Cultures Of Normal Cells Research Articles

Digoxin plus trametinib therapy of BRAF wild type metastatic melanoma patients.

9527Background: A 200,000 small molecule library was screened for cytotoxicity with an in vitro 2D and 3D culture of primary human melanoma cells and other normal human cells. Several cardiac glyco...

Lipoxin A4 prevents tight junction disruption and delays the colonization of cystic fibrosis bronchial epithelial cells by Pseudomonas aeruginosa.

The specialized proresolution lipid mediator lipoxin A4 (LXA4) is abnormally produced in cystic fibrosis (CF) airways. LXA4 increases the CF airway surface liquid height and stimulates airway epithelial repair and tight junction formation. We report here a protective effect of LXA4 (1 nM) against tight junction disruption caused by Pseudomonas aeruginosa bacterial challenge together with a delaying action against bacterial invasion in CF airway epithelial cells from patients with CF and immortalized cell lines. Bacterial invasion and tight junction integrity were measured by gentamicin exclusion assays and confocal fluorescence microscopy in non-CF (NuLi-1) and CF (CuFi-1) bronchial epithelial cell lines and in primary CF cultures, grown under an air/liquid interface, exposed to either a clinical or laboratory strains of P. aeruginosa LXA4 delayed P. aeruginosa invasion and transepithelial migration in CF and normal bronchial epithelial cell cultures. These protective effects of LXA4 were inhibited by the ALX/FPR2 lipoxin receptor antagonist BOC-2. LXA4 prevented the reduction in mRNA biosynthesis and protein abundance of the tight junction protein ZO-1 and reduced tight junction disruption induced by P. aeruginsosa inoculation. In conclusion, LXA4 plays a protective role in bronchial epithelium by stimulating tight junction repair and by delaying and reducing the invasion of CF bronchial epithelial cells by P. aeruginsosa.

LINE-1 hypomethylation and mutational status in cutaneous melanomas

Epigenetic dysregulation is an important emerging hallmark of cutaneous melanoma development. The global loss of DNA methylation in gene-poor regions and transposable DNA elements of cancer cells contributes to increased...

Preliminary study of spectral features of normal and malignant cell cultures

In this study the fluorescence emission spectra of normal and malignant cell cultures were recorded at an excitation wavelength of 290 nm, corresponding to the higher fluorescence intensity at 350 nm (due to tryptophan) of three malignant cells and normal cells. Similarly, Stokes shift spectra were recorded for normal and malignant cell cultures with a shift, Δλ, of 70 nm. The Stokes shift shows the existence of discriminating features between normal and carcinoma cell lines due to the higher concentration of phenylalanine and tryptophan in carcinoma cell lines which are completely absent in normal cell lines. Hence, both the emission spectra and the Stokes shift spectra showed considerably different spectral features between the normal and malignant cells.The preliminary studies indicate the potential application of fluorescence spectroscopy for cancer detection using the spectral features of biofluorophores.

Simultaneous Sensitive Determination of Eight Collagen-Related Bone Turnover Biomarkers in Diverse Clinical Samples by HPLC with Fluorescent Derivatization

The suitability of HPLC with fluorescent derivatization for simultaneous determination of eight collagen-related bone turnover biomarkers, 4-hydroxy-l-proline (OHP), pyridinoline (PYD), hydroxylysine (Hyl), deoxypyridinoline, and their glycosides, galactosyl-Pyd (Gal-Pyd, GP), galactosyl-glucosyl-Pyd, β-1-galactosyl-O-hydroxylysine (Gal-Hyl, GH), and α-1,2-glucosyl-galactosyl-O-hydroxylysine (GGH), in diverse clinical samples was investigated. 5-Carboxyfluorescein succinimidyl ester (CFSE) was chosen as the fluorescent labeling reagent. Various parameters affecting derivatization and separation were optimized. Under optimal conditions, maximum derivatization could be achieved in 25 min at 30 °C, and complete baseline separation could be completed within 25 min, with relative standard deviation values for corrected peak areas less than 3.5 % for intraday assay and 4.2 % for interday assay, respectively. The limits of detection were determined to be 0.1–1.7 nM, which are well below the expected concentrations in the real samples. The method has been used for the quantitative determination of eight target analytes in various clinical samples, including cell cultures of normal human osteoclast, preosteoblast, osteoclastoma cells and serum and urine fluids from patients with fracture, osteitis fibrosa, or osteoclastoma as well as healthy subjects, with spiked recoveries of 96–103 %. This original application of HPLC–fluorescence detection represents a powerful tool for investigating the dynamics of metabolic imbalance of these biomarkers for bone turnover under physiological and pathophysiological conditions.

Cadmium induces mucin 8 expression via Toll-like receptor 4-mediated extracellular signal related kinase 1/2 and p38 mitogen-activated protein kinase in human airway epithelial cells.

Inhalation of cadmium can lead to development of inflammatory airway diseases such as acute pulmonary edema and chronic obstructive pulmonary disease. In inflammatory airway diseases, expression of mucins is increased, which leads to increased morbidity and mortality of the affected patients. However, no study on the effect of cadmium on expression of mucin genes in airway epithelial cells has been reported. Therefore, this study was conducted in order to investigate the effect and the brief signaling pathway of cadmium on expression of mucin genes in human airway epithelial cells. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effect and signaling pathway of cadmium on expression of mucin genes were investigated using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). Cadmium increased mucin 8 (MUC8) expression and Toll-like receptor (TLR) 4 messenger RNA (mRNA) expression. Cadmium significantly activated phosphorylation of extracellular signal related kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) and p38 MAPK. ERK1/2 MAPK inhibitor, p38 MAPK inhibitor, TLR4 siRNA, ERK1/2 MAPK siRNA, and p38 MAPK siRNA significantly blocked cadmium-induced MUC8 mRNA expression. TLR4 siRNA significantly blocked cadmium-activated phosphorylation of ERK1/2 MAPK and p38 MAPK. The results of this study suggest for the first time that cadmium induces MUC8 expression via TLR4-mediated ERK1/2 and p38 MAPK signaling pathway in human airway epithelial cells.

Abstract C172: Concomitant disruption of WEE1 and AurKA synergistically enhances anti-cancer effects in p53 mutated head and neck squamous cell carcinoma

Abstract Head and neck cancer is a devastating disease which annually affects over half a million patients worldwide. Squamous cell carcinoma of the head and neck (SCCHN), which accounts for approximately 90% of all head and neck cancers, often harbors a disruptive mutation of the TP53 gene. Cells harboring TP53 mutations evade cisplatin-induced cellular senescence with cell cycle arrest in the G2 phase, allowing for DNA repair. Wee1 and Aurora Kinase A (AurKA) are crucial regulators of the mitotic cell cycle including G2/M phases and are significantly activated in SCCHN harboring TP53 mutations. Therefore, we hypothesized that a combination therapy aimed at these synthetic lethal targets, Wee1 and AurKA, would enhance anti-cancer effects and cisplatin sensitivity due to abrogation of G2 arrest concomitant with defects in tumor cell mitotic machinery, which is indeed caused by cisplatin. We tested this hypothesis using assays of cell viability, colony formation, anchorage independent cell growth, and finally a three-dimensional oncosphere formation assay in vitro. The combination index (CI) was calculated according to the Chou-Talalay method (CI<0.8, synergistic; 0.8<CI<1.2, additive; CI>1.2, antagonistic). The cell viability assay indicated that the combination of the Wee1 inhibitor MK-1775 with the AurKA inhibitor MLN8237 exhibited striking synergistic inhibitory effects on the cell growth of SCCHN cell lines including FaDu (TP53 mutation, CI = 0.397004), Detroit562 (TP53 mutation, CI = 0.202677) and UNC-7 (TP53 WT, CI = 0.587106). Consistent with the cell viability data we obtained, we also confirmed this synergistic effect under anchorage dependent and independent cell growth conditions in FaDu and UNC-7 cell lines. Interestingly, this combination dramatically reduced the numbers of a stem cell-like oncosphere formation, showing a more effective combination in comparison to MK-1775 with cisplatin (inhibitory effect (% Vehicle): MK-1775+MLN8238 vs MK-1775+Cisplatin; FaDu = 74% vs 69%; UNC-7 = 43% vs 36%). Moreover, all spheroids exposed to the combination of MK-1775 with MLN8237 did not survive following further incubation under normal cell culture conditions. Induction of apoptosis, as shown gradually increased PARP cleavage at Asp214-Gly215 in FaDu and UNC-7 cells, was demonstrated. Furthermore, this synergistic synthetic lethal effect was not seen in normal human tracheobronchial epithelial (NHTBE) cells in a three-dimensional organotypic (air-liquid interface culture condition) culture system, predicting low toxicity. These results suggest that SCCHN cells, including high-risk TP53 mutations, are sensitive to the combination of MK-1775 and MLN8237 in vitro, thereby supporting the preclinical and further clinical evaluation of MK-1775 in combination with MLN8237 for the treatment of patients with TP53 inactivated HNSCC. Citation Format: Jong Woo Lee, Janaki Parameswaran, Ja Seok Koo, Barbara Burtness. Concomitant disruption of WEE1 and AurKA synergistically enhances anti-cancer effects in p53 mutated head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C172.

Development of β Type Ti23Mo-45S5 Bioglass Nanocomposites for Dental Applications.

Titanium β-type alloys attract attention as biomaterials for dental applications. The aim of this work was the synthesis of nanostructured β type Ti23Mo-x wt % 45S5 Bioglass (x = 0, 3 and 10) composites by mechanical alloying and powder metallurgy methods and their characterization. The crystallization of the amorphous material upon annealing led to the formation of a nanostructured β type Ti23Mo alloy with a grain size of approximately 40 nm. With the increase of the 45S5 Bioglass contents in Ti23Mo, nanocomposite increase of the α-phase is noticeable. The electrochemical treatment in phosphoric acid electrolyte resulted in a porous surface, followed by bioactive ceramic Ca-P deposition. Corrosion resistance potentiodynamic testing in Ringer solution at 37 °C showed a positive effect of porosity and Ca-P deposition on nanostructured Ti23Mo 3 wt % 45S5 Bioglass nanocomposite. The contact angles of glycerol on the nanostructured Ti23Mo alloy were determined and show visible decrease for bulk Ti23Mo 3 wt % 45S5 Bioglass and etched Ti23Mo 3 wt % 45S5 Bioglass nanocomposites. In vitro tests culture of normal human osteoblast cells showed very good cell proliferation, colonization, and multilayering. The present study demonstrated that porous Ti23Mo 3 wt % 45S5 Bioglass nanocomposite is a promising biomaterial for bone tissue engineering.

A104: Notch-dependent crosstalk between stromal and neoplastic cells

Malignant tumors consist not only of neoplastic cells, but also of various normal cells, for example fibroblasts, macrophages or endothelial cells. These normal cells stand under constant pressure of transformed cells, regulating their properties and converting them into tumor-promoting cells. Tumor-stroma interaction takes place during all stages of carcinogenesis. Notch activation upon receptor binding with the ligand is a way of direct intercellular communication during embryo- and histogenesis, determining various processes like differentiation, proliferation, etc. It has been previously shown that in tumors this signaling cascade regulates not only properties of transformed cells, but also stromal cells activities, i.e. neoangiogenesis. The Notch role in communication between neoplastic cells and stromal fibroblasts is underinvestigated. Cancer-associated stromal fibroblasts (CAFs) are an important component of tumors secreting growth factors and proteases, modulating immune reactions and contributing to cancer stem cells niches formation. CAFs resemble myofibroblasts and express α Smooth-Muscle Actin ( α SMA). We obtained and characterized cultures of normal mesenchymal cells: a myofibroblasts-like (MF), and fibroblasts-like MC1 and MC2, differing by Notch1 expression. These cell cultures variously influenced growth of colon cancer notch- ligand Jagged2-positive HCT116 xenographs in nude mice. MF cells were characterized by the strongest while Notch1-deficient MC2 by the weakest tumor-promoting activity. MC1 but not MC2 started to express α SMA upon co-cultivation in vitro with neoplastic HCT116 cells. Such co-cultivation also lead to Notch activation according to a luciferase reporter. NICD (Notch Intracellular Domain) expression activated MC1 and MC2, while Notch1 silencing in MC1 abrogated both HCT116-mediated activation of the fibroblasts in vitro and their tumor-promoting activity in vivo. Notch signaling in mesenchymal cells stimulated TGFb production that lead to both autocrine and paracrine receptors stimulation. We believe that this cytokine activates fibroblasts in our experimental system. We also revealed that this process was p53-dependent. So we have shown Notch1 to be involved in tumor-stroma interaction, particularly its activation leading to fibroblasts transdifferentiation. Notch1-stimulated fibroblasts are able to produce TGFb and to promote tumor growth in xenographs. The tumor-stimulating potency of various fibroblasts in our experimental system depends on their ability to transdifferentiate to myofibroblasts upon Notch activation.

Characterization of a novel metastatic prostate cancer cell line of LNCaP origin.

The LNCaP cell line was originally isolated from the lymph node of a patient with metastatic prostate cancer. Many cell lines have been derived from LNCaP by selective pressures to study different aspects of prostate cancer progression. When injected subcutaneously into male athymic nude mice, LNCaP and its derivatives rarely metastasize. Here, we describe the characteristics of a new LNCaP derivative, JHU-LNCaP-SM, which was generated by long term passage in normal cell culture conditions. Short tandem repeat (STR) analysis and genomic sequencing verified JHU-LNCaP-SM derivation from parental LNCaP cells. JHU-LNCaP-SM cells express the same mutated androgen receptor (AR) but unlike LNCaP, are no longer androgen dependent for growth. The cells demonstrate an attenuated androgen responsiveness in transcriptional assays and retain androgen sensitive expression of PSA, AR, and PSMA. Unlike parental LNCaP, JHU-LNCaP-SM cells quickly form subcutaneous tumors in male athymic nude mice, reliably metastasize to the lymph nodes and display a striking intra-tumoral and spreading hemorrhagic phenotype as tumor xenografts. The JHU-LNCaP-SM cell line is a new isolate of LNCaP, which facilitates practical, preclinical studies of spontaneous metastasis of prostate cancer through lymphatic tissues.

Monolayer Graphene-Directed Growth and Neuronal Differentiation of Mesenchymal Stem Cells.

The development of an efficient platform for the growth and neuronal differentiation of stem cells is crucial for autologous cell therapy and tissue engineering to treat various neuronal disorders and neurodegenerative diseases. In this study, we describe the use of highly uniform graphene platforms that provide unique environments where unusual three-dimensional spheroids of human mesenchymal stem cells (hMSCs) are formed, which is advantageous for the differentiation of hMSCs into neurons. We suppose that graphene regulates the interactions at cell-substrate or cell-cell interfaces, consequently promoting the neurogenesis of hMSCs as well as the outgrowth of neurites, which was evidenced by the graphene-induced upregulation of early neurogenesis-related genes. We also demonstrated that the differentiated neurons from hMSCs on graphene are notably sensitive to external ion stimulation, and their neuronal properties can be maintained even after detaching and re-seeding onto a normal cell culture substrate, suggesting the enhanced maturity of resulting neuronal cells. Thus, we conclude that monolayer graphene is capable of regulating the growth and neural differentiation of hMSCs, which would provide new insight and strategy not only for autologous stem cell therapy but for tissue engineering and regenerative medicine based on graphene scaffolds.

Synergistic Proinflammatory Responses by IL-17A and Toll-Like Receptor 3 in Human Airway Epithelial Cells.

Viral respiratory infections activate the innate immune response in the airway epithelium through Toll-like receptors (TLRs) and induce airway inflammation, which causes acute exacerbation of asthma. Although increases in IL-17A expression were observed in the airway of severe asthma patients, the interaction between IL-17A and TLR activation in airway epithelium remains poorly understood. In this study, we demonstrated that IL-17A and polyI:C, the ligand of TLR3, synergistically induced the expression of proinflammatory cytokines and chemokines (G-CSF, IL-8, CXCL1, CXCL5, IL-1F9), but not type I interferon (IFN-α1, -β) in primary culture of normal human bronchial epithelial cells. Synergistic induction after co-stimulation with IL-17A and polyI:C was observed from 2 to 24 hours after stimulation. Treatment with cycloheximide or actinomycin D had no effect, suggesting that the synergistic induction occurred without de novo protein synthesis or mRNA stabilization. Inhibition of the TLR3, TLR/TIR-domain-containing adaptor-inducing interferon β (TRIF), NF-κB, and IRF3 pathways decreased the polyI:C- and IL-17A/polyI:C-induced G-CSF and IL-8 mRNA expression. Comparing the levels of mRNA induction between co-treatment with IL-17A/polyI:C and treatment with polyI:C alone, blocking the of NF-κB pathway significantly attenuated the observed synergism. In western blotting analysis, activation of both NF-κB and IRF3 was observed in treatment with polyI:C and co-treatment with IL-17A/polyI:C; moreover, co-treatment with IL-17A/polyI:C augmented IκB-α phosphorylation as compared to polyI:C treatment alone. Collectively, these findings indicate that IL-17A and TLR3 activation cooperate to induce proinflammatory responses in the airway epithelium via TLR3/TRIF-mediated NF-κB/IRF3 activation, and that enhanced activation of the NF-κB pathway plays an essential role in synergistic induction after co-treatment with IL-17A and polyI:C in vitro.

Abstract 707: Distinct roles of Na,K-ATPase function and expression in medulloblastoma

Abstract Medulloblastoma, which usually arises in the cerebellum, is the most common malignant brain cancer in children and is still associated with substantial mortality and survivors often suffer from serious life-long therapy-related side effects. In recent years several medulloblastoma subtypes with distinct developmental origins, genetic profiles, pathway signatures, and clinicopathological features have been identified. The current consensus divides medulloblastoma tumors into four subgroups, WNT, sonic hedgehog (Shh), Group 3 and Group 4 allowing for different targeted therapeutic approaches. Not specifically restricted to a defined subgroup, aberrant activation of the epidermal growth factor receptor family (EGFR/ErbB) has been reported in a large percentage of medulloblastoma and has been associated with poor outcome. The Shh pathway is an important signaling pathway involved in cerebellar development. Shh is secreted from the Purkinje cells, and acts as a mitogen for cerebellar granule precursor (CGP) cells. Mutations leading to hyperactive Shh signaling have been associated with improper migration and differentiation of CGP cells and medulloblastoma formation. Still, the molecular targets contributing to Shh-mediated proliferation in these cells are poorly understood. Na,K-ATPase is a membrane protein that maintains intracellular ion homeostasis, and is responsible for generating ion gradients across cell membranes. Consisting of a catalytic alpha subunit and a beta subunit, the enzyme not only pumps sodium ions out and potassium ions into the cell using ATP but also functions a signaling scaffold and a cell adhesion molecule. Changes in Na,K-ATPase function and expression have been reported in various cancers and may contribute to tumor development and progression. We now show that the Na,K-ATPase beta1-subunit is drastically reduced in medulloblastoma tumors of mice with aberrant activation of Shh signaling. In addition, Shh activation prevented the upregulation of the beta1-subunit in primary cultures of normal cerebellar granule cells and the polycomb transcription factor Bmi1 that is induced upon activation of Shh signaling repressed beta1-subunit levels. Furthermore, shRNA-mediated knockdown of the beta1-subunit increased cell proliferation and tumorigenicity of medulloblastoma cells. Nevertheless, cardiac glycosides that inhibit the pump function of Na,K-ATPase inhibited EGF-induced signaling and cell motility suggesting that Na,K-ATPase alpha and beta subunit may have dual functions in CGP cells and medulloblastoma. Citation Format: Zhiqin Li, Alisa Litan, Seung Joon Lee, Bruce Graves, Sonali P. Barwe, Sigrid A. Langhans. Distinct roles of Na,K-ATPase function and expression in medulloblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 707. doi:10.1158/1538-7445.AM2015-707

Abstract 5138: New technique generates cell cultures from rare adenoid cystic carcinomas of the salivary gland

Abstract Introduction: Historically it has been difficult to generate and maintain cell cultures derived directly from a patient's tumor and virtually impossible to generate continuous cultures of normal cells. Preclinical models provide an essential tool to study both basic biology as well as aid in translational research, including drug target identification and drug discovery efforts. Even today, one of the greatest challenges in cancer research has been the generation of stable cancer cell lines that can genocopy and phenocopy the respective primary tumors. Recently, a new cell culture method was developed by our research group (Liu et al, AJP 2012) called “conditionally reprogrammed cells” (CRCs). Cells derived from primary tumors and corresponding normal tissues can be grown indefinitely under defined conditions without using any extrinsic genetic immortalization technique. No authentic cell culture line currently exists for the rare and aggressive adenocystic carcinoma (ACC) of the salivary gland and the few cell lines described have proven to be contaminated with conventional cell lines such as HeLa. Here we show that our CRC technology can be successfully applied to generate cell cultures from this rare subtype of salivary gland tumor. Method: The CRC technique utilizes irradiated mouse J2 feeder cells and a ROCK inhibitor to rapidly grow and expand each patient's epithelial cells. All PDx tissue samples were provided by the Adenoid Cystic Carcinoma Research Foundation (ACCRF) and processed in our laboratory for generating CRC lines. STR analysis indicated that these cultures are unique and show no similarity to cell lines in the ATCC data base. Results: We have successfully established CRC cultures from three different PDx tumor biopsies. Preliminary analysis on one CRC culture, ACC11, shows that the Myb-NF1B translocation is maintained, similar to the primary tumor and PDx. The sequencing of RT-PCR products using primers for Myb and NF1B revealed the exact break point for both genes. The cell cultlure maintains the overexpression of the Myb protein as assessed by Western blot when compared with a non-ACC salivery gland culture. Next generation sequencing of 48 TruSeq cancer panel identified mutations in key genes including ATM and FGFR2 that were further confirmed by RT-PCR followed by sequencing. Conclusion: The CRC technology represents a new approach to culturing cells from tumors and normal cells, including tumors that have been historically challenging to grow such as ACC of salivary gland. The resulting cells maintained the key genetic translocation and corresponding overexpression of the Myb protein. Newly identified mutations were also discovered in this tumor. Future studies will explore the genetic alterations in the two additional ACC tumor cell cultures and to generate a biobank of ACC tumor cultures for basic biological studies and to identify the key drivers of malignancy. Citation Format: Chen Chen, Sujata Choudhury, Xuefeng Liu, Richard Schlegel, Seema Agarwal. New technique generates cell cultures from rare adenoid cystic carcinomas of the salivary gland. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5138. doi:10.1158/1538-7445.AM2015-5138

Small molecule and RNAi induced phenotype transition of expanded and primary colonic epithelial cells.

Recent progress in mammalian intestinal epithelial cell culture led to novel concepts of tissue modeling. Especially the development of phenotypically stable cell lines from individual animals enables an investigation of distinct intestinal loci and disease states. We here report primary and prolonged culture of normal porcine epithelial cells from colon for cell line development. In addition, a novel primary three-dimensional intestinal culture system is presented, which generated organoids composed of a highly polarized epithelial layer lining a core of subepithelial tissue. Cellular characterization of monolayer cell lines revealed epithelial identity and pointed to a proliferative crypt cell phenotype. We evaluated both RNAi and chemical approaches to induce epithelial differentiation in generated cell lines by targeting promoters of epithelial to mesenchymal transition (EMT). By in silico prediction and ectopic expression, miR-147b was proven to be a potent trigger of intestinal epithelial cell differentiation. Our results outline an approach to generate phenotypically stable cell lines expanded from primary colonic epithelial cultures and demonstrate the relevance of miR-147b and chemical inhibitors for promoting epithelial differentiation features.

Involvement of pro-inflammatory cytokines and growth factors in the pathogenesis of Dupuytren's contracture: a novel target for a possible future therapeutic strategy?

Dupuytren's contracture (DC) is a benign fibro-proliferative disease of the hand causing fibrotic nodules and fascial cords which determine debilitating contracture and deformities of fingers and hands. The present study was designed to characterize pro-inflammatory cytokines and growth factors involved in the pathogenesis, progression and recurrence of this disease, in order to find novel targets for alternative therapies and strategies in controlling DC. The expression of pro-inflammatory cytokines and of growth factors was detected by immunohistochemistry in fibrotic nodules and normal palmar fascia resected respectively from patients affected by DC and carpal tunnel syndrome (CTS; as negative controls). Reverse transcription (RT)-PCR analysis and immunofluorescence were performed to quantify the expression of transforming growth factor (TGF)-β1, interleukin (IL)-1β and vascular endothelial growth factor (VEGF) by primary cultures of myofibroblasts and fibroblasts isolated from Dupuytren's nodules. Histological analysis showed high cellularity and high proliferation rate in Dupuytren's tissue, together with the presence of myofibroblastic isotypes; immunohistochemical staining for macrophages was completely negative. In addition, a strong expression of TGF-β1, IL-1β and VEGF was evident in the extracellular matrix and in the cytoplasm of fibroblasts and myofibroblasts in Dupuytren's nodular tissues, as compared with control tissues. These results were confirmed by RT-PCR and by immunofluorescence in pathological and normal primary cell cultures. These preliminary observations suggest that TGF-β1, IL-1β and VEGF may be considered potential therapeutic targets in the treatment of Dupuytren's disease (DD).

Single cell measurement of telomerase expression and splicing using microfluidic emulsion cultures.

Telomerase is a reverse transcriptase that maintains telomeres on the ends of chromosomes, allowing rapidly dividing cells to proliferate while avoiding senescence and apoptosis. Understanding telomerase gene expression and splicing at the single cell level could yield insights into the roles of telomerase during normal cell growth as well as cancer development. Here we use droplet-based single cell culture followed by single cell or colony transcript abundance analysis to investigate the relationship between cell growth and transcript abundance of the telomerase genes encoding the RNA component (hTR) and protein component (hTERT) as well as hTERT splicing. Jurkat and K562 cells were examined under normal cell culture conditions and during exposure to curcumin, a natural compound with anti-carcinogenic and telomerase activity-reducing properties. Individual cells predominantly express single hTERT splice variants, with the α+/β− variant exhibiting significant transcript abundance bimodality that is sustained through cell division. Sub-lethal curcumin exposure results in reduced bimodality of all hTERT splice variants and significant upregulation of alpha splicing, suggesting a possible role in cellular stress response. The single cell culture and transcript abundance analysis method presented here provides the tools necessary for multiparameter single cell analysis which will be critical for understanding phenotypes of heterogeneous cell populations, disease cell populations and their drug response.

Toxicity and dyeing properties of dyes obtained through laccase-mediated synthesis

The use of enzymes in textile processes has many advantages as far as environmentally friendly processes are concerned. These advantages include water and energy savings, the use of lower amounts of chemicals, and mild and more sustainable production processes. Several orange–red colored bio-dyes were synthesized during laccase-mediated homomolecular or heteromolecular transformations of simple aromatic precursors. Due to the presence of many various substituents in the structure of these precursors, ensuring their laccase-mediated transformation into colored products, the dyes obtained were tested for their toxicity effect against humans and the environment. The evaluation of environmental toxicity employed the bioluminescent marine bacterium (Vibrio fischeri) as a test organism according to the Microtox® basic test protocol. The effect of dyes on human cells was tested in culture of normal human colon epithelial cells, using two independent tests. Despite the presence of the reactive amino group in the structure of precursors, a majority of the dyes obtained were non-toxic. The presence of a carboxylic and/or sulfonic group in the formula of the precursors ensures a good solubility of the dyes in water. The dyes obtained possess good dyeing activity towards natural and synthetic fibers. Mild conditions of synthesis thereof, as well as a lack of toxic by-products and effluents, constitute advantages of the application of laccase-mediated biocatalysis in the synthesis of textile dyes. Due to the environmental impact associated with the production of textile dyes, the potential application of fungal laccase as a biocatalyst for the synthesis of valuable colorants has been demonstrated in this paper.

Temperature responsive hydrogels enable transient three-dimensional tumor cultures via rapid cell recovery.

Recovery of live cells from three-dimensional (3D) culture would improve analysis of cell behaviors in tissue engineered microenvironments. In this work, we developed a temperature responsive hydrogel to enable transient 3D culture of human glioblastoma (GBM) cells. N-isopropylacrylamide was copolymerized with hydrophilic grafts and functionalized with the cell adhesion peptide RGD to yield the novel copolymer poly(N-isopropylacrylamide-co-Jeffamine(®) M-1000 acrylamide-co-hydroxyethylmethacrylate-RGD), or PNJ-RGD. This copolymer reversibly gels in aqueous solutions when heated under normal cell culture conditions (37°C). Moreover, these gels redissolve within 70 s when cooled to room temperature without the addition of any agents to degrade the synthetic scaffold, thereby enabling rapid recollection of viable cells after 3D culture. We tested the efficiency of cell recovery following extended 3D culture and were able to recover more than 50% of viable GBM cells after up to 7 days in culture. These data demonstrate the utility of physically crosslinked PNJ-RGD hydrogels as a platform for culture and recollection of cells in 3D.

Development of full-thickness human skin equivalents with blood and lymph-like capillary networks by cell coating technology.

We developed a human skin equivalent (HSE) containing blood and lymph-like capillary networks using a cell coating technique, which is a rapid fabrication technology of three-dimensional cellular constructs by cell surface coating using layer-by-layer assembled nanofilms of extracellular matrices. The thickness of dermis consisting of normal human dermal fibroblasts was easily controlled from approximately 5 to 100 µm by altering the seeded cell number. Keratinocytes as a major cell population showed homogeneous differentiation on the surface of the dermis by lifting to air-liquid interface. Histological analysis revealed four distinct layers such as basal layer, spinous layer, granular layer, and cornified cell layer in the epidermis. Interestingly, the measurement of transepithelial electrical resistance (TEER) indicated prolongation of the attainment time for maximum value by increasing the number of the dermal fibroblasts, and the HSEs with six layers of dermis revealed the longest period maintaining over 500 Ω cm(2) of TEER. The co-sandwich culture of human umbilical vein endothelial cells and normal human dermal lymphatic microvascular endothelial cells within eight-layered dermis showed in vitro co-network formation of individual blood and lymph-like capillaries inside the dermis. This is the report for homogeneous full-thickness HSEs with blood and lymph capillary networks, which will be useful for biomedical and pharmaceutical applications.