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Primary Cells (primary + cell)

Distribution by Scientific Domains

Medical Sciences	42%
Life Sciences	34%
Chemistry	15%
Polymers and Materials Science	7%

Terms modified by Primary Cells

primary cell culture

primary cell wall

Selected Abstracts

Co-Cultures of Primary Cells on Self-Supporting Nanoporous Alumina Membranes,

ADVANCED ENGINEERING MATERIALS, Issue 7 2010
Andreas Hoess
Due to their unique properties, self-supporting nanoporous aluminum oxide (alumina) membranes are useful substrates for the indirect co-cultivation of cells. The membrane can act as a physical barrier between different cell types, whereas the cell-to-cell communication is guaranteed by the diffusion of soluble molecules or factors through the pores. With the help of such membranes the mRNA expression of hepatic genes can be induced in human adipose-derived mesenchymal stem cells (hASCs) during an indirect co-cultivation with primary mouse hepatocytes under different culture conditions. This proof of concept shows that such a cultivation approach is beneficial for different issues in the field tissue engineering or cell therapy. [source]

ORIGINAL ARTICLE: Urocortin Increases IL-4 and IL-10 Secretion and Reverses LPS-induced TNF-, Release from Human Trophoblast Primary Cells

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 4 2009
Michela Torricelli
Problem, As urocortin (Ucn) is a placental peptide belonging to the corticotrophin-releasing hormone (CRH) family that modulates immune function in other biological models, this study evaluated Ucn effects on cytokines secretion from cultured human trophoblast cells. Method of study, Placentas were collected from normal term pregnancies after elective caesarean section, and primary trophoblast culture was prepared followed by the treatment of Ucn and/or CRH selective antagonists, antalarmin and astressin 2b. The anti-inflammatory cytokines IL-4 and IL-10 and the pro-inflammatory cytokine TNF-, were measured by ELISA. Results, Urocortin treatment induced a significant and dose-dependent increase of IL-4 and IL-10, whereas it did not affect TNF-, secretion. When incubated in the presence of LPS, Ucn reversed LPS-induced TNF-, release from cultured trophoblast cells, an effect that was blocked by the CRH-R2 selective antagonist, astressin 2b. Conclusion, Urocortin stimulates IL-4 and IL-10 secretion and reverses LPS-induced TNF-, release from trophoblast cells through action on CRH-R2 receptors, suggesting that this peptide may play a possible role as an anti-inflammatory agent. [source]

Oncoapoptosis: A novel molecular therapeutic for cancer treatment

IUBMB LIFE, Issue 2 2010
John A. Blaho
Abstract Many cancer cells refractory to radiation treatment and chemotherapy proliferate due to loss of intrinsic programmed cell death (apoptosis) regulation. Consequently, the resolution of these cancers are many times outside the management capabilities of conventional therapeutics. We have developed a replication defective herpes simplex virus system which triggers apoptosis specifically in transformed human cells, termed oncoapoptosis. Susceptibility to virus induced cell death is dependent on the p53 protein status in the tumor cells, indicating specific targeting of the treatment. Primary cells which produce functional p53 are resistant to oncoapoptotic killing but not to apoptosis induced by nonviral environmental factors. Thus, induction of apoptosis by nonreplicating virus is a feasible molecular therapeutic approach for killing human cancer cells. Our findings have important implications in designing novel virus-based anticancer strategies. © 2009 IUBMB IUBMB Life, 62(2): 87,91, 2010 [source]

Analysis of genomic dose-response information on arsenic to inform key events in a mode of action for carcinogenicity

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2010
P. Robinan Gentry
Abstract A comprehensive literature search was conducted to identify information on gene expression changes following exposures to inorganic arsenic compounds. This information was organized by compound, exposure, dose/concentration, species, tissue, and cell type. A concentration-related hierarchy of responses was observed, beginning with changes in gene/protein expression associated with adaptive responses (e.g., preinflammatory responses, delay of apoptosis). Between 0.1 and 10 ,M, additional gene/protein expression changes related to oxidative stress, proteotoxicity, inflammation, and proliferative signaling occur along with those related to DNA repair, cell cycle G2/M checkpoint control, and induction of apoptosis. At higher concentrations (10,100 ,M), changes in apoptotic genes dominate. Comparisons of primary cell results with those obtained from immortalized or tumor-derived cell lines were also evaluated to determine the extent to which similar responses are observed across cell lines. Although immortalized cells appear to respond similarly to primary cells, caution must be exercised in using gene expression data from tumor-derived cell lines, where inactivation or overexpression of key genes (e.g., p53, Bcl-2) may lead to altered genomic responses. Data from acute in vivo exposures are of limited value for evaluating the dose-response for gene expression, because of the transient, variable, and uncertain nature of tissue exposure in these studies. The available in vitro gene expression data, together with information on the metabolism and protein binding of arsenic compounds, provide evidence of a mode of action for inorganic arsenic carcinogenicity involving interactions with critical proteins, such as those involved in DNA repair, overlaid against a background of chemical stress, including proteotoxicity and depletion of nonprotein sulfhydryls. The inhibition of DNA repair under conditions of toxicity and proliferative pressure may compromise the ability of cells to maintain the integrity of their DNA. Environ. Mol. Mutagen., 2010. © 2009 Wiley-Liss, Inc. [source]

Surface morphology of eggs of Euproctis chrysorrhoea (Linnaeus, 1758)

ACTA ZOOLOGICA, Issue 2 2008
Selami Candan
Abstract Candan, S., Suludere, Z. and Bayrakdar, F. 2007. Surface morphology of eggs of Euproctis chrysorrhoea (Linnaeus, 1758). ,Acta Zoologica (Stockholm) 88: 000,000. Filaments covering the egg batches and chorion structure were studied both by light and scanning electron microscopy in the brown-tailed moth Euproctis chrysorrhoea (Linnaeus, 1758). Females lay eggs in masses on the underside of apple leaves. The egg batches are covered with brown hairs derived from the bodies of the female. Each female lays about 200,400 eggs. The spherical eggs are about 0.84 mm long and 0.47 mm wide. Newly deposited eggs are golden-yellow and darken after the onset of embryonic development. The micropylar area appears somewhat depressed and has a circular outline. The region is surrounded by a rosette of 10,12 petal-shaped primary cells, which are completely surrounded by a series of secondary and tertiary cells. The remainder of the egg is largely smooth, but shows aeropyles. These are located in the corners of ill-defined polygons. [source]

Development of Live Cell Chips to Monitor Cell Differentiation Processes

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 1 2008
C. Maercker
Abstract A big demand exists for high-throughput functional in vitro assays which can measure cellular phenotypes by molecular methods and therefore improve the resources of primary cells for cell therapy, tissue engineering and high-content screenings in drug development. This approach focuses on cellular adhesion which is an important differentiation process during homing of stem cells. Moreover, it is a promising method especially for adherent cells which are not accessible by classical cell sorting methods. The chip design includes a housing with electrodes to measure electric field densities and impedance, respectively. Moreover, specific coatings of the wells permit a perfect growth of the selected cell types. In parallel, protein biomarkers can be followed by light microscopy. So far, experiments have been started to discriminate between different cell densities and cell types. In addition, after stimulating human cardiac fibroblasts and human umbilical vein endothelial cells, concentrations of proteins involved in adhesion had been increased, and proteins were translocated within the cells. In ongoing experiments, different human cell lines and fibroblastoid mesenchymal stem cells isolated from fat tissue, umbilical cord, or bone marrow are tested in the chip. To optimize the adhesion conditions, the surfaces within the vials of the chip were specifically activated. Microscopy was adjusted to be able to measure cellular morphology in parallel. This concept allows to identify the behavior of mesenchymal stem cells, which cannot be described so far by standard biomarkers. In addition, simulation of the homing process of the cells within its stem cell niche in an in vitro assay is a promising setup for large-scale gain-of-function or loss-of-function screenings in functional genomics as well as for generating precursor cells relevant for the therapy of various diseases. [source]

Analysis of genomic dose-response information on arsenic to inform key events in a mode of action for carcinogenicity

Nucleofection: a new, highly efficient transfection method for primary human keratinocytes,

EXPERIMENTAL DERMATOLOGY, Issue 4 2005
Jörg H. W. Distler
Abstract:, Transfection is an essential tool for numerous in vitro applications including studies of gene expression, promoter analysis, and intracellular signaling pathways and also for therapeutic strategies such as tissue engineering and gene therapy. However, transfection of primary cells including keratinocytes with common methods such as calcium phosphate, DEAE-dextran, liposome-mediated transfer, electroporation or viral vectors is problematic because of low transfection efficiency and the induction of terminal differentiation. Here we analyzed the use of nucleofection, a new, electroporation-based transfection method that enables the DNA to enter directly the nucleus, for the transfection of keratinocytes. Several different conditions were tested and optimized, resulting in a final transfection efficiency of 56% in primary human epidermal keratinocytes. This efficiency is superior to all non-viral transfection methods reported so far. The number of non-viable keratinocytes after nucleofection was low, varying between 14 and 16%. In contrast to other transfection protocols, nucleofection did not induce terminal differentiation in the transfected keratinocytes. In addition, nucleofection is a fast method, because the results can be analyzed within 7 h. In summary, nucleofection is a fast, easy and highly effective alternative for the transfection of primary human keratinocytes, which offers new opportunities for various research applications. [source]

Cell type-specific transgene expression of the prion protein in Xenopus intermediate pituitary cells

FEBS JOURNAL, Issue 4 2006
Jos W. G. Van Rosmalen
The cellular form of prion protein (PrPC) is anchored to the plasma membrane of the cell and expressed in most tissues, but predominantly in the brain, including in the pituitary gland. Thus far, the biosynthesis of PrPC has been studied only in cultured (transfected) tumour cell lines and not in primary cells. Here, we investigated the intracellular fate of PrPCin vivo by using the neuroendocrine intermediate pituitary melanotrope cells of the South-African claw-toed frog Xenopus laevis as a model system. These cells are involved in background adaptation of the animal and produce high levels of its major secretory cargo proopiomelanocortin (POMC) when the animal is black-adapted. The technique of stable Xenopus transgenesis in combination with the POMC gene promoter was used as a tool to express Xenopus PrPC amino-terminally tagged with the green fluorescent protein (GFP,PrPC) specifically in the melanotrope cells. The GFP,PrPC fusion protein was expressed from stage-25 tadpoles onwards to juvenile frogs, the expression was induced on a black background and the fusion protein was subcellularly located mainly in the Golgi apparatus and at the plasma membrane. Pulse,chase metabolic cell labelling studies revealed that GFP,PrPC was initially synthesized as a 45-kDa protein that was subsequently stepwise glycosylated to 48-, 51-, and eventually 55-kDa forms. Furthermore, we revealed that the mature 55-kDa GFP,PrPC protein was sulfated, anchored to the plasma membrane and cleaved to a 33-kDa product. Despite the high levels of transgene expression, the subcellular structures as well as POMC synthesis and processing, and the secretion of POMC-derived products remained unaffected in the transgenic melanotrope cells. Hence, we studied PrPC in a neuroendocrine cell and in a well-defined physiological context. [source]

Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene

FEBS JOURNAL, Issue 7 2001
Muriel Erent
The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase C,. NDP kinase C, had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase C,, based on the crystal structure of NDP kinase B, indicated that NDP kinase C, had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase C, readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo. [source]

Calcium influx mechanisms underlying calcium oscillations in rat hepatocytes,

HEPATOLOGY, Issue 4 2008
Bertina F. Jones
The process of capacitative or store-operated Ca2+ entry has been extensively investigated, and recently two major molecular players in this process have been described. Stromal interacting molecule (STIM) 1 acts as a sensor for the level of Ca2+ stored in the endoplasmic reticulum, and Orai proteins constitute pore-forming subunits of the store-operated channels. Store-operated Ca2+ entry is readily demonstrated with protocols that provide extensive Ca2+ store depletion; however, the role of store-operated entry with modest and more physiological cell stimuli is less certain. Recent studies have addressed this question in cell lines; however, the role of store-operated entry during physiological activation of primary cells has not been extensively investigated, and there is little or no information on the roles of STIM and Orai proteins in primary cells. Also, the nature of the Ca2+ influx mechanism with hormone activation of hepatocytes is controversial. Hepatocytes respond to physiological levels of glycogenolytic hormones with well-characterized intracellular Ca2+ oscillations. In the current study, we have used both pharmacological tools and RNA interference (RNAi)-based techniques to investigate the role of store-operated channels in the maintenance of hormone-induced Ca2+ oscillations in rat hepatocytes. Pharmacological inhibitors of store-operated channels blocked thapsigargin-induced Ca2+ entry but only partially reduced the frequency of Ca2+ oscillations. Similarly, RNAi knockdown of STIM1 or Orai1 substantially reduced thapsigargin-induced calcium entry, and more modestly diminished the frequency of vasopressin-induced oscillations. Conclusion: Our findings establish that store-operated Ca2+ entry plays a role in the maintenance of agonist-induced oscillations in primary rat hepatocytes but indicate that other agonist-induced entry mechanisms must be involved to a significant extent. (HEPATOLOGY 2008.) [source]

A novel direct aerodynamically assisted threading methodology for generating biologically viable microthreads encapsulating living primary cells

JOURNAL OF APPLIED POLYMER SCIENCE, Issue 2 2008
Sumathy Arumuganathar
Abstract In a recent discovery, coaxial electrospinning was explored to encapsulate living organisms within a continuous bio-polymeric microthread from which active biological scaffolds were fabricated (Townsend-Nicholson and Jayasinghe, Biomacromolecules 2006, 7, 3364). The cells were demonstrated to have gone through all expected cellular activity without their viability being compromised. These biologically active threads and scaffolds have direct and tremendous applicability from regenerative to therapeutic medicine. Currently these post-processed cells as composite threads and scaffolds are being investigated in-depth at a cellular level to establish if the processing methodology has any affect on the cellular make-up. We now demonstrate a competing non-electric field driven approach for fabricating composite threads and scaffolds influenced only by a differential pressure. We refer to this novel composite thread to scaffold fabrication methodology as coaxial aerodynamically assisted bio-threading (CAABT). Our investigations firstly, demonstrate that this technique can process handle living organisms without biologically perturbing them in anyway. Secondly the process is elucidated as possessing the ability to form composite active threads from which biologically viable scaffolds are formed. Finally our study employs florescent activated cell sorting (FACScan), a method by which the cellular dynamics and viability are quantified on control and threaded cellular samples at two prescribed time points. In parallel with FACScan, optical comparison of cellular morphology at three time points within a period of three weeks is carried out to photographically observe any changes in the post-processed cellular phenotype. Our developmental investigations into this novel aerodynamically assisted threading methodology has unearthed a unique biomicrofabrication approach, which joins cell electrospinning in the cell threading to scaffold fabrication endeavor. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source]

Mechanically Strained Cells of the Osteoblast Lineage Organize Their Extracellular Matrix Through Unique Sites of ,V,3 -Integrin Expression

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2000
Magdalena Wozniak
Abstract Bone cells transduce mechanical signals into anabolic biochemical responses. However, the mechanisms of mechanotransduction are unknown. To address this issue, we performed studies in primary cells of the human osteoblast lineage grown on collagen/vitronectin-coated supports. We discovered that mechanical strain stimulated a redistribution of the ,v,3 -integrin to irregular plaque-like areas at the cell-extracellular matrix surface. Proteins involved in integrin-matrix interactions in focal adhesions, vinculin and talin, did not localize to the plaque-like areas of ,v,3 -expression, but signaling molecules such as focal adhesion kinase (FAK) did. Mechanical strain increased the number and size of the plaques defined by surface expression of ,v,3 -integrin. Osteopontin was secreted as a cross-linked macromolecular complex, likely through the action of tissue transglutaminase that also was found in the plaques of ,v,3 -integrin cell-matrix interaction. Mechanical strain increased mineralization of the extracellular matrix that developed in these plaques in ,v,3 -integrin-dependent manner. Because the plaque-like areas of cell-matrix interaction exhibit macromolecular assembly and mineralization, we conclude that they may represent subcellular domains of bone formation and that ,v,3 -integrin activation represents one mechanism by which mechanical strain stimulates bone formation. [source]

Effluxing ABC transporters in human corneal epithelium

JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2010
Kati-Sisko Vellonen
Abstract ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1,6 (MRP1,6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed over-expression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1087,1098, 2010 [source]

Quantification of the expression level of integrin receptor ,v,3 in cell lines and MR imaging with antibody-coated iron oxide particles

MAGNETIC RESONANCE IN MEDICINE, Issue 4 2006
Sabrina Benedetto
Abstract Targeted imaging requires site-specific accumulation of a contrast agent (CA), and the properties of that agent must be selected according to the abundance of the target to obtain a signal above the detection limit of the instrument. However, numerical estimates of receptors per cell are rarely found in the literature. Integrin receptors would be particularly promising targets because of their accessibility from the blood stream and expression on activated neovascular endothelial cells. We systematically estimated the number of integrin receptors of cell lines and primary cells by flow cytometry analysis. Since integrin receptors are heterodimeric molecules, and ,v forms complexes with various , subunits, the numbers of ,v and ,3 subunits are therefore dissimilar. The observed values are 3 · 103,1.4 · 104/cell for ,v, and 5.3 · 102,1.1 · 104/cell for ,3. Despite the low number of exposed receptors, we show that up to single-cell MR visualization can be achieved with the use of iron oxide beads complexed with antibodies as CAs. Magn Reson Med, 2006. © 2006 Wiley-Liss, Inc. [source]

Gene expression changes in human cells after exposure to mobile phone microwaves

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2006
Daniel Remondini
Abstract Possible biological effects of mobile phone microwaves were investigated in,vitro. In this study, which was part of the 5FP EU project REFLEX (Risk Evaluation of Potential Environmental Hazards From Low-Energy Electromagnetic Field Exposure Using Sensitive in,vitro Methods), six human cell types, immortalized cell lines and primary cells, were exposed to 900 and 1800,MHz. RNA was isolated from exposed and sham-exposed cells and labeled for transcriptome analysis on whole-genome cDNA arrays. The results were evaluated statistically using bioinformatics techniques and examined for biological relevance with the help of different databases. NB69 neuroblastoma cells, T,lymphocytes, and CHME5 microglial cells did not show significant changes in gene expression. In EA.hy926 endothelial cells, U937,lymphoblastoma cells, and HL-60 leukemia cells we found between 12 and 34,up- or down-regulated genes. Analysis of the affected gene families does not point towards a stress response. However, following microwave exposure, some but not all human cells might react with an increase in expression of genes encoding ribosomal proteins and therefore up-regulating the cellular metabolism. [source]

Engineered Streptomyces quorum-sensing components enable inducible siRNA-mediated translation control in mammalian cells and adjustable transcription control in mice

THE JOURNAL OF GENE MEDICINE, Issue 4 2005
Wilfried Weber
Abstract Background Recent advances in functional genomics, gene therapy, tissue engineering, drug discovery and biopharmaceuticals production have been fostered by precise small-molecule-mediated fine-tuning of desired transgenes. Methods Capitalizing on well-evolved quorum-sensing regulatory networks in Streptomyces coelicolor we have designed a mammalian regulation system inducible by the non-toxic butyrolactone SCB1. Fusion of the S. coelicolor SCB1 quorum-sensing receptor ScbR to the human Kox-1-derived transsilencing domain reconstituted a mammalian transsilencer (SCS) able to repress transcription from SCS-specific operator-containing promoters in a reverse SCB1-adjustable manner. Results This quorum-sensing-derived mammalian transgene control system (Q-ON) enabled precise SCB1-specific fine-tuning of (i) desired transgene transcription in a variety of mammalian/human cell lines and human primary cells, (ii) small interfering RNA-mediated posttranscriptional knockdown (siRNA) in mammalian cells, and (iii) dosing of a human glycoprotein in mice. Conclusions As exemplified by Q-ON technology, bacterial quorum-sensing regulons may represent a near-infinite source for the design of mammalian gene control systems compatible with molecular interventions relevant to future gene therapy and tissue engineering scenarios. Copyright © 2004 John Wiley & Sons, Ltd. [source]

Gene therapy for HIV/AIDS: the potential for a new therapeutic regimen

THE JOURNAL OF GENE MEDICINE, Issue 8 2003
Greg Fanning
Abstract Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). HIV/AIDS is a disease that, compared with the not so distant past, is now better held in check by current antiretroviral drugs. However, it remains a disease not solved. Highly active antiretroviral therapy (HAART) generally uses two non-nucleoside and one nucleoside reverse transcriptase (RT) inhibitor or two non-nucleoside RT and one protease inhibitor. HAART is far more effective than the mono- or duo-therapy of the past, which used compounds like the nucleoside reverse transcriptase inhibitor AZT or two nucleoside reverse transcriptase inhibitors. However, even with the relatively potent drug cocktails that comprise HAART, there are the issues of (i) HIV escape mutants, (ii) an apparent need to take the drugs in an ongoing manner, and (iii) the drugs' side effects that are often severe. This review speaks to the potential addition to these potent regimens of another regimen, namely the genetic modification of target hematopoietic cells. Such a new treatment paradigm is conceptually attractive as it may yield the constant intracellular expression of an anti-HIV gene that acts to inhibit HIV replication and pathogenicity. A body of preclinical work exists showing the inhibition of HIV replication and decreased HIV pathogenicity by anti-HIV genetic agents. This preclinical work used hematopoietic cell lines and primary cells as the target tissue. More recently, several clinical trials have sought to test this concept in vivo. Copyright © 2003 John Wiley & Sons, Ltd. [source]

Autocitrullination of human peptidyl arginine deiminase type 4 regulates protein citrullination during cell activation

ARTHRITIS & RHEUMATISM, Issue 6 2010
Felipe Andrade
Objective To address mechanisms that control the activity of human peptidyl arginine deiminase type 4 (PAD-4). Methods PAD-4 autocitrullination was determined by anti,modified citrulline immunoblotting, using purified recombinant and endogenous PAD-4 from activated human primary neutrophils and cell lines expressing PAD-4. The citrullination sites in PAD-4 were determined by mass spectrometry. Mechanisms of autocitrullination-induced inactivation and the functional consequences of autocitrullination in PAD-4 polymorphic variants were addressed using purified components and cell lines expressing PAD-4 wild-type, PAD-4 mutant, and PAD-4 polymorphic variants relevant to rheumatoid arthritis (RA). Results PAD-4 is autocitrullinated in vitro and during activation of primary cells and cell lines expressing PAD-4. Interestingly, this modification inactivated the function of the enzyme. The efficiency of inactivation differed among genetically defined PAD-4 variants relevant to RA. PAD-4 was citrullinated at 10 sites, which are clustered into 3 distinct regions, including a cluster of arginines around the active site cleft where Arg-372 and -374 were identified as the potential autocitrullination targets that inactivate the enzyme. Autocitrullination also modified the structure of PAD-4, abrogating its recognition by multiple rabbit antibodies, but augmenting its recognition by human anti,PAD-4 autoantibodies. Conclusion Our findings suggest that autocitrullination regulates the production of citrullinated proteins during cell activation, and that this is affected by structural polymorphisms in PAD-4. Autocitrullination also influences PAD-4 structure and immune response. [source]

Invariant natural killer T cells are natural regulators of murine spondylarthritis

ARTHRITIS & RHEUMATISM, Issue 4 2010
Peggy Jacques
Objective To investigate the role of invariant natural killer T (iNKT) cells in TNF,ARE/+ mice, an animal model of spondylarthritis (SpA) with both gut and joint inflammation. Methods The frequency and activation of iNKT cells were analyzed on mononuclear cells from the lymph nodes and livers of mice, using flow cytometry with ,-galactosylceramide/CD1d tetramers and quantitative polymerase chain reaction for the invariant V,14,J,18 rearrangement. Bone marrow,derived dendritic cells (DCs) were obtained by expansion of primary cells with granulocyte,macrophage colony-stimulating factor followed by coculture with iNKT cell hybridomas, and interleukin-2 release into the cocultures was then measured by enzyme-linked immunosorbent assay (ELISA). Cytokine levels were determined by ELISA or cytometric bead array analyses of freshly isolated DCs and iNKT cells in mixed cocultures. TNF,ARE/+ mice were backcrossed onto J,18,/, and CD1d,/, mice, and disease onset was evaluated by clinical scoring, positron emission tomography, and histology. CD1d levels were analyzed on mononuclear cells in paired blood and synovial fluid samples from patients with SpA compared with healthy control subjects. Results In the absence of iNKT cells, symptoms of gut and joint inflammation in TNF,ARE/+mice were aggravated. Invariant NKT cells were activated during the course of the disease. This was linked to an enrichment of inflammatory DCs, characterized by high levels of CD1d, particularly at draining sites of inflammation. A similar increase in CD1d levels was observed on DCs from patients with SpA. Inflammatory DCs from TNF,ARE/+ mice stimulated iNKT cells to produce immunomodulatory cytokines, in the absence of exogenous stimulation. Prolonged, continuous exposure, but not short-term exposure, to tumor necrosis factor (TNF) was found to be responsible for the enhanced DC,NKT cell crosstalk. Conclusion This mode of iNKT cell activation represents a natural counterregulatory mechanism for the dampening of TNF-driven inflammation. [source]

Mature antigen-experienced T helper cells synthesize and secrete the B cell chemoattractant CXCL13 in the inflammatory environment of the rheumatoid joint

ARTHRITIS & RHEUMATISM, Issue 11 2008
Antonio Manzo
Objective Synovial B cells play a critical role in rheumatoid arthritis (RA), being involved in autoantibody synthesis, T cell activation, and cytokine production. CXCL13 is a B cell chemoattractant that is instrumental in synovial B cell organization; the regulatory determinants of CXCL13 in inflammation are poorly characterized. This study was undertaken to investigate the functional involvement of synovial T cells in the ectopic expression of CXCL13 in RA. Methods CXCL13 production and regulation were addressed using immunohistochemistry, in situ hybridization, quantitative polymerase chain reaction, multicolor flow cytometry, and enzyme-linked immunosorbent assay, by in situ,ex vivo analysis and in vitro functional assays with rheumatoid synovial tissue and primary cells. Results CXCL13 messenger RNA and protein expression and spontaneous CXCL13 secretion were detected in RA synovial fluid T cells but were not detected (or were detected only occasionally) in peripheral blood T cells. Analysis of tissue expression confirmed cytoplasm localization of CXCL13 in T lymphocytes infiltrating B cell follicles and small perivascular aggregates. Multicolor characterizations in synovial fluid demonstrated CXCL13 expression in antigen-experienced T helper cells, frequently characterized by terminal differentiation and the lack of the follicular helper T cell markers CXCR5 and BCL6 protein. In vitro functional assays revealed the enhancing effect of T cell receptor,CD28 engagement on CXCL13 production and secretion in primary cells. Conclusion Our findings define a new functional property of synovial T cells, demonstrating their active involvement in the local production of B cell chemoattractants, and support a direct contribution of the adaptive immune system and antigen-dependent signals in the mechanisms of B cell localization in RA. [source]

Lidocaine/Monoethylglycinexylidide Test, Galactose Elimination Test, and Sorbitol Elimination Test for Metabolic Assessment of Liver Cell Bioreactors

ARTIFICIAL ORGANS, Issue 6 2010
Jörg C. Gerlach
Abstract Various metabolic tests were compared for the performance characterization of a liver cell bioreactor as a routine function assessment of cultures in a standby for patient application in clinical studies. Everyday quality assessment (QA) is essential to ensure a continuous level of cellular functional capacity in the development of hepatic progenitor cell expansion systems providing cells for regenerative medicine research; it is also of interest to meet safety requirements in bioartificial extracorporeal liver support systems under clinical evaluation. Quality criteria for the description of bioreactor cultures were developed using primary porcine liver cells as a model. Porcine liver cells isolated by collagenase perfusion with an average of 3 × 109 primary cells were used in 39 bioreactors for culture periods up to 33 days. Measurements of monoethylglycinexylidide synthesis and elimination of lidocaine, galactose elimination, and sorbitol elimination proved to be useful for routine QA of primary liver cell cultures. We demonstrate two methods for dispensing test substances, bolus administration and continuous, steady-state administration. Bolus test data were grouped in Standard, Therapy, Infection/Contamination, and Cell-free control groups. Statistical analyses show significant differences among all groups for every test substance. Post hoc comparisons indicated significant differences between Standard and Cell-free groups for all elimination parameters. For continuous tests, results were categorized according to number of culture days and time-dependent changes were analyzed. Continuous administration enables a better view of culture health and the time dependency of cellular function, whereas bolus administration is more flexible. Both procedures can be used to define cell function. Assessment of cellular function and bioreactor quality can contribute significantly to the quality of experimental or clinical studies in the field of hepatic bioreactor development. [source]

Enhanced differentiation of embryonic stem cells using co-cultivation with hepatocytes

BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2008
Rebecca N. Moore
Abstract We examined the effects of co-cultivated hepatocytes on the hepatospecific differentiation of murine embryonic stem (ES) cells. Utilizing an established mouse ES cell line expressing high or low levels of E-cadherin, that we have previously shown to be responsive to hepatotrophic growth factor stimulation (Dasgupta et al., 2005. Biotechnol Bioeng 92(3):257,266), we compared co-cultures of cadherin-expressing ES (CE-ES) cells with cultured rat hepatocytes, allowing for either paracrine interactions (indirect co-cultures) or both juxtacrine and paracrine interactions (direct co-cultures, random and patterned). Hepatospecific differentiation of ES cells was evaluated in terms of hepatic-like cuboidal morphology, heightened gene expression of late maturation marker, glucose-6-phosphatase in relation to early marker, alpha-fetoprotein (AFP), and the intracellular localization of albumin. Hepatocytes co-cultured with growth factor primed CE-ES cells markedly enhanced ES cell differentiation toward the hepatic lineage, an effect that was reversed through E-cadherin blockage and inhibited in control ES cells with reduced cadherin expression. Comparison of single ES cell cultures versus co-cultures show that direct contact co-cultures of hepatocytes and CE-ES cells maximally promoted ES cell commitment towards hepatodifferentiation, suggesting cooperative effects of cadherin-based juxtacrine and paracrine interactions. In contrast, E-cadherin deficient mouse ES (CD-ES) cells co-cultured with hepatocytes failed to show increased G6P expression, confirming the role of E-cadherin expression. To establish whether albumin expression in CE-ES cells was spatially regulated by co-cultured hepatocytes, we co-cultivated CE-ES cells around micropatterned, pre-differentiated rat hepatocytes. Albumin localization was enhanced "globally" within CE-ES cell colonies and was inhibited through E-cadherin antibody blockage in all but an interfacial band of ES cells. Thus, stem cell based cadherin presentation may be an effective tool to induce hepatotrophic differentiation by leveraging both distal/paracrine and contact/juxtacrine interactions with primary cells of the liver. Biotechnol. Bioeng. © 2008 Wiley Periodicals, Inc. [source]

Quantitative real-time analysis of HIV-1 gene expression dynamics in single living primary cells

BIOTECHNOLOGY JOURNAL, Issue 6 2006
Asier Sáez-Cirión
Abstract Studies on the regulation of viral transcription upon infection of the target cells have provided important information on the viral and host factors that influence pathogenesis. However, these studies have been limited so far to steady-state analysis of gene expression. Here we report an image based photon-counting method that allows real-time quantitative imaging of viral gene expression in infected single cells. Employing an HIV-1 vector bearing the firefly luciferase reporter gene, we exploited a single cell photon imaging methodology (a customized and highly sensitive imaging microscope) to measure viral gene expression following integration into a host genome in situ. Our approach reveals real-time dynamics of viral gene expression in living HIV natural target cells (primary human CD4 T cells and macrophages), and promises itself as a powerful tool for quantitative studies on a wide variety of virus-host cell interactions. [source]

Development and functional characterization of human bone marrow mesenchymal cells immortalized by enforced expression of telomerase

BRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2003
Keichiro Mihara
Summary. To create immortal mesenchymal cell lines, we transduced primary human bone marrow mesenchymal cells with telomerase reverse transcriptase (TERT). TERT+ mesenchymal cells continued to grow for >,2 years; parallel TERT, cultures underwent senescence after 15 weeks. TERT+ mesenchymal cells did not form foci in soft agar, had a normal karyotype and could differentiate into osteoblasts and chondrocytes. Their capacity to support leukaemic lymphoblasts and normal CD34+ haematopoietic cells was equal to or greater than that of primary cells; 42 TERT+ mesenchymal cell clones varied in their supporting capacity. Immortalized mesenchymal cells offer a promising tool for identifying molecules that regulate human haematopoiesis. [source]

Adhesion pattern and growth of primary human osteoblastic cells on five commercially available titanium surfaces

CLINICAL ORAL IMPLANTS RESEARCH, Issue 7 2010
Giovanni Passeri
Abstract Objective: The aim of this study is to analyze the morphology and proliferation of human osteoblastic cells in vitro on five commercially available titanium surfaces. Materials and methods: Human primary cells of the osteoblastic lineage were obtained from bone explants. The cells were plated on polished (T1), machined (T2), sand-blasted/acid-etched (T3), sand-blasted/acid-etched, modified with hydrogen peroxide rinse (T4), and plasma-sprayed titanium (T5) disks. Cell morphology was studied after 6, 24, 72 h, 7 and 14 days of culture by scanning electron microscopy. The formation and distribution of focal adhesions was investigated by immunocytochemical staining at 3, 6 and 24 h. Cell growth was measured by an MTT assay after 3, 7 and 9 days of culture. Moreover, the production of osteocalcin and osteoprotegerin (OPG) was evaluated in the supernatants by ELISA. Results: Morphological analysis revealed that substrate topography profoundly affected cells' shape and their anchoring structures. Large lamellipodia were formed on polished and machined surfaces, while thin filopodia were more frequently observed on T3 and T4 samples. Moreover, cells formed stronger focal adhesions on T3 and T4 surfaces, and cell proliferation was higher on rough surfaces. Osteocalcin production was higher on the T4 surface, whereas OPG steadily increased on every surface. Conclusions: Taken together, these data show that all the surfaces allowed cell attachment, adhesion and proliferation, but T4 and T5 surfaces appeared to be a better substrate for the adhesion, proliferation and differentiation of cells of the osteoblastic lineage. To cite this article: Passeri G, Cacchioli A, Ravanetti F, Galli C, Elezi E, Macaluso GM. Adhesion pattern and growth of primary human osteoblastic cells on five commercially available titanium surfaces. Clin. Oral Impl. Res. 21, 2010; 756,765. doi: 10.1111/j.1600-0501.2009.01906.x [source]