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Primary Human Cells (primary + human_cell)
Selected AbstractsInteraction between the CCR5 chemokine receptors and microbial HSP70EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2006Trevor Whittall Abstract Evidence is presented that the microbial 70-kD heat shock protein (HSP70) binds to CCR5 chemokine receptors in CCR5-transfected cell lines and in primary human cells. Significant CCR5-mediated calcium mobilization was stimulated by HSP70 and inhibited with TAK,779, which is a specific CCR5 antagonist. HSP70-mediated activation of the p38 MAPK phosphorylation signaling pathway was also demonstrated in CCR5-transfected HEK 293 cells. Direct binding of three extracellular peptides of CCR5 to HSP70 was demonstrated by surface plasmon resonance. Functional evidence of an interaction between HSP70, CCR5 and CD40 was shown by enhanced production of CCL5 by HEK 293 cells transfected with both CD40 and CCR5. Primary monocyte-derived immature DC stimulated with HSP70 produced IL-12 p40, which showed dose-dependent inhibition of >90% on treatment with both TAK 779 and anti-CD40 mAb. Stimulation of IL-12 p40 or TNF-, by HSP70 was related to the differential cell surface expression of CCR5 in primary human immature and mature DC, and those with the homozygous ,,32 CCR5 mutation. These findings may be of significance in the interaction between HSP70 and immune responses of CCR5+ T cells in HIV-1 infection, as well as in inflammatory bowel disease. See accompanying commentary: http://dx.doi.org/10.1002/eji.200636551 [source] Combinatorial Modification of Degradable Polymers Enables Transfection of Human Cells Comparable to Adenovirus,ADVANCED MATERIALS, Issue 19 2007J. Green End-modified poly(,-amino ester)s, easy-to-synthesize degradable polymers, are able to deliver DNA to primary human cells at levels comparable to adenovirus and two orders of magnitude better than the commonly used non-viral vector, polyethylenimine. Small structural changes are found to affect multiple steps of gene delivery including the DNA binding affinity, nanoparticle size, intracellular DNA uptake, and final protein expression. In vivo, these polymer modifications enhance DNA delivery to ovarian tumors. [source] Site-specific proteolysis of cyclooxygenase-2: A putative step in inflammatory prostaglandin E2 biosynthesisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2007Arturo Mancini Abstract Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in inflammatory prostanoid biosynthesis. Transcriptional, post-transcriptional, and post-translational covalent modifications have been defined as important levels of regulation for COX-2 gene expression. Here, we describe a novel regulatory mechanism in primary human cells involving regulated, sequence-specific proteolysis of COX-2 that correlates with its catalytic activity and ultimately, the biosynthesis of prostaglandin E2 (PGE2). Proinflammatory cytokines induced COX-2 expression and its proteolysis into stable immunoreactive fragments of 66, 42,44, 34,36, and 28 kDa. Increased COX-2 activity (PGE2 release) was observed coincident with the timing and degree of COX-2 proteolysis with correlation analysis confirming a linear relationship (R2,=,0.941). Inhibition of induced COX-2 activity with non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 selective inhibitors also abrogated cleavage. To determine if NSAID inhibition of proteolysis was related to drug-binding-induced conformational changes in COX-2, we assayed COX-inactive NSAID derivatives that fail to bind COX-2. Interestingly, these compounds suppressed COX-2 activity and cleavage in a correlated manner, thus suggesting that the observed NSAID-induced inhibition of COX-2 cleavage occurred through COX-independent mechanisms, presumably through the inhibition of proteases involved in COX-2 processing. Corroborating this observation, COX-2 cleavage and activity were mutually suppressed by calpain/cathepsin protease inhibitors. Our data suggest that the nascent intracellular form of COX-2 may undergo limited proteolysis to attain full catalytic capacity. J. Cell. Biochem. 101: 425,441, 2007. © 2006 Wiley-Liss, Inc. [source] Establishment and characterization of immortalized human gingival keratinocyte cell linesJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2008S. Gröger Background and Objective:, Primary human keratinocytes are used to analyze the properties of the oral epithelium and the early stages of oral bacterial infections. In vitro, these cells are characterized by their short life span and restricted availability. Approaches for culturing these cells will end after approximately 6,10 passages as a result of entry into apoptosis. For this reason, it is important to generate cell lines suitable for obtaining an unlimited source of cells. Therefore, the aim of the present study was to generate gingival keratinocyte cell lines and to compare their in vitro behaviour with those of primary human gingival keratinocytes. Material and Methods:, Primary human gingival keratinocytes were immortalized with a combination of the human papilloma virus onkoproteins E6 and E7. The pattern of the cytokeratins, involucrin and filaggrin was investigated by intracellular staining using flow cytometry. This method allows quantitative analysis of the expression of a variety of intracellular or extracellular markers. Results:, The immortalized cell lines showed many morphological similarities, expressing a cytokeratin pattern that is comparable with that of primary gingival keratinocytes. Furthermore, they developed transepithelial electrical resistance, which is a marker for the generation of tight junctions. These results indicate that the cells might be able to act as an epithelial barrier, reflecting the reaction of primary human cells. Conclusion:, The establishment of a continuous line of human gingival epithelial cells with functional characteristics of the epithelial barrier provides a valuable in vitro model for using to study the early steps of gingival/periodontal infections. [source] SIGIRR/TIR-8 is an inhibitor of toll-like receptor signaling in primary human cells and regulates inflammation in models of rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 8 2010Stefan K. Drexler Objective Single-immunoglobulin interleukin-1 receptor,related (SIGIRR), which is also known as Toll/interleukin-1 receptor 8 (TIR-8), is a member of the TIR domain,containing family of receptors and was first characterized as an inhibitor of interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) signaling. In the Dextran sulfate sodium,induced colitis model, SIGIRR,/, mice were shown to have increased inflammation and to be more susceptible to endotoxin challenge. Increasing evidence implicates TLR and IL-1R signaling in the pathology of rheumatoid arthritis (RA). Therefore, the purpose of this study was to investigate the involvement of SIGIRR in regulating inflammation in disease-relevant models. Methods Primary human monocyte-derived macrophages and dendritic cells (DCs) were used to overexpress SIGIRR as well as to knock down endogenously expressed SIGIRR using small interfering RNAs. SIGIRR was also overexpressed in synovial cells derived from RA patients. To investigate the role of SIGIRR in vivo, zymosan-induced arthritis (ZIA) and collagen antibody,induced arthritis (CAIA) were induced in SIGIRR-knockout mice. Results SIGIRR overexpression inhibited TLR-induced cytokine production in macrophages and DCs, while SIGIRR knockdown resulted in increased cytokine production following TLR stimulation. Moreover, SIGIRR overexpression inhibited the spontaneous release of cytokines by human RA synovial cells. The role of SIGIRR as an inhibitor of inflammation was confirmed in vivo, since SIGIRR,/, mice developed a more severe disease in both the ZIA and CAIA models. Conclusion Our study is the first to show the expression pattern and function of SIGIRR in primary human cells. Furthermore, this investigation defines the role of SIGIRR in disease-relevant cell types and demonstrates that SIGIRR is a potential therapeutic target for RA. [source] | ||||||||||