scholarly journals Functional characterization of adherent synovial fluid cells in rheumatoid arthritis: Destructive potential in vitro and in vivo

2003 ◽  
Vol 48 (7) ◽  
pp. 1873-1880 ◽  
Author(s):  
Michel Neidhart ◽  
Christian A. Seemayer ◽  
Klaus M. Hummel ◽  
Beat A. Michel ◽  
Renate E. Gay ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Functional Characterization ◽  
Synovial Fluid Cells ◽  
Destructive Potential

scholarly journals AB0100 PHENOTYPIC AND FUNCTIONAL CHARACTERIZATION OF SYNOVIAL FLUID-DERIVED FIBROBLAST-LIKE SYNOVIOCYTES IN RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1349.1-1349
Author(s):  
D. Køster ◽  
J. H. Egedal ◽  
M. Hvid ◽  
M. R. Jakobsen ◽  
U. Müller-Ladner ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Synovial Tissue ◽  
Mononuclear Cells ◽  
Functional Characterization ◽  
Human Cartilage ◽  
Fibroblast Like Synoviocytes

Background:Fibroblast-like synoviocytes (FLS) are central cellular components in persistent inflammatory joint diseases such as rheumatoid arthritis (RA). Pathological subsets of FLS have been identified from synovial tissue. However, the synovial tissue obtained from arthroplasty procedures is acquired at late disease stages and the cellular yield obtained from synovial tissue biopsies is fairly low. Collectively, challenging the robustness of human RAin vivoandin vitromodels. FLS obtained from the synovial fluid (SF-FLS) are proposed as an alternative source of FLS, but a detailed phenotypical and functional characterization of FLS subsets from the synovial fluid has not been performed.Objectives:The aim of this study was to determine the phenotypical and functional characteristics of synovial fluid-derived fibroblast-like synoviocytes in rheumatoid arthritis.Methods:In the present study, paired peripheral blood mononuclear cells (PBMC) and SF-FLS from patients with RA were obtained (n=7). FLS were isolated from the synovial fluid by a strict trypsinization protocol1and their cellular characteristics and functionality were evaluated at passage 4. Monocultures (SF-FLS) and autologous co-cultures (SF-FLS and PBMC) were established from five patients with RA and subsequently evaluated by flow cytometry, Western blotting and multiplex immunoassays. Human cartilage-sponges (n=3) with SF-FLS and without SF-FLS (n=3) were co-implanted subcutaneously in SCID mice (n=15), mice with only cell-free human cartilage-sponges were used as controls (n=12). After 45 days, the implants were evaluated using stained sections to determine the SF-FLS invasion score based on perichondrocytic cartilage degradation. Data are expressed as median (25-75 percentile). P-values <0.05 were considered statistically significant.Results:The homogeneous subpopulations of FLS, isolated from the synovial fluid, were negative for CD34 and CD45 [98.9%, (97.5-99.7]) and positive for Thy-1 and PDPN [94.6%, (79.9-97.4]). Without stimulation, RA SF-FLS showed high and comparable levels of NFκB related pathway proteins and secreted multiple pro-inflammatory cytokines and chemokines dominated by IL-6 [2648 pg/mL, (1327-6116)] and MCP-1 [2458 pg/mL, (692-8719)]. SF-FLS increased their ICAM-1 and HLA-DR expression after encountering autologous PBMCs (p<0.01), (p<0.05). Further, SF-FLS and PBMC interacted synergistically in a co-culture model of RA and significantly increasing the secretion of several cytokines (IL-1β, IL-2, IL-6, (p<0.01)) and a chemokine (MCP-1, (p<0.01)). The invasion score of the human SF-FLSin vivowas at primary site, [1.6, (1.3-1.7)] and contralateral implantation site [1.5, (1.1-2.2)]. The invasion score of the human SF-FLS-containing implants both at primary and contralateral site were significantly higher compared with cartilage-sponges evaluated from SF-FLS-free control mice (p<0001).Conclusion:This phenotypical and functional characterization of SF-FLS, acquired and activated at the site of pathology, lays a foundation for establishingin vivoandin vitroFLS models. These FLS models will be beneficial in our understanding of the role of this cellular subset in arthritis and for characterization of drugs specifically targeting this pathological RA FLS subset.References:[1]Nielsen M. A. et al. Responses to Cytokine Inhibitors Associated with Cellular Composition in Models of Immune-Mediated Inflammatory Arthritis. ACR Open Rheumatology, 2(1):3-10.http://doi.org/10.1002/acr2.11094Disclosure of Interests:Ditte Køster: None declared, Johanne Hovgaard Egedal: None declared, Malene Hvid: None declared, Martin Roelsgaard Jakobsen: None declared, Ulf Müller-Ladner Speakers bureau: Biogen, Bent Deleuran: None declared, Tue Wenzel Kragstrup Shareholder of: iBio Tech ApS, Consultant of: Bristol-Myers Squibb, Speakers bureau: TWK has engaged in educational activities talking about immunology in rheumatic diseases receiving speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, and UCB., Elena Neumann: None declared, Morten Aagaard Nielsen: None declared

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals Omics profiling identifies MAPK/ERK pathway as a gatekeeper of nephron progenitor metabolism

2021 ◽  
Author(s):  
Hyuk Nam Kwon ◽  
Kristen Kurtzeborn ◽  
Xing Jin ◽  
Bruno Reversade ◽  
Sunghyouk Park ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Mitogen Activated Protein Kinase ◽  
Metabolic Effects ◽  
Erk Pathway ◽  
Double Knockout ◽  
Nephron Progenitor ◽  
Progenitor Maintenance

Nephron endowment is defined by fetal kidney growth and it critically dictates renal health in adults. Despite the advances in understanding the molecular regulation of nephron progenitor maintenance, propagation, and differentiation, the causes for low congenital nephron count and contribution of basic metabolism to nephron progenitor regulation remain poorly studied. Here we have analyzed the metabolic effects that depend on and are triggered by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, which is an essential intracellular cascade required for nephron progenitor maintenance. Our combined approach utilizing LC/MS-based metabolomics and transcriptional profiling of MAPK/ERK-deficient cells identified 18 out of total 46 metabolites (38 untargeted and 8 targeted) that were down-regulated. These represent glycolysis, gluconeogenesis, pentose phosphate, glycine, and proline pathways among others. We focused our functional characterization of identified metabolites on pyruvate and proline. Use of in vitro kidney cultures revealed dosage-specific functions for pyruvate in not only controlling ureteric bud branching but also determining progenitor and differentiated (tip-trunk) cell identities. Our in vivo characterization of Pycr1/2 double knockout kidneys revealed functional requirement for proline metabolism in nephron progenitor maintenance. In summary, our results demonstrate that MAPK/ERK cascade regulates energy and amino acid metabolism in developing kidney where these metabolic pathways specifically regulate progenitor preservation.

scholarly journals Molecular and functional characterization of somatostatin-type signalling in a deuterostome invertebrate

Open Biology ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 200172
Author(s):  
Ya Zhang ◽  
Luis Alfonso Yañez Guerra ◽  
Michaela Egertová ◽  
Cleidiane G. Zampronio ◽  
Alexandra M. Jones ◽  
...  
Keyword(s):  
Functional Characterization ◽  
The Body ◽  
Pharmacological Characterization ◽  
Cardiac Stomach ◽  
Physiological Processes ◽  
Pharmacological Tests ◽  
The Central Nervous System

Somatostatin (SS) and allatostatin-C (ASTC) are structurally and evolutionarily related neuropeptides that act as inhibitory regulators of physiological processes in mammals and insects, respectively. Here, we report the first molecular and functional characterization of SS/ASTC-type signalling in a deuterostome invertebrate—the starfish Asterias rubens (phylum Echinodermata). Two SS/ASTC-type precursors were identified in A. rubens (ArSSP1 and ArSSP2) and the structures of neuropeptides derived from these proteins (ArSS1 and ArSS2) were analysed using mass spectrometry. Pharmacological characterization of three cloned A. rubens SS/ASTC-type receptors (ArSSR1–3) revealed that ArSS2, but not ArSS1, acts as a ligand for all three receptors. Analysis of ArSS2 expression in A. rubens using mRNA in situ hybridization and immunohistochemistry revealed stained cells/fibres in the central nervous system, the digestive system (e.g. cardiac stomach) and the body wall and its appendages (e.g. tube feet). Furthermore, in vitro pharmacological tests revealed that ArSS2 causes dose-dependent relaxation of tube foot and cardiac stomach preparations, while injection of ArSS2 in vivo causes partial eversion of the cardiac stomach. Our findings provide new insights into the molecular evolution of SS/ASTC-type signalling in the animal kingdom and reveal an ancient role of SS-type neuropeptides as inhibitory regulators of muscle contractility.

scholarly journals Functional and Genomic Characterization of Ligilactobacillus salivarius TUCO-L2 Isolated From Lama glama Milk: A Promising Immunobiotic Strain to Combat Infections

2020 ◽  
Vol 11 ◽  
Author(s):  
Sandra Quilodrán-Vega ◽  
Leonardo Albarracin ◽  
Flavia Mansilla ◽  
Lorena Arce ◽  
Binghui Zhou ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Functional Characterization ◽  
Comparative Genomic ◽  
Cytokines And Chemokines ◽  
Isolation And Characterization ◽  
Intestinal Pathogens ◽  
Lama Glama

Potential probiotic or immunobiotic effects of lactic acid bacteria (LAB) isolated from the milk of the South American camelid llama (Lama glama) have not been reported in published studies. The aim of the present work was to isolate beneficial LAB from llama milk that can be used as potential probiotics active against bacterial pathogens. LAB strains were isolated from llama milk samples. In vitro functional characterization of the strains was performed by evaluating the resistance against gastrointestinal conditions and inhibition of the pathogen growth. Additionally, the adhesive and immunomodulatory properties of the strains were assessed. The functional studies were complemented with a comparative genomic evaluation and in vivo studies in mice. Ligilactobacillus salivarius TUCO-L2 showed enhanced probiotic/immunobiotic potential compared to that of other tested strains. The TUCO-L2 strain was resistant to pH and high bile salt concentrations and demonstrated antimicrobial activity against Gram-negative intestinal pathogens and adhesion to mucins and epithelial cells. L. salivarius TUCO-L2 modulated the innate immune response triggered by Toll-like receptor (TLR)-4 activation in intestinal epithelial cells. This effect involved differential regulation of the expression of inflammatory cytokines and chemokines mediated by the modulation of the negative regulators of the TLR signaling pathway. Moreover, the TUCO-L2 strain enhanced the resistance of mice to Salmonella infection. This is the first report on the isolation and characterization of a potential probiotic/immunobiotic strain from llama milk. The in vitro, in vivo, and in silico investigation performed in this study reveals several research directions that are needed to characterize the TUCO-L2 strain in detail to position this strain as a probiotic or immunobiotic that can be used against infections in humans or animals, including llama.

scholarly journals Functional characterization of human Kindlin-2 core promoter identifies a key role of SP1 in Kindlin-2 transcriptional regulation

2011 ◽  
Vol 16 (4) ◽  
Author(s):  
Ammad Khan ◽  
Takashi Shimokawa ◽  
Staffan Strömblad ◽  
Hongquan Zhang
Keyword(s):  
Transcriptional Regulation ◽  
Core Promoter ◽  
Functional Characterization ◽  
Ph Domain ◽  
Interacting Protein ◽  
Normal Tissues ◽  
Functional Aspects

AbstractKindlin-2 is a recently identified FERM and PH domain containing integrin interacting protein. Kindlin-2 is ubiquitously expressed in normal tissues. So far, much effort has been spent exploring the functional aspects of Kindlin-2. However, the transcriptional regulation of Kindlin-2 has not yet been investigated. In this study we identified and functionally characterized the promoter of the human Kindlin-2 gene. We show that the core promoter of Kindlin-2 is a 39 base pair long GC rich fragment located −122/-83 upstream of the Kindlin-2 transcription start site. Functional characterization of this core promoter region by both in silico as well as in vitro/in vivo analysis shows that the transcription factor SP1 plays an important role in regulation of Kindlin-2 expression.

scholarly journals Formulation, High Throughput In Vitro Screening and In Vivo Functional Characterization of Nanoemulsion-Based Intranasal Vaccine Adjuvants

PLoS ONE ◽  
2015 ◽  
Vol 10 (5) ◽  
pp. e0126120 ◽  
Author(s):  
Pamela T. Wong ◽  
Pascale R. Leroueil ◽  
Douglas M. Smith ◽  
Susan Ciotti ◽  
Anna U. Bielinska ◽  
...  
Keyword(s):  
High Throughput ◽  
Functional Characterization ◽  
In Vitro Screening ◽  
Vaccine Adjuvants ◽  
Intranasal Vaccine

scholarly journals Functional characterization of a stable, noncytolytic stage of macrophage activation in tumors.

1977 ◽  
Vol 146 (6) ◽  
pp. 1511-1520 ◽  
Author(s):  
S W Russell ◽  
W F Doe ◽  
A T McIntosh
Keyword(s):  
Macrophage Activation ◽  
Functional Characterization ◽  
Cytolytic Activity ◽  
Target Cells ◽  
Tumor Target ◽  
Total Absence

The state in which macrophages (Mphi) from regressing Moloney sarcomas could kill tumor target cells was a highly labile one which decayed rapidly in vitro. Thereafter, regressor Mphi were noncytolytic. Mphi from several different progressing sarcomas failed to kill, even when challenged with target cells immediately after explantation. Similarly, thioglycollate-induced peritoneal Mphi (TG-Mphi) did not kill. Noncytolygic Mphi derived either from progressing sarcomas or from long-term (up to 96 h) cultures of regressor Mphi were exquisitely sensitive to stimulation by bacterial lipopolysaccharide (LPS); picogram/milliliter amounts induced killing. Similar concentrations of LPS had no demonstrable effect on TG-Mphi. Thus, tumor Mphi generally appeared to have been primed in vivo, with those in regressing sarcomas having additionally acquired cytolytic activity. Inability of progressor Mphi to kill apparently stemmed from lack of, or failure to respond to, the signal needed in vivo to trigger cytolytic activity, rather than the total absence of activation.

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