Flow Cytometry Fluorescence-activated Cell Sorting Research Articles

Quality by design-assisted development of D-α-tocopherol polyethylene glycol 1000 succinate-incorporated gefitinib-loaded cationic liposome(s).

Aim: Gefitinib-loaded D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)-coated cationic liposomes (GEF-TPGS-LIPO+) were developed and optimized by the quality by design (QbD) approach for its potential anticancer effect. Methods/materials: Box-Behnken design (BBD) a systematic design of experiments was added to screen and optimize the formulation variables. Results: GEF-TPGS-LIPO+ shows vesicle size (210±4.82nm), polydispersity index(0.271±0.002), zeta potential (22.2±0.84mV) and entrapment efficiency (82.3±1.95). MTT result shows the enhanced cytotoxicity and higher intracellular drug uptake with highest and lowest levels of the reactive oxygen species and NF-κBexpressions on A549 lung cancer cells, determined by fluorescence-activated cell sortingflow cytometry. Conclusion: Potential anticancer effect on A549 cells might be found due to cationic liposomal interaction with cancer cells.

UCA1 executes an oncogenic role in pancreatic cancer by regulating miR-582-5p/BRCC3.

As a fatal disease, the mechanism of pancreatic cancer is unclear. Urothelial carcinoma antigen 1(UCA1), a long noncoding RNA (lncRNA) that was first reported in bladder cancer, acts as an oncogene. However, the regulatory role and mechanism of UCA1 in pancreatic cancer remain unknown. This study aims to investigate the expression level and prognostic value of UCA1 in pancreatic cancer tissues, the effects and mechanism of UCA1 in regulating cell proliferation, apoptosis and metastasis. UCA1 expression levels in tissues were detected by in situ hybridization (ISH) and the prognostic value was evaluated by univariate and multivariate survival analysis. For in vitro experiments, proliferation was evaluated by a cell count kit assay, Edu experiments, and a clone formation assay. Apoptosis was evaluated by fluorescence-activated cell sorting flow-cytometry. Cell migration and invasion capacities were detected by wound healing and transwell assays. Western blots were performed to detect apoptotic associated molecules and epithelial-mesenchymal transition (EMT) markers. For the in vivo experiment, subcutaneous transplantation models of pancreatic cancer in nude mice were established to observe the tumor growth. The regulatory mechanism of UCA1 was explored by proteomics, bioinformatic analysis, luciferase reporter assays, and rescue experiments. ISH staining revealed that UCA1 levels between cancer tissues (n=94) and tumor-adjacent tissues (n=73) did not show significant differences. Survival analysis indicated that high expression of UCA1 was an unfavorable prognosis factor for pancreatic cancer. Downregulation of UCA1 by siRNA significantly inhibited cell proliferation, decreased the capacities of cell migration and invasion, induced cell apoptosis, and inhibited EMT. Furthermore, we demonstrated that UCA1 positively regulated the expression of BRCC3 by inhibiting miR-582-5p. Rescue experiments indicated that either inhibiting the expression of miR-582-5p or enhancing expression of BRCC3 could partly attenuate the antitumor effects of downregulation of UCA1. UCA1 acted as an oncogene in pancreatic cancer by partly regulating miR-582-5p/BRCC3, which could be a new therapeutic target for pancreatic cancer.

Abstract 5982: Single-cell RNA sequencing (scRNA-Seq) from mice model reveals metastatic and stemness signatures in esophageal squamous cell carcinoma (ESCC)

Abstract Remote oligometastases are the predominant roots of the unsatisfactory 5-year survival rate in esophageal squamous cell carcinoma (ESCC) patients. The title of metastatic-initiating cells (MICs) has been designated to describe tumor subpopulation with heterogeneous genetic background and behaviors that potentially displays stemness features and favors metastatic lesion formation. However, preceding studies have overpassed the early surviving MICs due to limitations in bulk transcriptomic sequencing at macrometastatic stage. With the aim of denoting and characterizing MICs in ESCC, we established the pulmonary metastatic mice model via intravenous injection and followed by multiplex single-cell RNA sequencing (scRNA-Seq) of tumor cells collected from lung tissues along longitudinal end-points. The scRNA-Seq results uncover a 7-gene surface marker signature panel that could depict the early surviving MICs of ESCC, including CD44, TACSTD2, TM4SF1, S100A14, RHOD, CST6, and C19orf33. In silico gene ontology (GO) enrichment of the early surviving MICs subpopulation reveals numerous migration and adhesion-related biological processes (p<0.05). In addition to scRNA-Seq, immunohistochemistry (IHC) staining of tumor cells in mice lungs collected in sequential order evidenced the survival heterogeneity of tumor subpopulations. Likewise, flow cytometry fluorescence-activated cell sorting (FACS) of signature-enriched tumor cells was performed to resemble the target MICs. CD44Pos ESCC cells are found to show distinctively greater wound-healing (p<0.01), foci formation (p<0.05), migration (p<0.01) and invasion (p<0.01) ability than CD44Neg cells in vitro, and longer survival patterns (p<0.05) upon implantation in vivo. Multi-color immunofluorescence (mIF) staining of these gene signatures is underway in mice models and clinical samples for comprehensive validation. We anticipate this surface marker signature to accelerate development for clinical diagnosis together with molecular targeting in metastatic ESCC patients. Citation Format: Ching Ngar Wong, Yu Zhang, Beibei Ru, Hong Yu Zhou, Kwong Yiu Lam, Xin Yuan Guan. Single-cell RNA sequencing (scRNA-Seq) from mice model reveals metastatic and stemness signatures in esophageal squamous cell carcinoma (ESCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5982.

Highly Stable Red-Emissive Ratiometric Probe for Monitoring β-Galactosidase Activity Using Fluorescence Microscopy and Flow Cytometry.

β-Galactosidase (β-gal), well known as a useful reporter enzyme, is a potent biomarker for various diseases such as colorectal and ovarian cancers. We have developed a highly stable red-emissive ratiometric fluorescent probe (CCGal1) for quantitatively monitoring the β-gal enzyme activity in live cells and tissues. This ratiometric probe showed a fast emission color change (620-662 nm) in response to β-gal selectively, which was accompanied by high enzyme reaction efficacy, cell-staining ability, and outstanding stability with minimized cytotoxicity. Confocal fluorescence microscopy ratiometric images, combined with fluorescence-activated cell sorting flow cytometry, demonstrated that CCGal1 could provide useful information for the diagnosis, prognosis, and treatment of β-gal enzyme activity-related diseases such as colorectal and ovarian cancers. Further, it may yield meaningful strategies for designing and modifying multifunctional bioprobes with different biomedical applications.

Examining the Developmental Trajectory of an in Vitro Model of Mouse Primordial Germ Cells following Exposure to Environmentally Relevant Bisphenol A Levels.

Background:Animal-based studies indicate that bisphenol A (BPA) exposure is detrimental to reproductive health, but its impact on the earliest stages of germ cell development remains poorly defined.Objectives:Using a murine in vitro model of early germ cell specification and differentiation, we sought to assess whether exposure to low levels of BPA prior to formation of primordial germ cells (PGCs) alters their differentiation trajectory and unique molecular program.Methods:We used an established method of in vitro differentiation of mouse embryonic stem cells (ESCs) into epiblast-like cells (EpiLCs) followed by PGC-like cells (PGCLCs), which together recapitulate defined stages of early germ cell development. Cellular consequences were determined using hemocytometer-based cell counting, fixation, and intracellular staining, followed by flow cytometry/fluorescence-activated cell sorting (FACS) of cells exposed to increasing concentrations (range: ) of BPA. To interrogate and characterize gene expression differences resulting from BPA exposure, we also generated RNA-seq libraries from RNA extracted from FACS-purified PGCLCs and performed transcriptome analysis using bioinformatics-based approaches.Results:Exposure of EpiLCs to BPA resulted in higher numbers of cells that were associated with a higher proportion of cells in S-phase as well as a lower proportion undergoing apoptosis; this difference occurred in a concentration-dependent manner. Exposure also resulted in a greater fraction of EpiLCs showing signs of DNA damage. Remarkably, EpiLC exposure did not negatively affect PGC specification and resulted in a concentration-dependent effect on PGCLC proliferation in XX but not XY cells. PGCLC transcriptome analysis revealed an aberrant program with significant deregulation of X-linked genes and retrotransposon expression. Differential gene expression analysis also revealed the deregulation of genes associated with lipid metabolism as well as deregulated expression of genes associated with later stages of gametogenesis.Conclusions:To the best of our knowledge our findings represent the first characterization of the consequences of early BPA exposure on a model of mammalian PGC development, highlighting altered cell behavior, altered underlying pathways, and altered molecular processes. https://doi.org/10.1289/EHP8196

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat.

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Higher frequency electrical stimulation enhanced myloglossus satellite cell differentiation by upregulating expression of Pax7 mRNA, MyoD, myogenin and MyHC protein.

We investigated the effect of electrical stimulation (ES) of varying pulse frequency on differentiation and proliferation of canine myloglossus satellite cells in vitro. Cellular viability and proliferation were assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometry fluorescence-activated cell sorting analysis. Cellular differentiation and expression of mark molecule were assayed by Real Time-PCR and Western blot. With increasing frequency ES, we found a significant increase in Myod (r=0.988, p<0.0001), myogenin (r=0.988, p<0.0001), MyHC-slow (r=0.988, p<0.0001), MyHC-fast (r=0.875, p<0.0001) protein expression, and Pax7 mRNA expression (r=0.712, p=0.001). Pax7 mRNA expression and MyoD, myogenin, and MyHC protein expression were increased with increment of electrical stimulation frequency in myloglossus muscle satellite. Higher frequency ES enhanced myloglossus satellite cell differentiation, not proliferation and viability.

Antifibrotic role of low-dose mitomycin-c-induced cellular senescence in trabeculectomy models

PurposeWe assessed whether mitomycin-C (MMC) has different antifibrotic mechanisms in trabeculectomy wound healing.MethodsWe identified 2 concentrations of MMC as “low-dose” by using WST-1 assay, Lactic dehydrogenase assay, and fluorescence-activated cell sorting flow cytometry. Senescence-associated β-galactosidase (SA-β-gal) and fibrotic gene expression was examined through immunocytochemistry, flow cytometry, real-time quantitative reverse transcription polymerase chain reaction, Western blotting, zymography, and modified scratch assay in vitro. In vivo, 0.1 mL of MMC or normal saline was injected to Tenon’s capsule before trabeculectomy in a rabbit model. SA-β-gal expression, apoptotic cell death, and collagen deposition in sites treated and not treated with MMC were evaluated using terminal dUTP nick end labeling assay and histochemical staining. Bleb function and intraocular pressure (IOP) levels were examined 3, 7, 14, 21, 28, and 35 days after trabeculectomy.ResultsIn vitro, human Tenon’s fibroblast (HTF) senescence was confirmed by observing cell morphologic change, SA-β-gal accumulation, formation of senescence-associated heterochromatin, increased p16INK4a and p21CIP1/WAF1 expression, lower percentage of Ki-67-positive cells, and decreased COL1A1 release. Increased expression of α-SMA, COL1A1, and Smad2 signaling in TGF-β1-induced stress fibers were passivated in senescent HTFs. In addition, cellular migration enhanced by TGF-β1was inactivated. In vivo, histological examination indicated increased SA-β-gal accumulation, lower apoptosis ratios, and looser collagen deposition in sites treated with 0.2 μM MMC. Low-dose MMC-induced cellular senescence prolonged trabeculectomy bleb survival and reduced IOP levels in a rabbit model.ConclusionLow-dose MMC-induced cellular senescence is involved in the antifibrotic mechanism of trabeculectomy wound healing.

Isolation of canine adipose-derived mesenchymal stem cells from falciform tissue obtained via laparoscopic morcellation: A pilot study.

To evaluate the feasibility of stem cell isolation from falciform fat harvested via laparoscopic morcellation. Pilot study. Eleven client-owned dogs. Falciform was harvested traditionally via laparotomy and laparoscopically via tissue morcellation. Harvested tissue was processed with a commercially available adipose tissue dissociation kit to obtain a stromal vascular fraction (SVF). Cells were subsequently labeled for CD90, CD45, and CD44 cell surface antigens by using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting flow cytometry. CD90+ cells were quantitated, and their viability was assessed with a hemocytometer and a trypan blue exclusion test of cell viability. No perioperative complications occurred in dogs undergoing laparoscopic morcellation. Laparoscopically and traditionally harvested samples yielded an average of 0.39 (±0.1) × 106 and 0.33 (±0.1) × 106 CD90+ cells, respectively, per 10 million SVF cells. CD90+ cell viability after MACS was 89% (±11%) for morcellated and 86% (±7%) for traditionally harvested samples. Neither CD90+ cell quantity nor viability was different between samples obtained via traditional laparotomy vs laparoscopic morcellation (P = .38 and P = .63, respectively). Populations of CD90+ cells isolated with each harvest technique had similar CD44 and CD45 expression profiles. Viable populations of CD90+ cells with similar CD44/CD45 expression profiles were isolated from laparoscopically morcellated and traditionally harvested falciform tissue. No appreciable morbidity was associated with laparoscopic falciform morcellation. Laparoscopic morcellation is a safe and effective minimally invasive approach to falciform tissue harvest for adipose-derived mesenchymal stem cell isolation.

High-Resolution Recording of the Circadian Oscillator in Primary Mouse α- and β-Cell Culture.

Circadian clocks have been developed in evolution as an anticipatory mechanism allowing for adaptation to the constantly changing light environment due to rotation of the Earth. This mechanism is functional in all light-sensitive organisms. There is a considerable body of evidence on the tight connection between the circadian clock and most aspects of physiology and metabolism. Clocks, operative in the pancreatic islets, have caught particular attention in the last years due to recent reports on their critical roles in regulation of insulin secretion and etiology of type 2 diabetes. While β-cell clocks have been extensively studied during the last years, α-cell clocks and their role in islet function and orchestration of glucose metabolism stayed unexplored, largely due to the difficulty to isolate α-cells, which represents a considerable technical challenge. Here, we provide a detailed description of an experimental approach for the isolation of separate mouse α- and β-cell population, culture of isolated primary α- and β-cells, and their subsequent long-term high-resolution circadian bioluminescence recording. For this purpose, a triple reporter ProGlucagon-Venus/RIP-Cherry/Per2:Luciferase mouse line was established, carrying specific fluorescent reporters for α- and β-cells, and luciferase reporter for monitoring the molecular clockwork. Flow cytometry fluorescence-activated cell sorting allowed separating pure α- and β-cell populations from isolated islets. Experimental conditions, developed by us for the culture of functional primary mouse α- and β-cells for at least 10 days, will be highlighted. Importantly, temporal analysis of freshly isolated α- and β-cells around-the-clock revealed preserved rhythmicity of core clock genes expression. Finally, we describe the setting to assess circadian rhythm in cultured α- and β-cells synchronized in vitro. The here-described methodology allows to analyze the functional properties of primary α- and β-cells under physiological or pathophysiological conditions and to assess the islet cellular clock properties.

Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening.

When produced biologically, especially by photosynthetic organisms, hydrogen gas (H2) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H2-producing enzymes is the absence of methods for the high-throughput screening of H2 production in biological systems. Here, we re-engineered the natural H2 sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H2. A lacZ gene was placed under the control of the hupA H2-inducible promoter in a strain lacking the uptake hydrogenase and the nifH nitrogenase gene. This system was then used in combination with fluorescence-activated cell sorting flow cytometry to screen large libraries of nitrogenase Fe protein variants generated by random mutagenesis. Exact correlation between fluorescence emission and H2 production levels was found for all automatically selected strains. One of the selected H2-overproducing Fe protein variants lacked 40% of the wild-type amino acid sequence, a surprising finding for a protein that is highly conserved in nature. We propose that this method has great potential to improve microbial H2 production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.

Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway.

BackgroundCombinations of adjuvant sensitizers with anticancer drugs is a promising new strategy to reverse chemoresistance. Ursolic acid (UA) is one of the natural pentacyclic triterpene compounds known to have many pharmacological characteristics such as anti-inflammatory and anticancer properties. This study investigates whether UA can sensitize hepatocellular carcinoma cells to cisplatin.Materials and methodsCells were transfected with nuclear factor erythroid-2-related factor 2 (Nrf2) small interfering RNA and Nrf2 complementary DNA by using Lipofectin 2000. The cytotoxicity of cells was investigated by Cell Counting Kit 8 assay. Cell apoptosis, cell cycle, reactive oxygen species, and mitochondrial membrane potential were detected by flow cytometry fluorescence-activated cell sorting. The protein level of Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and heme oxygenase-1 (HO-1) was detected by Western blot analysis.ResultsThe results showed that the reverse index was 2.9- and 9.69-fold by UA of 1.125 μg/mL and 2.25 μg/mL, respectively, for cisplatin to HepG2/DDP cells. UA–cisplatin combination induced cell apoptosis and reactive oxygen species, blocked the cell cycle in G0/G1 phase, and reduced the mitochondrial membrane potential. Mechanistically, UA–cisplatin dramatically decreased the expression of Nrf2 and its downstream genes. The sensibilization of UA–cisplatin combination was diminished in Nrf2 small interfering RNA-transfected HepG2/DDP cells, as well as in Nrf2 complementary DNA-transfected HepG2/DDP cells.ConclusionThe results confirmed the sensibilization of UA on HepG2/DDP cells to cisplatin, which was possibly mediated via the Nrf2/antioxidant response element pathway.

Monocyte Expression Levels of Cytokines in Patients With Fibromyalgia Syndrome

Objectives: This study aims to determine monocyte expression levels of interleukin 1 beta (IL-1β), IL-6, IL-8, and tumor necrosis factor alpha in fibromyalgia syndrome (FMS) patients and healthy females. Patients and methods: A total of 80 female FMS patients and 50 healthy females were evaluated. Twelve of the FMS patients who did not meet the 1990 American College of Rheumatology criteria and 10 healthy controls were excluded. After exclusion 68 participants as FMS patient (mean age 41.41±7.14 years; range 18 to 55 years) and 40 participants as control group (mean age 39.12±10.9 years; range 18 to 55 years) were included in the study. For both groups, a detailed form was filled out which included information on age, body mass index, marital status, educational status, visual analog scale, fibromyalgia impact questionnaire, Hamilton anxiety scale, Hamilton depression scale, modified fatigue impact scale, and Nottingham health profile. Whole blood peripheral blood monocyte cells were drawn in ethylenediaminetetraacetic acid tubes from patient and control groups between 08:30 to 9:30 hours. Tumor necrosis factor alpha, IL-6, IL-8, and IL-1β expressions were evaluated using fluorescence-activated cell sorting flow cytometry device. Results: No statistically significant difference was detected between the two groups in terms of average age, body mass index, and other demographic data (p>0.05). Visual analog scale, Hamilton anxiety scale, Hamilton Depression Scale, fibromyalgia impact questionnaire, modified fatigue impact scale total score and Nottingham health profile total-subscore values were statistically significantly higher in the patient group than the control group (p 0.05). Conclusion: Although FMS is not traditionally considered an inflammatory disorder, evidence for elevated inflammatory processes has been noted in some studies. Our results do not support any role of the inflammatory cytokines in the pathogenesis of FMS.

Effect of formaldehyde inactivation on poliovirus.

Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treating live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors, which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analyzed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescence-activated cell sorting flow cytometry and real-time reverse transcription-PCR (RT-PCR). The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion in vitro, as judged by measuring changes in antigenicity, the ability to bind to hPVR, and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, since cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells. Importance: This study represents a novel insight into the molecular mechanisms responsible for poliovirus inactivation. We show that inactivation with formaldehyde has an effect on early steps of viral replication as it reduces the ability of PV to bind to hPVR, decreases the sensitivity of PV to convert to 135S particles, and abolishes the infectivity of its viral RNA. These changes are likely responsible for the loss of infectivity shown by PV following inactivation. Techniques used in this study represent new approaches for the characterization of inactivated PV products and could be useful in developing improved methods for the production and quality control testing of inactivated polio vaccines. Measuring the antigenicity, capsid stability, and RNA integrity of inactivated PV samples could help establishing the optimal balance between the loss of infectivity and the preservation of virus antigenicity during inactivation.

Ezrin Gene Expression and Protein Production in the CD44+ Subpopulation of SCC-9 Cells in a Malignant Oral Cancer Cell Line In Vitro

The primary objective of this study was to examine ezrin in the CD44(+) subpopulation of SCC-9 cells in a malignant oral cancer cell line. The CD44(+) subpopulation of SCC-9 cells was sorted using CD44 fluorescence-activated cell sorting flow cytometry. Expressions of the ABCG2, CD133, Oct-4, BMI-1, an ezrin genes and proteins were detected by immunofluorescence and real-time polymerase chain reaction, and their differential expressions in the CD44(+) and CD44(-) cell populations and embryonic stem cells were compared. The location of the ezrin protein expressed in SCC-9 cells was determined using laser scanning confocal microscopy. The expression of ezrin in the CD44(+) subpopulation was 38 times higher than in the CD44(-) subpopulation based on real-time polymerase chain reaction, and levels of ABCG2, CD133, Oct-4, and BMI-1 in the CD44(+) subpopulation were higher than those in the CD44(-) subpopulation (P < .05). The overexpression of ezrin in the CD44(+) subpopulation of SCC-9 cells might affect the functions of CD44(+) tumor cells, which contribute to the metastatic behavior of malignant oral squamous cell carcinoma.

Removing the cells from adult bone marrow derived stem cell therapy does not eliminate cardioprotection†

The debate as to whether adult stem cell therapy is regenerative or not continues. The non-regenerative benefits of adult bone marrow-derived stem cell therapy were investigated by testing whether the supernatant derived from unfractionated bone marrow mononuclear cells might be cardioprotective in an animal model of myocardial ischaemia-reperfusion injury. Regional myocardial reperfusion injury was acquired by 25 min reversible left anterior descending coronary artery (LAD) occlusion followed by 2 h reperfusion, in anaesthetized Wistar male rats. Unfractionated bone marrow mononuclear cells (BMMNC) isolated from sibling Wistar male rat whole bone marrow were phenotyped by fluorescence activated cell sorting flowcytometry for the haematopoietic stem cell surface markers c-kit, CD34, CD45 and CD133. Animals subjected to regional myocardial reperfusion injury received either 10 million BMMNC or BMMNC supernatant (BMS); both were collected in 0.5 ml phosphate-buffered saline and delivered by intravenous bolus at the onset of reperfusion. The left ventricular region distal to the LAD occlusion point was excised for measurement of myocardial infarct size and proteomic analysis, which was used to identify whether there were any differences in myocardial proteins associated with intravenous injection of either BMMNC or BMS. BMMNC were phenotyped to be c-kit(+) (7 ± 1%), CD34(+) (7 ± 1%), CD45(+) (54 ± 6%), CD133(+) (15 ± 1%). The supernatant reduced myocardial infarct size (BMS 34 ± 2%, n = 15 vs control 57 ± 2%, n = 7, P < 0.0001), which was comparable to the reduction in infarct size afforded by the injection of cells (BMMNC 33 ± 3% vs control 57 ± 2%, n = 10, P < 0.0001). Proteomics of hearts treated with either BMS or BMMNC demonstrated higher expression of (i) anti-apoptotic signal transduction protein: 14-3-3-epsilon (1.5-fold); (ii) anti-oxidants: peroxiredoxin-6 (2.1-fold); (iii) heat shock proteins: alpha B-crystallin (1.7-fold), heat shock protein 72 (2.8-fold), tumour necrosis factor receptor-1 associated protein (2.3-fold), ischaemia responsive protein-94 (1.6-fold); (iv) glycolytic protein: glyceraldehyde-3-phosphate dehydrogenase (2.3-fold); (v) mitochondrial respiratory proteins: mitochondrial aconitase (4.7-fold), voltage-dependent anion-selective channel protein-1 (VDAC-1) (2.7-fold). Regional myocardial reperfusion injury can be attenuated by intravenous administration of either BMMNC or BMS at the onset of reperfusion, which suggests adult stem cells mediate non-regenerative cardioprotection.

Forkhead Proteins Are Critical for Bone Morphogenetic Protein-2 Regulation and Anti-tumor Activity of Resveratrol

Osteoporosis is a major public health problem and the most obvious preventive strategy, hormone replacement therapy, has lost favor due to recent findings of the Women's Health Initiative regarding increased risks of breast cancer and cardiovascular disease. Resveratrol, a naturally occurring compound possessing estrogenic activity, is thought to have considerable potential for therapy of osteoporosis. In the present study, resveratrol was found to exhibit bone-protective effects equivalent to those exerted by hormone replacement therapy and decrease the risk of breast cancer in the in vivo and in vitro models. Forkhead proteins were found to be essential for both effects of resveratrol. The bone-protective effect was attributable to induction of bone morphogenetic protein-2 through Src kinase-dependent estrogen receptor activation and FOXA1 is required for resveratrol-induced estrogen receptor-dependent bone morphogenetic protein-2 expression. The tumor-suppressive effects of resveratrol were the consequence of Akt inactivation-mediated FOXO3a nuclear accumulation and activation. Resveratrol is therefore anticipated to be highly effective in management of postmenopausal osteoporosis without an increased risk of breast cancer.

Evaluation and significance of circulating epithelial cells in patients with hormone‐refractory prostate cancer

To determine the feasibility of using flow cytometry fluorescence-activated cell sorting (FACS) analysis for detecting circulating epithelial cells (CECs) in patients with hormone-refractory prostate cancer (HRPC), and to determine whether CECs can be used to predict survival in these patients. Several prognostic models that include routinely used clinical and laboratory variables for predicting survival in men with HRPC have been reported; the presence of CECs measured by reverse transcriptase-polymerase chain reaction for prostate-specific antigen (PSA) in patients with HRPC is an independent prognostic factor for survival. CECs detected by FACS analysis correlate with advanced stage and poor survival outcome. A retrospective study was conducted to assess the presence of CECs by FACS analysis in metastatic HRPC patients initiating systemic chemotherapy with a taxane-based regimen. The association between clinical variables previously described and the presence of CECs along with the effect of the magnitude of CECs on survival was calculated, in 41 patients with HRPC, all of whom had peripheral blood collected for FACS analysis. Except for four patients, all those with metastatic HRPC had detectable CECs. Among these patients, the number of CECs/mL was correlated with age, serum PSA level and serum alkaline phosphatase (ALP). Higher serum levels of PSA and ALP predicted a poor survival outcome. Similarly, patients with < or =1.8 CECs/mL had a significantly longer survival than those with more CECs/mL (P = 0.02). With a median follow-up of 15.4 months, the median overall survival for all patients was 18.4 months. The presence of more CECs in patients with metastatic HRPC was associated with a poorer survival outcome; levels of > or =1.8 CECs/mL were associated with a shorter survival in patients with metastatic HRPC.

Heparanase: A New Metastasis-Associated Antigen Recognized in Breast Cancer Patients by Spontaneously Induced Memory T Lymphocytes

Increased expression and secretion of heparanase (Hpa) by tumor cells promotes tumor invasion through extracellular matrices, tissue destruction, angiogenesis, and metastasis. Here, we show the existence in breast cancer patients of Hpa-specific T lymphocytes by fluorescence-activated cell sorting flow cytometry using Hpa peptide-MHC class I tetramers. We furthermore show memory T-cell responses in a high proportion of breast cancer patients to Hpa-derived HLA-A2-restricted peptides, leading to production of IFN-gamma and to generation of antitumor CTLs lysing breast cancer cells. Such CTLs recognized endogenously processed respective Hpa peptides on Hpa-transfected and Hpa-expressing untransfected breast carcinoma cells. According to these results and to the fact that such cells were not found in healthy people, Hpa seems to be an attractive new tumor-associated antigen and its HLA-A2-restricted peptides ought to be good candidates for peptide vaccination to reactivate memory immune responses to invasive and metastatic cancer cells.

Surface display of Zymomonas mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae.

The ice-nucleation protein (Inp) is a glycosyl phosphatidylinositol-anchored outer membrane protein found in some Gram-negative bacteria. Using Pseudomonas syringae inp as an anchoring motif, we investigated the functional display of a foreign protein, Zymomonas mobilis levansucrase (LevU), on the surface of Escherichia coli. The cells expressing Inp-LevU were found to retain both the ice-nucleation and whole-cell levansucrase enzyme activities, indicating the functional expression of Inp-LevU hybrid protein on the cell surface. The surface localization was further verified by immunofluorescence microscopy, fluorescence-activated cell sorting flow cytometry and immunogold electron microscopical examination. No growth inhibition or changes in the outer membrane integrity were observed upon the induction of fusion protein synthesis. Viability of the cells was also maintained over 48 hours in the stationary phase. Surface-displayed levansucrases were found to be resistant to the externally added proteases unless the cells were treated with EDTA. When the levansucrase-displayed cells were used as the enzyme source, levan (44 g/L) was efficiently synthesized from sucrose (130 g/L) with 34% (wt/wt) conversion yield, generating glucose (65 g/L) as a by-product.