Use of a sample injection loop for an accurate measurement of particle number concentration by flow cytometry.

Flow cytometry plays a pivotal role in biotechnology by providing quantitative measurements for a wide range of applications. Nonetheless, achieving precise particle quantification, particularly without relying on counting beads, remains a challenge. In this study, we introduce a novel exhaustive counting method featuring a sample loop-based injection system that delivers a defined sample volume to a detection system to enhance quantification in flow cytometry. We systematically assess the performance characteristics of this system with micron-sized polystyrene beads, addressing issues related to sample introduction, adsorption, and volume measurement. Results underscore the excellent analytical performance of the proposed method, characterized by high linearity and repeatability. We compare our approach to counting bead-based measurements, and while an approximate bias value was observed, the measured values were found to be similar between the methods, demonstrating its comparability and reliability. This method holds great promise for improving the accuracy and precision of particle quantification in flow cytometry, with implications for various fields including healthcare and environmental monitoring.

Quantifying fluorescent nanoparticle uptake in mammalian cells using a plate reader.

In keeping with the rapid expansion of nanoparticle applications, various tools are required to investigate how nanoparticles interact with biological entities. Many assays have been developed to measure the cellular uptake of nanoparticles, but so far most of the methods are laborious and often non-quantitative. Here we developed an easily accessible and robust quantitative measurement method of the level of cellular uptake of fluorescently labeled nanoparticles using a plate reader. In the experimental design, potential issues that could lead to measurement variation were identified and addressed. For example, the variation in fluorescence intensity of samples due to differences in cell number was normalized to optical density, which is a physical value corresponding to the cell number. Number of washings and sample handling temperature were optimized to minimize the interference by residual nanoparticles and possible efflux of nanoparticles from cells, respectively. The developed assay was demonstrated with the lymphocyte cell line Jurkat to measure the cellular uptake of fluorescently labeled 50 nm polystyrene beads, and its applicability was further confirmed with the lung carcinoma cell line A549 and another lymphocyte cell line RPMI8226.

Blockade of transforming growth factor ß2 by anti-sense oligonucleotide improves immunotherapeutic potential of IL-2 against melanoma in a humanized mouse model.

BACKGROUND AIMS: IL-2 is a potent cytokine that activates natural killer cells and CD8+ cytotoxic T lymphocytes (CTLs) and has been approved for the treatment of metastatic renal cell carcinoma and metastatic melanoma. However, the medical use of IL-2 is restricted because of its narrow therapeutic window and potential side effects, including the expansion of regulatory T cells (Tregs). METHODS: In this study, the authors investigated the complementary effects of transforming growth factor-ß2 (TGF-ß2) anti-sense oligodeoxynucleotide (TASO) on the immunotherapeutic potential of IL-2 in a melanoma-bearing humanized mouse model. RESULTS: The authors observed that the combination of TASO and IL-2 facilitated infiltration of CTLs into the tumor, thereby potentiating the tumor killing function of CTLs associated with increased granzyme B expression. In addition, TASO attenuated the increase in Tregs by IL-2 in the peripheral blood and spleen and also inhibited infiltration of Tregs into the tumor, which was partly due to decreased CCL22. Alteration of T-cell constituents at the periphery by TGF-ß2 inhibition combined with IL-2 might be associated with the synergistic augmentation of serum pro-inflammatory cytokines (such as interferon Î³ and tumor necrosis factor α) and decreased ratio of Tregs to CTLs in tumor tissues, which consequently results in significant inhibition of tumor growth CONCLUSIONS: These results indicate that the application of TASO improves IL-2-mediated anti-tumor immunity, thus implying that blockade of TGF-ß2 in combination with IL-2 may be a promising immunotherapeutic strategy for melanoma.

Antithyroid cancer effects of human neural stem cells expressing therapeutic genes on anaplastic thyroid cancer cells.

Stem cells that express therapeutic proteins have been identified to have an anticancer effects on various types of cancer. In the present study study, human neural stem cells (hNSCs) that were genetically engineered to express cytosine deaminase (CD) and human interferon-ß (IFN-ß) were used for anaplastic thyroid cancer (ATC) treatment owing to their tumor-tropic properties and therapeutic effects. CD is an enzyme that converts 5-fluorocytosine (5-FC), a prodrug, to 5-fluorouracil (5-FU) which is a medication to suppress tumor growth through DNA synthesis inhibition. Also, IFN-ß suppresses tumor growth by the induction of apoptotic process. In water soluble tetrazolium salt (WST) assay, SNU-80 cells which are human female ATC cells were cocultured with three cell types including engineered hNSCs such as HB1.F3, HB1.F3.CD, and HB1.F3.CD.IFN-ß cells on transwells and treated with 5-FC for 72 hours. Finally, the SNU-80 cell viability was reduced by the coculture with HB1.F3.CD and HB1.F3.CD.IFN-ß cells. In dichlorofluorescein diacetate (DCF-DA) and TdT-mediated dUTP nick-end labeling (TUNEL) assays, the production of reactive oxygen species (ROS) and the number of apoptotic cells were increased by HB1.F3.CD and HB1.F3.CD.IFN-ß cells in the presence of 5-FC. In Western blot assay, ROS, and apoptosis-related genes were increased in SNU-80 cells when they were cocultured with HB1.F3.CD and HB1.F3.CD.IFN-ß cells. In transwell migration assay, hNSCs selectively migrated to SNU-80 cells because hNSCs interacted with chemoattractant factors like SDF-1α, uPAR, and CCR2 secreted by SNU-80 cells. Taken together, engineered hNSCs were revealed to selectively migrate to ATC cells and to inhibit growth as well as to induce apoptosis of ATC cells via ROS production through the actions of transgenes such as CD and IFN-ß. Therefore, these engineered hNSCs can be promising candidates for the treatment of metastatic ATC.

Antitumor Effect of Various Phytochemicals on Diverse Types of Thyroid Cancers.

Thyroid cancers developed from the tissues of the thyroid gland are classified into papillary (PTC), follicular (FTC), medullary (MTC), and anaplastic thyroid cancer (ATC). Although thyroid cancers have been generally known as mild forms of cancer, undifferentiated MTC and ATC have a more unfavorable prognosis than differentiated PTC and FTC because they are more aggressive and early metastatic. A variety of therapies such as surgery, radiotherapy, and chemotherapy have been currently used to treat thyroid cancer, but they still have limitations including drug resistance or unfavorable side effects. Phytochemicals are plant-derived chemicals having various physiological activities that are expected to be effective in cancer treatment. In this review, anticancer efficacy of phytochemicals, such as resveratrol, genistein, curcumin, and other substances in each type of thyroid cancer was introduced with their chemopreventive mechanisms. English articles related with thyroid cancer and anti-thyroid cancer of phytochemicals were searched from PubMed and Google Scholar. This article mainly focused on in vitro or animal studies on phytochemicals with anti-thyroid cancer activity. These various phytochemicals have been shown to induce apoptosis in all types of thyroid cancer cells, inhibit cell proliferation and invasion, and to be helpful in enhancing the effect of radioiodine therapy that is a typical therapy to thyroid cancer. These results suggest that thyroid cancer can be more effectively treated by the combinations of phytochemicals and the existing therapies or substances.

Root bark of Ulmus davidiana var. japonica restrains acute alcohol-induced hepatic steatosis onset in mice by inhibiting ROS accumulation.

Alcohol-induced hepatic steatosis and inflammation are key drivers of alcohol-induced liver injury, mainly caused by oxidative stress. The roots bark of Ulmus davidiana var. japonica is well known for its substantial antioxidative and antitumorigenic potency. In this study, we examined whether this plant can ameliorate alcohol-induced liver injuries characterized by hepatic steatosis and inflammation through its antioxidative activity. C57BL/6J mice were treated with the root bark extract of Ulmus davidiana var. japonica (RUE; 100 mg of extract/kg bodyweight; oral gavage) and alcohol (1 g/kg of bodyweight; oral gavage) for 5 days. Markers of acute alcohol-induced hepatic steatosis were determined and putative molecular mechanisms responsible for the protection of RUE were investigated. RUE noticeably protected against alcohol-induced hepatic steatosis and inflammation. Reactive oxygen species (ROS), over-produced by alcohol, negatively orchestrated various signaling pathways involved in the lipid metabolism and inflammation. These pathways were restored through the ROS scavenging activity of RUE in the liver. In particular, the expression of lipogenic genes (e.g., SREBP-1, ACC, and FAS) and inflammatory cytokines (e.g., IL-1ß, and NF-κB p65) significantly decreased with RUE treatment. Conversely, the expression of fatty acid oxidation-related genes (e.g., SIRT1, AMPKα, and PGC1α) were increased in mice treated with RUE. Thus, the results indicate that RUE counteracts and thus attenuates alcoholic hepatic steatosis onset in mice, possibly by suppressing ROS-mediated steatosis and inflammation.