Roswell Park Memorial Institute Research Articles

Effects and mechanism of estrogen receptor β agonist on the migration and oxidative stress of human umbilical vein endothelial cell under high glucose condition

Objective: To investigate the effects and related mechanism of estrogen receptor β (ERβ) agonist on the migration and oxidative stress of human umbilial vein endothelial cells (HUVECs) under high glucose condition. Methods: The experimental research method was adopted. HUVECs were routinely cultured and passaged, and then cells of the logarithmic growth phase were collected for the subsequent experiments. The cells were divided into three groups according to the random number table, including normal control group (cultured with Roswell Park Memorial Institute 1640 cell culture medium (the same cell culture medium below) containing 5.5 mmol/L D-glucose), high glucose alone group (cultured with cell culture medium containing 25.0 mmol/L D-glucose alone), and high glucose+ERβ agonist diarylpropionitrile (DPN) group (cultured with cell culture medium containing 25.0 mmol/L D-glucose and 10 μmol/L DPN). Scratch test was conducted to detect the cell migration rate in the 3 groups at 24 h post scratching. Fluorescent probe method was used to detect the reactive oxygen species (ROS, denoted by red fluorescence intensity) of cells in the 3 groups on 5 d post culture. Western blotting was used to detect the protein expression levels of vascular endothelial growth factor (VEGF) and superoxide dismutase 2 (SOD2) of cells in the 3 groups on 5 d post culture. In the above-mentioned experiments, cells were grouped and cultured correspondingly as before, the number of samples in each group was 5. Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results: At 24 h post scratching, the cell migration rate in high glucose alone group was (36±5)%, which was significantly lower than (76±4)% of normal control group and (65±5)% of high glucose+DPN group (t=14.511, 9.603, P<0.01), and the cell migration rate in high glucose+DPN group was significantly lower than that in normal control group (t=3.943, P<0.01). On 5 d post culture, the level of ROS of cells in high glucose alone group (1.81±0.12) was significantly increased compared with normal control group and high glucose+DPN group (1.00±0.14, 0.91±0.15, t=9.679, 10.549, P<0.01), while the level of ROS of cells in normal control group and high glucose+DPN group were close (t=1.031, P>0.05). On 5 d post culture, the protein expression levels of VEGF and SOD2 of cells in high glucose alone group were significantly lower than the levels of normal control group (t=14.175, 13.787, P<0.01) and high glucose+DPN group (t=6.321, 17.750, P<0.01). The protein expression level of VEGF of cells in high glucose+DPN group was significantly lower than the level of normal control group (t=7.206, P<0.05), while the protein expression level of SOD2 of cells in high glucose+DPN group was significantly higher than the level of normal control group (t=2.890, P<0.05). Conclusions: The activation of ERβ can improve the inhibition of HUVECs migration induced by high glucose and alleviate oxidative stress injury induced by high glucose, which may be achieved by promoting the expression of VEGF and SOD2.

In vitro antiplasmodial evaluation of ethanolic and n-hexane extracts of Parinari curatellifolia stem bark

Parinari curatellifolia and other Parinari species are used traditionally in many parts of Africa as a remedy for malaria among other diseases. To ascertain this folkloric claim, the antiplasmodial potential of ethanol extract of Parinari curatellifolia stem bark (EEPCSB) and n-hexane extract of Parinari curatellifolia stem bark (HEPCSB) on Plasmodium falciparum was studied. Parasites were grown in a 96-well plate containing Roswell Park Memorial Institute-1640. The wells were grouped into: control (untreated), artemether-treated, EEPCSB-treated and HEPCSBtreated groups. Treatments were administered to the tune of 10, 20, 40 and 80 μg/ml. Parasitemia was observed by microscopy after 24, 48 and 72h of incubation. EEPCSB and HEPCSB elicited dose and duration-dependent reduction (p&lt;0.05) in parasitemia when compared with the untreated group. The recorded percentage parasite inhibition by the extracts was lower (p&lt;0.05) when compared with artemether. There was no difference (p&gt;0.05) in plasmodium lactate dehydrogenase activity of EEPCSB-treated and artemether-treated groups. Findings from this study show that extracts of P. curatellifolia stem bark, especially EEPCSB, demonstrated excellent inhibitory activities against P. falciparum and can be a good source of compounds for the development of novel antimalarial drugs.&#x0D; Keywords: Parinari curatellifolia; Extracts; Plasmodium falciparum; Parasitemia; Antiplasmodial

Speciation of platinum nanoparticles in different cell culture media by HPLC-ICP-TQ-MS and complementary techniques: A contribution to toxicological assays

Toxicological studies of nanoparticles (NPs) are highly demanded nowadays but they are very challenging. In the in vitro assays, the understanding of the role of cell culture media is crucial to derive a proper interpretation of the toxicological results and to do so, new analytical tools are necessary. In this context, an analytical strategy based on reversed-phase liquid chromatography hyphenated to inductively coupled plasma-triple quadrupole mass spectrometry (HPLC-ICP-TQ-MS) has been developed for the first time for the detection and characterization of both 5 and 30 nm PtNPs, as well as ionic platinum species, in commonly used cell culture media. For this purpose, Dulbecco's Modified Eagle Medium, DMEM-high glucose, DMEM-F12, DMEM 31053–028, and Roswell Park Memorial Institute, RPMI-1640 (supplemented with 10% fetal bovine serum (FBS) and antibiotics) at several incubation times (24, 48, and 96 h at 37 °C) were tested. After a careful optimization and analytical performance, the developed method allows to simultaneously study the oxidation process, leading to the release of ionic species, and the increase in the hydrodynamic volume of PtNPs, probably related to the formation of new biological entities (protein corona). The magnitude of both processes was found to be dependent on the tested cell culture media and incubation times. Dynamic light scattering (DLS) and high-resolution scanning electron microscopy (HR-SEM) were used as complementary techniques to study the important process of both soft and hard protein corona formation. The feasibility of the HPLC-ICP-TQ-MS to get relevant information for toxicological studies has been demonstrated and in light of our results, the influence of the cell culture media on the behavior of PtNPs should not be underestimated.

Features of obtaining metaphase chromosomes Caprovinea

Obtaining cultures of lymphocyte cells for studying of animal karyotypes is an easily accessible, relatively cheap method and allows obtaining results in a short time. We optimized the usual method with corrected some adjustments to the protocol to obtain metaphase plates of chromosomes of various animal species of good quality. At the same time, such criteria as the use of different media for the cultivation of blood cell cultures and the possibility of using them in different species of animals were taken into account. Blood samples were collected from the jugular vein in goats (n=15), sheep (n=30) and round (n=1). The cell culture was cultivated for 72 h; colchicine was added for 70 h in an amount of 0.5 ?l / ml. The study used nutrient media such as DMEM (Dulbecco?s Modified Eagle?s Medium) и RPMI 1640 (Roswell Park Memorial Institute) and mitogens phytohemagglutinin and Concanavalin A in different concentrations. Optimal parameters for the preparation of metaphase plates with good chromosome distribution were obtained using RPMI 1640 medium supplemented with dry L-glutamine and the simultaneous use of mitogens such as conconavalin and PHA (phytohemagglutinin). The number of plates of good quality when obtaining a culture of lymphocyte cells, depending on the type of animals, ranged from 2.17 to 6.52% in sheep, 33.3-66.7% in goats, and reached 100% in tur. The modified technique made it possible to reduce the loss of cells during the preparation of chromosome preparations. Thus, the results of our study show that the growth and division of lymphocyte cells isolated from the peripheral blood of farm animals depends on the composition of the environment and the species characteristics of the animals.

Arbutin exerts anticancer activity against rat C6 glioma cells by inducing apoptosis and inhibiting the inflammatory markers and P13/Akt/mTOR cascade.

Gliomas are a type of brain cancer that occurs in the supporting glial cells of the brain. It is highly malignant and accountsfor 80% of brain tumors with high mortality and morbidity. Phytomedicines are potent alternatives for allopathic drugs which cause side effects. They have beenused from ancient times by traditional Chinese, Ayurveda, and Siddha medicine. Arubtin is a glycoside phytochemical extracted from plants and belongs to the family of Ericaceae. Arbutin possesses various pharmacological properties such as anti-inflammatory, antioxidant, antitumor, and so on. Hence in the present study, we analyzed the anticancer potency of arbutin against rat C6 glioma cells. Rat C6 glioma cells were procured from American Type Culture Collection and the cells were cultured in Roswell Park Memorial Institute-1640 medium. To assess the cytotoxicity effect of the arbutin against C6 glioma cells, an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test was performed with different doses from 10 to 60 µM. Arbutin effectively induced apoptosis in the cells and the IC50 dose was obtained at 30 µM. For further studies, we selected the 30 µM IC50 dose and a higher dose of 40 µM. Reactive oxygen species (ROS) generated were analyzed with DCFDA/H2DCFDA stain and the destruction of mitochondrial membrane permeability which is the initiator of apoptosis was analyzed with a cationic stain Rhodamine 123. Dual staining with acridine orange and ethidium bromide was performed to assess the viable and dead cells. Cell adhesion propertiesof glioma cells were analyzed with Matrigel assay. The apoptotic, inflammatory, and phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling molecules were analyzed with quantitative polymerase chain reaction (qPCR) analysis to confirm the anticancer effect of arbutin. Arbutin generated excessive ROS and disrupted the mitochondrial membrane, which induced apoptosis in cells, it also inhibited the cell adhesion property of C6 glioma cells. qPCR analysis clearly indicates arbutin increases the apoptotic genes and decreased the inflammatory and PI3K/mTOR signaling molecules. Overall, our results authentically confirm that arbutin can be a potent alternative for treating glioma.

Naringenin and Ellagic Acid Reduce Tetrazolium Salt in the Absence of Cells

Background: Plant derived compounds, naringenin and ellagic acid might interfere in MTT (3-(4, 5- Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide) assay in the form of reducing the tetrazolium salt in the absence of cells. Objective: Effect of naringenin and ellagic acid in MTT assay. Methods: MTT assay has been performed at different conditions like, varying the concentration of phytocompounds, incubation time, and changing the media. Results: Ellagic acid at the concentration of 80 μg/ml showed maximum absorbance of 0.579 in the presence of RPMI media (Roswell Park Memorial Institute Medium) that has 10% newborn calf serum. Naringenin showed higher absorbance of 2.74 at 80 μg/ml in RPMI media that has 10% newborn calf serum. Naringenin and Ellagic acid had better ability to reduce the MTT in the presence of RPMI media than anhydrous ethanol. Conclusion: Naringenin reduced the MTT better than ellagic acid in RPMI-1640 medium. These results show the need to be cautious of choosing MTT assay to measure the metabolic activity of cells treated with phytocompounds.

Liraglutide inhibits the progression of prediabetes in rats by reducing Raf-1 kinase inhibitor protein.

BackgroundThe cleavage product of Raf-1 kinase inhibitor protein (RKIP), hippocampal cholinergic neurostimulating peptide (HCNP) is involved in the promotion of insulin secretion. Studies have shown that liraglutide can inhibit the progression of prediabetes. This study aims to investigate whether the above effects of liraglutide are related to RKIP and HCNP.MethodsInsulin-1 (INS-1) cells were divided into control group (CON), HCNP group, and HCNP + darifenacin group (H-DAR). The three groups were cultured with Roswell Park Memorial Institute (RPMI) 1640, synthetic HCNP (50 pg/mL) and RPMI 1640, and HCNP + RPMI 1640 + darifenacin respectively. Subsequently, twelve 12- to 14-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were randomly divided into 2 groups: the placebo group (PBO) and the liraglutide treatment group (LIRA). Six Long Evans Tokushima Otsuka (LETO) rats were used as the control group (CON). The LIRA group was given liraglutide 200 µg/kg intraperitoneally twice a day. After 12 weeks, body weight, fasting blood glucose, 2 hours postprandial blood glucose, and insulin resistance index were recorded. Western blot was used to detect expression level of C-RKIP, N-RKIP, and extracellular signal-regulated kinase of phosphorylation (p-ERK). Real-time quantitative polymerase chain reaction (qRT-PCR) to detect pancreatic tissue choline acetyltransferase (ChAT) and M3 cholinergic receptor (M3R) gene expression levels.ResultsAt glucose concentrations of 5.6 and 16.7 mmol/L, the insulin content in the HCNP group was higher than that in the CON and H-DAR groups (all P<0.01). The body weight and fasting serum insulin (FINS) of rats in the PBO group were higher than those in the LIRA group and the CON group (P<0.01). The relative content of C-RKIP protein in the PBO group was higher than that in the LIRA and CON groups (P<0.01). The relative content of N-RKIP protein and p-ERK protein was lower than that in the LIRA and CON group (P<0.05 and P<0.01, respectively). ChAT and M3R gene expression levels in PBO group were lower than those in LIRA and CON group (P<0.01).ConclusionsLiraglutide promotes the production of HCNP, can increase ChAT activity, activate M3R, and further promote the secretion of insulin.

Evaluation of different protocols for culturing mesenchymal stem cells derived from murine bone marrow

IntroductionCulturing bone marrow mesenchymal stem cells (BM-MSCs) is a key point in different fields of research, including tissue engineering and regenerative medicine and studies of the bone marrow microenvironment. However, isolating and expanding murine BM-MSCs in vitro has challenged researchers due to the low purity and yield of obtained cells. In this study, we aimed to evaluate five different protocols to culture murine BM-MSCs in vitro.MethodsAll protocols were based on the adhesion capacity of BM-MSCs to the tissue culture plastic surface and varied in the types of plate, culture media, serum, additional supplementation and initial cell density. Flow cytometry analysis was used to investigate lineage purity after expansion.ResultsThe expression of CD45 and CD11b was detected in the cultures generated according to all protocols, indicating low purity with the presence of hematopoietic cells and macrophages. The cellular growth rate and morphology varied between the cultures performed according to each protocol. Cells cultured according to protocol 5 (8 × 107cells/plate, Roswell Park Memorial Institute (RPMI) culture medium during first passage and then Iscove's Modified Delbecco's Medium (IMDM) culture medium, both supplemented with 9% fetal bovine serum, 9% horse serum, 12µM L-glutamine) presented the best performance, with a satisfactory growth rate and spindle-shape morphology.ConclusionOur results point out that the purity and satisfactory growth rate of murine BM-MSC cultures are not easily achieved and additional approaches must be tested for a proper cell expansion.

Dendritic cell TLR4 induces Th1-type immune response against Cryptosporidium parvum infection.

The objective of this study was to investigate the mechanism of Toll-like receptor (TLR4)- mediated dendritic cell (DC) immune against Cryptosporidium parvum infection. C. parvum sporozoites were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester. Murine bone marrow-derived DCs were isolated, and divided into TLR4 antibody blocking (TAB; infected with 2 × 105 labeled sporozoites and 0.5 μg TLR4 blocking antibody), TLR4 antibody unblocking (TAU; infected with 2 × 105 labeled sporozoites), and blank control (BC; with 1.5 mL Roswell Park Memorial Institute 1640 medium) groups. The adhesion of Cryptosporidium sporozoites to DCs and CD11c+ levels were examined by fluorescence microscopy and flow cytometry. Male KM mice were orally injected with C. parvum. The proliferation of T lymphocytes in spleen, expression of cytokines in peripheral blood, and TLR4 distribution features in different organs were further determined by immunohistochemistry. A significantly higher expression of CD11c+ and higher C. parvum sporozoite adhesion were found in the TAU group compared with other groups. The expression of CD4+CD8- /CD8+CD4- in the spleen were obviously differences between the TAB and TAU groups. The expression of TLR4, interleukin IL-4, IL-12, IL-18 and IFN-γ improved in the TAU group compared with TAB group. Higher expression of TLR4 was detected in the lymph nodes of mice in the TAU group, with pathological changes in the small intestine. Hence, TLR4 could mediate DCs to recognize C. parvum, inducing Th1 immune reaction to control C. parvum infection.

LncRNA transcription factor 7 is related to deteriorating clinical features and poor prognosis in multiple myeloma, and its knockdown suppresses disease progression by regulating the miR-203-mediated Jagged1-Notch1 signaling pathway.

Multiple myeloma (MM) remains a challenge to treat, and its precise pathogenic mechanisms have not been fully clarified. The present study aimed to evaluate the relation between long non-coding RNA transcription factor 7 (lnc-TCF7) and clinical features, as well as the prognosis of patients with MM, and to determine the effects of lnc-TCF7-knockdown on the regulation (and regulatory mechanisms) of MM progression. lnc-TCF7 expression was detected in the bone marrow plasma cells of 86 patients with MM and 30 healthy controls. In patients with MM, the clinical data were collected, and event-free survival (EFS) and overall survival (OS) analyses were conducted. In vitro, lnc-TCF7 expression was detected in MM cell lines and normal bone marrow plasma cells. Using Roswell Park Memorial Institute 8226 cells, functional experiments were conducted following lnc-TCF7 short hairpin (sh)RNA transfection, and compensation experiments were performed after lnc-TCF7 shRNA transfection alone and in combination with a microRNA (miR)-203 inhibitor. lnc-TCF7 expression was increased in patients with MM compared with the healthy controls and was positively related to β-2-microglobulin expression and International Staging System stage, while negatively associated with complete response, EFS and OS. In vitro, lnc-TCF7 was upregulated in MM cells compared with normal bone marrow plasma cells, and its knockdown suppressed MM cell proliferation while promoting apoptosis. Compensation experiments showed that miR-203 inhibition promoted MM progression by regulating the Jagged1-Notch1 signaling pathway in lnc-TCF7-knockdown cells. In conclusion, increased lnc-TCF7 expression was related to deteriorating clinical features and prognosis, and lnc-TCF7-knockdown inhibited disease progression by regulating the miR-203-mediated Jagged1-Notch1 signaling pathway activation in MM.

Anti-inflammatory and Antioxidant Activities of Teucrium polium Leaf Extract and its Phenolic and Flavonoids Content

Teucrium polium leaf is one of the folkloric medicinal plants used traditionally to treat many diseases in the Palestinian community. It has antibacterial antioxidant and anti-inflammatory consequences. Present study aims the evaluating the in vitro inhibitory effect of Teucrium polium leaf extracts on tumor necrosis factor-α (TNF-α) using polymorphonuclear cells (PMNCs), in addition to determine its antioxidant and total phenolic and flavonoids contents. Polymorphonuclear cells were withdrawn from whole blood according to Histopaque (Ficol-1077) method. Blood cells were cultured in an enriched Roswell Park Memorial Institute (RBMI) medium. The levels of tumor necrosis factor (TNF-α) were determined after 24 h using LPS stimulation. Total phenolic contents, flavonoids contents and antioxidant activity were measured using spectrophotometric method. The TNF-α concentrations were compared using paired-samples t test. The leaf extracts of Teucrium polium revealed significant reduction in terms of TNF-α levels. The extract contained high phenolic and flavonoids contents and its antioxidant activities were remarkable. The reduced values in the TNF-α levels as affected by Teucrium polium leaf extracts indicate its effect in anti-inflammation. The plant is rich with polyphenolic compounds and flavonoids and has strong antioxidant activity. The observed anti-inflammatory effect of the extracts under study may be discussed as the influence of the significant presence of the phenolic compounds and flavonoids.

Induction of Adipogenic Genes by Novel Serum-Free Conditions From Pre-adipocyte 3T3-L1 and ST2 Cells.

IntroductionObesity, defined as a condition of excessive fat accumulation in adipose tissue, is a global epidemic implicated in a myriad of processes deleterious to human health. It has become one of the leading impediments to public health globally. The study of obesity necessitates adipocyte models, which commonly employ a medium enriched with adipogenic hormones and fetal bovine serum (FBS) to culture terminal adipocytes. In the current study, we developed a novel protocol for serum-free differentiation of 3T3-L1 and ST2 pre-adipocytes using media enriched with free fatty acids (FFA) and bovine serum albumin (BSA). Differentiation was characterized by measuring FFA uptake and changes in expression of adipogenic genes. The novel protocol was also compared against the existing serum-inclusive method.MethodsThe National Institutes of Health (NIH)-3T3-L1 and ST2 pre-adipocyte cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% calf serum and 1% penicillin-streptomycin and Roswell Park Memorial Institute Medium (RPMI) with 10% FBS and 1% penicillin-streptomycin mixture, respectively, at 37℃, 5% CO2 in a humidified atmosphere. Differentiation was induced using a mixture of 0.25 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 10 µg/mL insulin, or 1% insulin-transferrin-selenium (ITS). Cells were cultured in serum-free media containing DMEM with BSA (2.5%) and lipid mixture 1 (LM1 1%) as well as serum-inclusive media enriched with 10% FBS. Total RNA was extracted, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using delta-delta Ct method, also known as the 2-∆∆Ct method. Ribosomal protein, large, P0 (RPLP0) was used as a house-keeping gene for quantitation of relative expressions.ResultsWe observed an increase in fatty acid accumulation relative to controls using Oil Red O neutral lipid staining and spectrophotometry. This result was consistent with the effects of the serum-inclusive method. Differentiation was further confirmed by increased gene expression of adipogenic transcription factors - peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα); adipogenic genes - fatty acid-binding protein 4 (FABP4/aP2) and fatty acid translocase (FAT/CD36); and the lipogenic gene - perilipin by using quantitative RT-PCR.ConclusionOur data suggest that serum-free differentiation can significantly enhance the free fatty acid accumulation as well as adipogenic gene expression in both NIH-3T3-L1 and ST2 pre-adipocyte cells. Given the shortcomings of FBS, this method may provide advantages to the serum-inclusive protocols described previously.

Using nutrient-rich solutions and adding multiple cytoprotective agents as new strategies to develop lung preservation solutions.

Commonly, donor lungs are preserved with low-potassium dextran glucose solution at low temperature. We hypothesized that adding nutrients and/or cytoprotective agents to preservation solutions improves donor lung quality. Human lung epithelial cells and human pulmonary microvascular endothelial cells cultured at 37°C with serum containing medium were switched to designated testing solutions at 4°C with 50% O2 for different cold ischemic time, followed by switching back to serum containing culture medium at 37°C to simulate reperfusion. We found that bicarbonate buffer system should be avoided in preservation solution. When pH was maintained at physiological levels, cell culture media showed better cell survival than in low-potassium dextran glucose solution. Phosphate-buffered cell culture media were further improved by adding colloid dextran 40. When rat donor lungs were preserved at 4°C for 24 h, phosphate-buffered Roswell Park Memorial Institute-1640 medium [RPMI-1640(p)] plus dextran 40 or adding cytoprotective agents (alpha 1 antitrypsin, raffinose, and glutathione) to low-potassium dextran glucose solution prevented alveolar wall swelling, apoptosis, activation of endothelial cells, and cellular edema. Using nutrient-rich solution and/or adding multiple cytoprotective agents is a new direction for designing and developing organ preservation solutions. Cell culture model, as a screening tool, reduces the use of animals and provides potential underlying mechanisms.

In-Depth Analysis of the Impact of Different Serum-Free Media on the Production of Clinical Grade Dendritic Cells for Cancer Immunotherapy.

Dendritic cell (DC)-based antitumor vaccines have proven to be a safe approach, but often fail to generate robust results between trials. Translation to the clinic has been hindered in part by the lack of standard operation procedures for vaccines production, namely the definition of optimal culture conditions during ex-vivo DC differentiation. Here we sought to compare the ability of three clinical grade serum-free media, DendriMACS, AIM-V, and X-VIVO 15, alongside with fetal bovine serum-supplemented Roswell Park Memorial Institute Medium (RPMI), to support the differentiation of monocyte-derived DCs (Mo-DCs). Under these different culture conditions, phenotype, cell metabolomic profiles, response to maturation stimuli, cytokines production, allogenic T cell stimulatory capacity, as well as priming of antigen-specific CD8+ T cells and activation of autologous natural killer (NK) cells were analyzed. Immature Mo-DCs differentiated in AIM-V or X-VIVO 15 presented lower levels of CD1c, CD1a, and higher expression of CD11c, when compared to cells obtained with DendriMACS. Upon stimulation, only AIM-V or X-VIVO 15 DCs acquired a full mature phenotype, which supports their enhanced capacity to polarize T helper cell type 1 subset, to prime antigen-specific CD8+ T cells and to activate NK cells. CD8+ T cells and NK cells resulting from co-culture with AIM-V or X-VIVO 15 DCs also showed superior cytolytic activity. 1H nuclear magnetic resonance-based metabolomic analysis revealed that superior DC immunostimulatory capacities correlate with an enhanced catabolism of amino acids and glucose. Overall, our data highlight the impact of critically defining the culture medium used in the production of DCs for clinical application in cancer immunotherapy. Moreover, the manipulation of metabolic state during differentiation could be envisaged as a strategy to enhance desired cell characteristics.

Tuning poly (L-lactic acid) scaffolds with poly(amidoamine) and poly(propylene imine) dendrimers: surface chemistry, biodegradation and biocompatibility

The aim of this comparative study was focused on the surface chemistry, in vitro biodegradation and biocompatibility of poly(L-lactic acid) (PLLA) scaffolds incorporated with poly(amidoamine) (PAMAM) and poly(propylene imine) (PPI) dendrimers. The functionality of these two types of dendrimers from second, third and fifth generations (G2, G3 and G5) onto the surface of PLLA film and electrospun scaffolds was investigated and characterized by a series of analyses including X-ray photoelectron spectroscopy, atomic force microscope, Fourier transform infrared spectroscopy, contact angle, scanning electron microscope, weight loss, water uptake and pH changes after degradation in different media. The successful incorporation of dendrimers into the surface of PLLA scaffolds was observed. PPI-G5 could further increase the hydrophilicity and surface roughness of PLLA scaffolds compared to PPI-G3 and PPI-G2. Further improvement on PLLA surface chemistry was observed when PAMAM-G5 was applied compared to PPI-G5. The pH value of Roswell Park Memorial Institute Medium (RPMI) media containing raw electrospun PLLA scaffold decreased to 5.2 from its original value of 7.2. However, the local acidity of RPMI medium caused by PLLA hydrolysis was greatly reduced on the surface of PLLA/PAMAM-G5 and PLLA/PPI-G5 with pH values of 7.8 and 7.5, respectively. Moreover, the viability of fibroblast cells on the surface of PLLA/dendrimer scaffolds was considerably increased compared to raw PLLA. Fibroblasts showed higher attachment and homogeneous spread morphology on the surface of PLLA/PAMAM-G5 and PLLA/PPI-G5. These observations underline the favorable surface properties of PLLA scaffolds incorporated with dendrimers, to be used as a biodegradable biocompatible scaffold tailored for biomedical applications.

Exosomal mRNAs and lncRNAs involved in multiple myeloma resistance to bortezomib.

The bone marrow microenvironment plays an essential role in multiple myeloma (MM) progression. We aimed to explore the alterations of levels of long noncoding RNAs and messenger RNAs (mRNAs), derived from exosomes in peripheral blood, in resistance to bortezomib (Btz) of MM patients. Peripheral blood samples were collected from five Btz‐resistant and five Btz‐sensitive MM patients. Exosomes in patients' peripheral blood were enriched, and the profiles of long noncoding RNAs (lncRNAs) and mRNAs in exosomes were determined using deep sequencing. Bioinformatics analysis was performed to explore biological function. MTS was employed to determine the viability of Roswell Park Memorial Institute (RPMI) 8226 and LP‐1 cells incubated with exosomes derived from Btz‐resistant patients. Quantitative polymerase chain reaction (qPCR) was used to evaluate the levels of exosomal FFAR1, SP9, HIST1H2BG, and ITIH2. Incubation with Btz‐resistant patient‐derived exosomes significantly increased the viability of Btz‐treated RPMI 8226 and LP‐1 cells in a dose‐dependent manner. We identified 482 lncRNAs and 2099 mRNAs deregulated in exosomes of the Btz‐resistance group; and 78 mRNAs were enriched in DR‐related pathways, including mammalian target of rapamycin, platinum drug resistance, and the cAMP and phosphoinositide 3‐kinase–Akt signaling pathways. qPCR results verified the increases in FFAR1 and SP9 and decreases in HIST1H2BG and ITIH2 in Btz‐resistant patient‐derived exosomes. Moreover, exosomal FFAR1 and SP9 exhibited potential as independent prognostic indicators of survival of MM patients. Our study reveals significant dysregulation of exosomal RNA components in the Btz‐resistant group of MM patients as well as several mRNAs that may be used as biomarkers of prognosis of MM patients that are resistant to Btz.

Maintenance of liver fluke, Dicrocoelium dendriticum, outside the body of its native host

Aims: In vitro cultivation of digenea would help the development of effective treatments and studies of the biology of the parasites. The goal of the present study was to optimize culture conditions for the maintenance liver fluke, Dicrocoelium dendriticum. Materials and Methods: Forty fresh D. dendriticum were collected from the sheep liver and washed three times with warm Roswell Park Memorial Institute (RPMI) 1640 Medium. The collected worms were transferred to 24-well Nunc-Immuno plates containing RPMI media supplemented with 50% of fetal bovine serum (FBS), 2% of sheep red blood cells (RBCs), 50 IU/ml of penicillin, and 50 mg/ml streptomycin. The mobility of the live/dead worms was observed by inverted microscope. The mean and median survival time was calculated by Kaplan–Meier model, and survival and hazard function graphs were also analyzed. Results: D. dendriticum was lived in vitro only for long periods of about 25 dyes. The 1st day of maintaining in culture media, one worm was dead and the number of dead worms was raised to 40 after 25 days of incubation. On the one hand, the mean survival time was 392 h with a confidence interval (CI) of 95% (384.8–400.03). On the other hand, the median survival time was 420 h with a CI of 95% (406.9–433.09). D. dendriticum was able to be alive in RPMI 1640 media for at least 25 days. Conclusion: RPMI 1640 supplemented with FBS, and RBCs can be used as short-term maintenance for the in vitro culture of D. dendriticum. The outcomes of the current study could be useful for many aspects of parasitological analysis.

Immunomodulatory effects of flavonoids: An experimental study on natural-killer-cell-mediated cytotoxicity against lung cancer and cytotoxic granule secretion profile

Background: A new approach involving immune-cell-mediated cancer therapy has been adopted extensively for the sake of lung cancer treatments by utilizing natural killer (NK) cells. NK cell activity can be enhanced with certain agents, and among them are flavonoids. Thus, this study was conducted to investigate the immunomodulatory roles of apigenin, luteolin and quercetin on NK cell activity against lung cancer cells and on the secretions of perforin and granulysin profile. Methods: The NK-92 cells were grown in complete α-Minimum Essential Medium (MEM). NCI-H460 lung cancer cells were cultured in Roswell Park Memorial Institute 1640 media. NK cell activity against lung cancer cells were done using MTT(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The secretions of perforin and granulysin profiles were then analysed using enzyme-linked immunosorbent assay. Results: Apigenin, luteolin and quercetin significantly increased the NK-cell-mediated cytotoxic activity against lung cancer cells at concentrations 12.5 µg/ml and 25µg/ml ( P &lt; 0.001). The secretion levels of perforin and granulysin from NK cells were also significantly enhanced with apigenin and luteolin treatment but not with quercetin. Conclusions: All three flavonoid compounds possessed some significant immunomodulatory actions on NK cell cytotoxic activity and granule secretion profiles towards lung cancer therapy.

Effect of Peganum Harmala Seeds Extract on Nitric Oxide in U937 Monocytes and Macrophages

Background and Aims: Nitric oxide (NO) has an essential role in inflammation and has been related to pathogenesis and the progress of numerous inflammatory-based diseases, including some cancers. Peganum harmala (P. harmala) is a medicinal plant used for the treatment of numerous diseases such as several infections. Also, anti-inflammatory effects of P. harmala extracts and its derivatives (harmaline and harmine) by suppressing myeloperoxidase, NO, and other mediators have been demonstrated in vivo. In this study, the effect of P. harmala seeds aqueous extract on NO production in U937 monocytic cells and peritoneal macrophages has been evaluated in vitro.&#x0D; Materials and Methods: U937 and mice peritoneal macrophages were cultured in Roswell Park Memorial institute-1640 with 10% fetal calf serum. Then, the cells at the logarithmic growth phase were incubated with different concentrations of aqueous extract of P. harmala seeds (0.1-1 mg/ml) for 24 hours. Next, NO production was assessed by the Griess method in the culture medium.&#x0D; Results: P. harmala seeds aqueous extract did not significantly affect lipopolysaccharide-induced NO production in U937 cells and peritoneal macrophages after 24 hours incubation time compared with untreated control cells.&#x0D; Conclusion: These results suggest that the anti-inflammatory effects of P. harmala may be mediated through NO-independent mechanism(s). However, further studies are warranted to define the P. harmala aqueous extract impact on NO expression in other related normal and cancerous cells.

Prostate Specific Antigen (PSA): The Historical Perspective

In 1970, shortly after joining Roswell Park Memorial Institute, the New York State institute for the study of malignant diseases, the author initiated investigations on the use of tumor cell products for diagnosis and therapy of cancer. Immunochemical approaches were used primarily to differentiate quantitatively or qualitatively normal cells from tumor cells. Prostate cancer was a major area of endeavor, with the goal to identify and characterize prostate tumor specific and associated antigens, and eventually to develop a simple but reliable blood test for prostate cancer. Prostate cancer research had not received much attention at the time this work was begun. The early studies focused upon, among others, prostatic acid phosphatase, alkaline phosphatase, and new prostate tumor markers. By the mid 1970s, three able investigators--Dr. Ching-Li Lee, Dr. Carl S. Killian, and Dr. Ming C. Wang--had joined the prostate cancer research team, and were invited to take charge of these three research projects, respectively.