Candida planktonic cells was assessed by the fractional inhibitory concentration index. Mono- and dual-species biofilms were developed during 24 h and then treated with the compounds for 3 days, with two daily treatments of 1 min each. After, the total biomass, metabolic activity and the number of cultivable cells were quantified. Planktonic cells of the two species showed a similar susceptibility to the drug combination, however, a synergistic effect was only verified for C. glabrata. Regarding biofilm susceptibility, significant reductions in C. glabrata biomass, metabolism of C. albicans and mixed biofilms, and cultivable cells of single biofilms were verified for the drug combination, indicating an additive effect. For all other experiments, the effects were classified as indifferent. The combined use of tyrosol and farnesol was advantageous for some of the analysed parameters against Candida species. These findings may contribute to the development of oral care products containing tyrosol and farnesol to combat oral infections caused by Candida species.

Stochasticity in the enterococcal sex pheromone response revealed by quantitative analysis of transcription in single cells.

In Enterococcus faecalis, sex pheromone-mediated transfer of antibiotic resistance plasmids can occur under unfavorable conditions, for example, when inducing pheromone concentrations are low and inhibiting pheromone concentrations are high. To better understand this paradox, we adapted fluorescence in situ hybridization chain reaction (HCR) methodology for simultaneous quantification of multiple E. faecalis transcripts at the single cell level. We present direct evidence for variability in the minimum period, maximum response level, and duration of response of individual cells to a specific inducing condition. Tracking of induction patterns of single cells temporally using a fluorescent reporter supported HCR findings. It also revealed subpopulations of rapid responders, even under low inducing pheromone concentrations where the overall response of the entire population was slow. The strong, rapid induction of small numbers of cells in cultures exposed to low pheromone concentrations is in agreement with predictions of a stochastic model of the enterococcal pheromone response. The previously documented complex regulatory circuitry controlling the pheromone response likely contributes to stochastic variation in this system. In addition to increasing our basic understanding of the biology of a horizontal gene transfer system regulated by cell-cell signaling, demonstration of the stochastic nature of the pheromone response also impacts any future efforts to develop therapeutic agents targeting the system. Quantitative single cell analysis using HCR also has great potential to elucidate important bacterial regulatory mechanisms not previously amenable to study at the single cell level, and to accelerate the pace of functional genomic studies.

Optimization of Methods for Articular Cartilage Surface Tissue Engineering: Cell Density and Transforming Growth Factor Beta Are Critical for Self-Assembly and Lubricin Secretion.

Lubricin/superficial zone protein (SZP)/proteoglycan4 (PRG4) plays an important role in boundary lubrication in articular cartilage. Lubricin is secreted by superficial zone chondrocytes and synoviocytes of the synovium. The specific objective of this investigation is to optimize the methods for tissue engineering of articular cartilage surface. The aim of this study is to investigate the effect of cell density on the self-assembly of superficial zone chondrocytes and lubricin secretion as a functional assessment. Superficial zone chondrocytes were cultivated as a monolayer at low, medium, and high densities. Chondrocytes at the three different densities were treated with transforming growth factor beta (TGF-β)1 twice a week or daily, and the accumulated lubricin in the culture medium was analyzed by immunoblots and quantitated by enzyme-linked immunosorbent assay (ELISA). Cell numbers in low and medium densities were increased by TGF-β1; whereas cell numbers in high-density cell cultures were decreased by twice-a-week treatment of TGF-β1. On the other hand, the cell numbers were maintained by daily TGF-β treatment. Immunoblots and quantitation of lubricin by ELISA analysis indicated that TGF-β1 stimulated lubricin secretion by superficial zone chondrocytes at all densities with twice-a-week TGF-β treatment. It is noteworthy that the daily treatment of TGF-β1 increased lubricin much higher compared with twice-a-week treatment. These data demonstrate that daily treatment is optimal for the TGF-β1 response in a higher density of monolayer cultures. These findings have implications for self-assembly of surface zone chondrocytes of articular cartilage for application in tissue engineering of articular cartilage surface.

Multiwalled Carbon Nanotube-Chitosan Scaffold: Cytotoxic, Apoptoti c, and Necrotic Effects on Chondrocyte Cell Lines.

Carbon nanotubes (CNTs) have been considered highly successful and proficient in terms of their mechanical, thermal and electrical functionalization and biocompatibility. In regards to their significant extent in bone regeneration, it has been determined that CNTs hold the capability to endure clinical applications through bone tissue engineering and orthopedic procedures. In the present study, we report on a composite preparation, involving the use of CNT-chitosan as scaffold for bone repair and regeneration. Through the use of water-soluble tetrazolium salt (WST-1) and double staining methods, the cytotoxic, necrotic, and apoptotic effects of chitosan-multiwalled carbon nanotube nanocomposites on the chondrocyte ATTC cell line have been exhibited. The chitosan-multiwalled carbon nanotube scaffolds were prepared. Chondrocytes differentiation tool (ATCC) cell line was prepared. WST-1 assay for cytotoxicity studies were performed by using chondrocytes cells in 12.5-200 μL concentration range. The samples of membranes (chitosan- multiwalled carbon nanotube scaffold) were measured at 2 mg/mL and further prepared amongst chitosan- multiwalled carbon nanotube scaffold's which were placed into separate wells. While in the process of incubation, in the four-hour time range, the plates were immediately read in an Elisa microplate Reader. To predict the number of apoptotic and necrotic cells in culture, the technique of double staining with Hoechst dye was performed with PI on the basis of scoring cell nuclei. The mechanical properties such as tensile strength and elongation at break values of the chitosan only and chitosan/CNT scaffolds were evaluated on Texture Analyzer. Based on the results of the WST-1 assay procedure, the amount of cell viability was not significantly affected by nanocomposite concentrations and the lowest mortality rate of cells was obtained at a concentration of 12.5 μg/mL, whereas the highest mortality rate was obtained at a rate of 200 μg/mL. In addition, the effects of chitosan-CNT nanocomposites were not found to cytotoxic on chondrocyte cells. The double staining method has been able to determine the apoptotic and necrotic effects of chitosan MWCNT nanocomposites. The apoptotic and necrotic effects of the combined compounds had varied within the concentrations. In a similar manner to the outcome of the control groups, apoptosis was obtained at a percentage of 2.67%. Under a fluorescent inverted microscope, the apoptotic cell nuclei were stained with a stronger blue fluorescence in comparison to non-apoptotic cells, which may have had an effect. We also compared the strain-stress curve measurements results. The results indicated that the mechanical properties of scaffold were not different. Elongation at break values increased by addition of CNT. CNTs as a biomaterial hold the potential to be used for applications in future regenerative medicine. By using the components of chondrocytes (ATTC) cell lines, the cytotoxicity evaluations were made for the chitosan-multiwalled carbon nanotube scaffold. The chitosan-MWCNT nanocomposites do not seem to induce drastic cytotoxicity to the chondrocyte cells.

Analysis of soluble factors in conditioned media derived from primary cultures of cirrhotic liver of biliary atresia.

Biliary atresia (BA) is a rare and serious liver disease in newborn infants. Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of BA may contain hepatic stem/progenitor cells in primary culture of NPC fractions. In this study, NPC fractions were subjected to primary or passage culture and found that clusters of hepatocyte-like cells appear even without adding hepatocyte growth factor (HGF) to the culture medium, but not in their passage culture used as a control. Based on these findings, conditioned media (CMs) were collected and soluble factors in the CMs were analyzed in order to elucidate the mechanism of the appearance of hepatocyte-like cells or their clusters. A large amount of active HGF consisting of α and β chains was detected in CMs derived from primary culture, but not in CMs from passage culture, as determined by western blot analysis, bone morphogenetic protein (BMP)-4, oncostatin M (OSM), and transforming growth factor (TGF)-β1 were not detected in any of the CMs. The number of hepatocyte-like cells in primary culture tended to decrease following treatment with the HGF receptor c-Met inhibitor, SU11274 in a dose-dependent manner. Furthermore, the clusters of hepatocyte-like cells tended to increase in size and number when freshly isolated NPC fractions were cultured in the presence of 10% of CMs collected after 3-4wk of primary culture. In conclusion, these findings indicate that CMs derived from primary culture of NPC fractions of BA liver contain a large amount of active HGF, which may activate hepatic stem/progenitor cells and promote the appearance of hepatocyte-like cells or their clusters through HGF/c-Met signaling. The present study would lead to cell therapy using the patient's own cells for the treatment of BA.

Screening Applications to Test Cellular Fitness in Transwell® Models After Nanoparticle Treatment.

Nanoparticles (NPs) in biotechnology hold great promise for revolutionizing medical treatments and therapies. In order to bring NPs into clinical application there is a number of preclinical in vitro and in vivo tests, which have to be applied before. The initial in vitro evaluation includes a detailed physicochemical characterization as well as biocompatibility tests, among others. For determination of biocompatibility at the cellular level, the correct choice of the in vitro assay as well as NP pretreatment is absolutely essential. There are a variety of assay technologies available that use standard plate readers to measure metabolic markers to estimate the number of viable cells in culture. Each cell viability assay has its own set of advantages and disadvantages. Regardless of the assay method chosen, the major factors critical for reproducibility and success include: (1) choosing the right assay after comparing optical NP properties with the read-out method of the assay, (2) verifying colloidal stability of NPs in cell culture media, (3) preparing a sterile and stable NP dispersion in cell culture media used in the assay, (4) using a tightly controlled and consistent cell model allowing appropriate characterization of NPs. This chapter will briefly summarize these different critical points, which can occur during biocompatibility screening applications of NPs.

Immune Privileged Features of Multipotent Mesenchymal Stromal Cells Are Lost after Co-Cultivation with Allogeneic Lymphocytes in Vitro

Multipotent mesenchymal stromal cells (MSCs) have immunomodulatory properties and have been successfully used for treatment of autoimmune diseases and acute or chronic graft-versus-host disease. Therapy with MSCs is not always effective. It has been shown that MSCs immunomodulatory properties can be improved by means of various agents, such as IFN-g, TNF-a, IL-17. After 4 hours of IFN-g exposure the expression level of immunomodulatory genes increased - IDO1 300, CSF1 - 7, and IL6 - 2.4 times. MSCs typically express low levels of MHC class I, and no MHC class II or co-stimulatory molecules (e.g., B7-1, B7-2, or CD40), making them partially immunoprivileged. However, treatment with IFN-g leads to increased expression of HLA-DR antigens on MSCs. After injection to the patient the characteristics of MSCs differ from those which have been studied in culture due to their interactions with other cells in the bloodstream and tissues. In this study the model of MSCs and MSCs treated with IFN-g (IFN-g-MSC) interactions with allogeneic lymphocytes in vitro was developed.The aim of the study was to identify the changes in MSCs and IFN-g-MSCs characteristics after co-cultivation with lymphocytes in vitro in dynamics.Materials and methodsMSCs were isolated from 13 bone marrow (BM) samples used for allogeneic hematopoietic cells transplantation and cultured by a standard method in aMEM with 10% fetal bovine serum (FBS). MSCs on 2-3-d passages were seeded 105 cells per flask with 25 cm2 bottom area and a day later 500 units/mL of IFN-g were added for 4 hours to half of the cultures. Then the media was changed on RPMI-1640 with 10% FBS. Some cultures were seeded with 106 allogeneic lymphocytes, to half of these cultures 5 mg/ml phytohemagglutinin (PHA) was added for lymphocytes activation. All flasks were cultured up to 4 days at 37°C and 5% CO2.After 1, 2, 3 and 4 days lymphocytes were washed from MSCs. MSCs were removed from the flasks with trypsin and the number of viable cells was determined by dye exclusion method (trypan blue). For each of the MSCs cultures the mean fluorescent signal intensity level (MFI) of HLA-DR was determined by direct immunofluorescent staining with anti-HLA-DR APC (BD Pharmingen) antibodies and measured on flow cytometer BD FACS Canto II (BD Biosciences, USA).Data are presented as mean ± standard error. Statistical analysis was performed using Student’s t-test (considered reliable p <0.05).ResultsThe number of cells in cultures of MSCs and IFN-g-MSCs did not differ significantly during the 4 days of observations. The presence of non-activated lymphocytes had no effect on the parameters of growth and viability of MSCs. Co-cultivation of activated lymphocytes with MSCs resulted in a reduction of viable MSCs, to 51.6 ± 5.5% compared with IFN-g-MSCs (68,1 ± 6,2%, p = 0.02). The viability of MSCs cultures without lymphocytes was 74.5% ± 4.8 on day 4 from the beginning of the experiment.HLA-DR expression level gradually increased in IFN-g-MSCs (see table). Co-culturing MSCs with lymphocytes also leads to a gradual increase in HLA-DR expression on MSCs apparently due to IFN-g, produced by lymphocytes. However, in IFN-g-MSCs co-cultured with lymphocytes HLA-DR MFI was significantly lower (p = 0.05) than without lymphocytes. HLA-DR MFI increased about 10 times in MSCs co-cultured with activated lymphocytes regardless of their IFN-g pretreatmentThe data obtained indicate that IFN-g-MSCs did not change in their growth characteristics and had increased immunomodulating properties. IFN-g-MSCs have a greater resistance to activated lymphocytes, which makes them more effective for cell therapy. The results suggest that injected to patient MSCs could have increased level of HLA-DR expression regardless of their initial treatment with IFN-g. The increase of HLA-DR expression on MSCs co-cultured with lymphocytes indicates a possibility of loss of immune privilege by these cells when injected to patient. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting Chronic Myeloid Leukemia Stem Cells through Pharmacological Inhibition of HIF-1α

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell (HSC) disorder due to the 9;22(q34.1;q11.2) reciprocal chromosomal translocation, which results in the generation of BCR/abl "fusion" oncogene and the expression of the BCR/Abl oncoprotein, a constitutively-active tyrosine kinase (TK). To date, chronic-phase CML patients are treated with TK inhibitors (TKi) such as imatinib- mesylate (IM) and dasatinib. However, to eradicate CML, it is necessary to eliminate Leukemia Stem Cells (LSC), which are largely insensitive to TKi. Thus, TKi, rather than curing CML definitively, induce a state of minimal residual disease (MRD) apparently sustained by LSC persistence. Therefore, the identification of non-TKi drugs capable to target and eradicate LSC responsible for MRD and therefore the risk of relapse of disease is of primary importance.LSC, as well as normal HSC, in vivo reside in low oxygen tension areas of the bone marrow (BM) called "HSC niche". Several previous studies from our group and others demonstrate that low oxygen tension maintains the survival and stem cell potential of HSC and LSC, favoring their self-renewal. Interestingly, we also found that the expression of BCR/Abl oncoprotein is suppressed in low oxygen, which well explains the refractoriness of LSC to TKi, provided they manage to survive in the absence of BCR/Abl signaling.Hypoxia-Inducible Factor-1α (HIF-1α) is a key regulator of cell adaptation to low oxygen and of LSC maintenance. This study addresses the in vitro and in vivo effects of the pharmacological inhibition of HIF-1α on the maintenance of CML stem cell potential.After screening several HIF-1α inhibitors, we found that Acriflavine (ACF) induced apoptosis and DNA damage in CML cells and identified ACF as a very effective inhibitor of CML cell survival and growth in low oxygen, as regards the number of viable cells in culture and colony formation efficiency of CML cell lines or human or murine primary cells. Interestingly, ACF did not affect the colony formation efficiency of normal human hematopoietic cells. We also demonstrated in this study that IM and ACF exhibited an at least additive effect as far as the reduction of the total number of viable cells in low oxygen is concerned. Moreover, we demonstrated that low-oxygen incubation of CML cells increased the expression of genes related to stem cell potential, such as Nanog, Sox2 and Oct4, and that ACF treatment reduced their expression. These results on one hand confirmed that hypoxia selects cells with SC potential, on the other hand they demonstrated that ACF treatment reduces their maintenance.Moreover, we evaluated the maintenance of CML stem cell potential using a stem cell assay, demonstrating that ACF suppressed the stem cell potential of CML cell lines or primary cells. On the contrary, IM or Dasatinib, were unable to affect the stem cell potential of CML cells also when administered to primary samples neither TKi-treated before nor carrying primary mutations of BCR/Abl kinase domain. This is in agreement with what observed before by us and others and confirms the refractoriness of LSC to TKi.The effects of ACF in vivo were evaluated using a murine CML model where the disease was induced by a BCR/abl-carrying retrovirus. A 10 day-long ACF treatment was sufficient to attenuate CML development in mice, as evidenced by lower spleen weight, fewer leukemia cells in peripheral blood and BM, and a markedly reduced leukemia cell infiltration into the lungs. Moreover, it was possible to confirm in vivo the effects of ACF on LSC observed in vitro. ACF treatment indeed reduced the maintenance of the Long-Term LSC-containing LSK cell subset (CD34- LSK) in the BM of CML mice. Interestingly and in keeping with the less-severe addiction of HSC to HIF-1α deficiency, we found that non-CML cells were significantly less sensitive to ACF treatment than CML cells. This points to a good therapeutic index of ACF in discriminating leukemic from normal hematopoietic cells.On the basis of all above, the ACF/TKi combination may represent a novel therapeutic approach to target at one time CML cell bulk and LSC, in order to induce remission and prevent MRD of CML. Interestingly, ACF is an already FDA-approved drug for non-oncological uses in humans. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Human Erythropoiesis during Inflammation Is Mediated By High Mobility Group Box Protein 1 (HMGB1) through Decreased Commitment of Hematopoietic Stem Cells to the Erythroid Lineage and By Increased Apoptosis of Terminally Differentiating Erythroblasts

Anemia plays a significant role in the morbidity and mortality of chronic inflammatory diseases and post-sepsis recovery. Inflammation-associated anemia is further complicated as these conditions are typically refractory to erythropoietin (EPO) administration, which suggests that circulating inflammatory molecules prevent effective erythropoiesis. High mobility group box protein 1 (HMGB1), a pro-inflammatory molecule involved in sepsis has been shown to mediate anemia in a murine model of sepsis survivors. However, the effects of soluble HMGB1 on human erythropoiesis remain poorly understood. To address this issue, we studied the effect of increasing concentrations of HMGB1 on human erythropoiesis in vitro using CD34+ hematopoietic stem and progenitor cells (CB HSPCs) and a previously described 3-phase erythroid differentiation culture system to asses the effects on erythroid cell proliferation as well as the semisolid methylcellulose assay to study effects on erythroid progenitors. HMGB1 markedly reduced erythroid cell proliferation in the 3-phase culture system in a dose-dependent manner. We evaluated the contributions of early- and late-stage erythropoiesis to decreased erythroid proliferation using using flow cytometric analyses of cell surface markers interleukin-3 receptor (IL-3R), glycophorin A (GPA), CD36, CD34 (BFU-E and CFU-E) and GPA, α4-integrin and band 3 (ProEB-orthoEB). HMGB1 increased the percentage of IL-3Rpos cells (vehicle v HMGB1: 8.6% vs 15.5%), and reduced the percentage of GPApos cells (vehicle v HMGB1: 32.2% v 12.8%) implying decreased commitment of CB HSPCs to erythroid lineage in the presence of HMGB1. Flow cytometric analysis confirmed an increased commitment to myeloid lineage and Giemsa-stained cytospins revealed increased numbers of myelo-monocytic cells in HMGB1 cultures. Furthermore, the decreased number of erythroid progenitors generated in culture was blocked in terminal differentiation leading to increased apoptosis of erythroblasts.Since we found increased numbers of myelo-monocytic cells and increased erythroblast apoptosis in HMGB1 treated cultures, we performed additional studies to investigate the role of soluble HMGB1 on HSPC lineage commitment and erythroblast survival signaling (i.e. EPO signaling). CB CD34+ HSPCs were seeded in fully enriched methylcellulose ± HMGB1, and hematopoietic colony numbers were quantified based on morphologic characteristics. HMGB1 increased the number of CFU-GM colonies and decreased the number of erythroid colonies (CFU-E, BFU-E). Further, erythroblasts treated with 3IU/mL EPO ± HMGB1 revealed that HMGB1 significantly reduced EPO-dependent STAT5 phosphorylation in a dose-dependent manner.In order to begin to identify the HMGB1 receptors responsible for HSPC lineage skewing and erythroblast apoptosis we used transcriptome-wide RNA-sequencing data to examine the expression patterns of various HMGB1 receptors during differentiation of HSPCs in our culture system. Toll-like receptor 2 (TLR2), TLR4 and CXCR4 are expressed during the early-stages of erythroid differentiation while the receptor for advanced glycation end products (RAGE) is expressed only during late stages of erythroid differentiation. These findings suggest that two discrete HMGB1-receptor signaling axes may underlie the noted changes in early- and late-stage cellular differentiation in our culture system.Taken together, our results identify HMGB1 as a potent inhibitor of human erythropoiesis. HMGB1 exerts this effect at two levels of erythroid differentiation, the first during the commitment of HSPC to erythroid differentiation and subsequently during terminal erythroid differentiation. The erythroid differentiation defect appears to be partially dependent on EPO signaling while it has no effect on the HSPC defect. We proposed that the elevated HMGB1 during inflammation could explain the persistent erythroid defect observed in patients with chronic inflammatory diseases and sepsis survivors. DisclosuresNo relevant conflicts of interest to declare.

Proteasome Inhibitors and a Fusicoccin Derivative (ISIR-042) Cooperatively Inhibit Proliferation of Myeloma Cells through Induction of C/EBP-Homologous Protein (CHOP/GADD153)

Combinations of bortezomib and novel targeted therapeutic agents may synergistically increase antitumor effect and may overcome specific cellular resistance and/or antiapoptotic processes. However, highly effective combinations of bortezomib with targeted therapeutic agents have not been developed to date. We examined the synergistic effects of various compounds and bortezomib on myeloma cells and identified fusicoccin derivative ISIR-042 as the most potent drug. We developed ISIR-042 that exerts higher cytotoxic effects on hypoxic cells than on normoxic cells and that preferentially inhibits stem/progenitor cells in pancreatic cancer cell lines compared with other chemotherapeutic agents (Kawakami et al, Anticancer Agents Med Chem 2012). In the present study, we determined the synergistic effects of bortezomib and ISIR-042 cotreatment on human multiple myeloma cells. Human multiple myeloma cell lines RPMI8226, KMS-11, and KMS-26 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2 in air. Viability of cells cultured with or without the test compounds for indicated time was examined by performing a modified MTT assay.Bone marrow specimens and ascitic fluids were collected from patients at diagnosis or relapse after obtaining written informed consent for sample collection in accordance with institutional policy. The study was approved by the Institutional Review Board of Shimane University. For performing colony-forming assay, the cells (1 × 104/dish) were plated in 1.1 ml semisolid methylcellulose medium supplemented with 0.8% methylcellulose and 20% FBS in triplicates for 10 days. CHOP-/- and CHOP+/+ MEF cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. Expression of cell surface antigens was analyzed by performing flow cytometry with anti-CD38 or anti-CD138 antibody. Combined effects of myeloma cells in primary culture were analyzed by performing flow cytometry with anti-CD19 and anti-CD138 antibodies. Expression of caspase-10, BCL-2, p62, BCLAF1, LC3, actin, and CHOP was determined by performing western blotting with respective monoclonal antibodies. mRNA expression of genes encoding XBP-1u and XBP-1s was analyzed by performing reverse transcription-PCR. CHOP mRNA levels were measured by performing real-time PCR.ISIR-042 inhibited the growth of RPMI8226 cells in a concentration-dependent manner and exerted synergistic effects with bortezomib. ISIR-042 also exerted synergistic effects with carfilzomib. Results of flow cytometry showed that the combination of ISIR-042 and bortezomib decreased the number of myeloma cells in the primary culture in a concentration-dependently. Colony-forming assay showed that RPMI8226 cells were more sensitive than normal mouse hematopoietic cells to ISIR-042 and bortezomib cotreatment. ISIR-042 decreased the number of CD38-CD138- cells and increased the number of CD38+CD138+ cells in KMS11 cell population. mRNA expression of genes encoding XBP-1u and XBP-1s was not affected in RPMI8226 cells treated with ISIR-042 or bortezomib alone. However, mRNA expression of the gene encoding XBP-1s significantly increased in RPMI8226 cells cotreated with ISIR-042 and bortezomib. Growth-inhibitory effects of ISIR-042 and bortezomib were not associated with caspase-10 which modulates autophagic response for survival. Expression of autophagic markers such as LC3 and p62 was also examined. ISIR-042 exerted modest effects in the presence or absence of bortezomib. ER stress-mediated CHOP induction promotes the cytotoxic effects of proteasome inhibitors on many cancer cells. Carfilzomib and ISIR-042 cotreatment significantly induced CHOP mRNA expression, suggesting that ISIR-042 and proteasome inhibitor cotreatment induced apoptosis by enhancing ER stress and activating CHOP. This was confirmed using CHOP-/- MEF cells. Our results showed that carfilzomib and ISIR-042 cotreatment did not exert cytotoxic effects on CHOP-/- MEF cells. These results suggest that treatment of multiple myelomas with ISIR-042-supplemented proteasome inhibitor-based chemotherapy exerts beneficial anticancer effects. DisclosuresSuzuki:Chugai: Honoraria; Bristol-Myers Squibb: Honoraria; Kyowa Hakko kirin: Honoraria. Suzumiya:Astellas: Research Funding; Takeda: Honoraria; Eisai: Honoraria, Research Funding; Kyowa Hakko kirin: Research Funding; Chugai: Honoraria, Research Funding; Toyama Chemical: Research Funding.