Single-cell Suspensions (single-cell + suspension)

Distribution by Scientific Domains


Selected Abstracts


GATA-3 protects against severe joint inflammation and bone erosion and reduces differentiation of Th17 cells during experimental arthritis

ARTHRITIS & RHEUMATISM, Issue 3 2009
Jan Piet van Hamburg
Objective Rheumatoid arthritis is associated with the infiltration of T helper cells into the joints. It is unclear whether interferon-, (IFN,),producing Th1 cells or the novel T helper subset, interleukin-17 (IL-17),producing Th17 cells, are the pathogenic mediators of joint inflammation in chronic nonautoimmune arthritis. Therefore, this study was aimed at examining whether the Th2-specific transcription factor GATA-3 can regulate arthritis, in an experimental murine model, by modulating Th1 and/or Th17 cell polarization. Methods Arthritis was induced with methylated bovine serum albumin (mBSA) in both wild-type and CD2 T cell,specific GATA-3 (CD2,GATA-3),transgenic mice. At days 1 and 7 after the induction of arthritis, knee joints were scored macroscopically for arthritis severity and for histologic changes. Single-cell suspensions were generated from the spleens, lymph nodes, and inflamed knee joints. Cytokine expression by CD4+ T cells was determined using flow cytometry, and IL-17 expression in the inflamed knee joints was determined by enzyme-linked immunosorbent assay. Analyses of gene expression were performed for Th17-associated factors. Results Wild-type mice developed severe joint inflammation, including massive inflammatory cell infiltration and bone erosion that increased significantly over time, reaching maximal arthritis scores at day 7. In contrast, only mild joint inflammation was observed in CD2,GATA-3,transgenic mice. This mild effect was further accompanied by systemic and local reductions in the numbers of IL-17+IFN,, and IL-17+IFN,+, but not IL-17,IFN,+, CD4+ T cells, and by induction of Th2 cytokine expression. Moreover, GATA-3 overexpression resulted in reduced gene expression of the Th17-associated transcription factor retinoic acid,related orphan receptor ,t. Conclusion These results indicate that enforced GATA-3 expression protects against severe joint inflammation and bone erosion in mice, accompanied by reduced differentiation of Th17 cells, but not Th1 cells, during mBSA-induced arthritis. [source]


Development of an ex vivo cellular model of rheumatoid arthritis: Critical role of cd14-positive monocyte/macrophages in the development of pannus tissue

ARTHRITIS & RHEUMATISM, Issue 9 2007
Toshiko Nozaki
Objective To establish an ex vivo cellular model of pannus, the aberrant overgrowth of human synovial tissue (ST). Methods Inflammatory cells that infiltrated pannus tissue from patients with rheumatoid arthritis (RA) were collected without enzyme digestion, and designated as ST-derived inflammatory cells. Single-cell suspensions of ST-derived inflammatory cells were cultured in medium alone. Levels of cytokines produced in culture supernatants were measured using enzyme-linked immunosorbent assay kits. ST-derived inflammatory cells were transferred into the joints of immunodeficient mice to explore whether these cells could develop pannus. CD14 and CD2 cells were depleted by negative selection. Results Culture of ST-derived inflammatory cells from 92 of 111 patients with RA resulted in spontaneous reconstruction of inflammatory tissue in vitro within 4 weeks. Ex vivo tissue contained fibroblasts, macrophages, T cells, and tartrate-resistant acid phosphatase,positive multinucleated cells. On calcium phosphate,coated slides, ST-derived inflammatory cell cultures showed numerous resorption pits. ST-derived inflammatory cell cultures continuously produced matrix metalloproteinase 9 and proinflammatory cytokines associated with osteoclastogenesis, such as tumor necrosis factor ,, interleukin-8, and macrophage colony-stimulating factor. More importantly, transferring ST-derived inflammatory cells into the joints of immunodeficient mice resulted in the development of pannus tissue and erosive joint lesions. Both in vitro development and in vivo development of pannus tissue by ST-derived inflammatory cells were inhibited by depleting CD14-positive, but not CD2-positive, cells from ST-derived inflammatory cells. Conclusion These findings suggest that overgrowth of inflammatory cells from human rheumatoid synovium simulates the development of pannus. This may prove informative in the screening of potential antirheumatic drugs. [source]


4414: Flow cytometry for the characterization of retinal neural populations and the quantification of retinal apoptosis

ACTA OPHTHALMOLOGICA, Issue 2010
PA TSOKA
Purpose The primary purpose of this study was to evaluate the potential to quantify the different retinal neuronal populations, as also the retinal detachment-induced apoptosis in Sprague,Dawley rats, in an accurate quantitative way by using flow cytometry. Methods Retinal detachment was performed on the right eye of deeply anesthetized animals. The detachment was induced by a sub-retinal injection of sodium hyaluronate. Rats were sacrifised and the eyes were enuclated to achieve retinal dissection. Tissue dissociation was accomplished with trypsin. The cells were mechanically dissociated into a single-cell suspension. At least 100.000 cells were analyzed with a FACScalibur and FlowJo software. The primary antibodies were anti-rhodopsin against rod photoreceptors, anti-PKC against rod bipolars, anti-calbindin against horizontals, anti-ChAT against cholinergic amacrine cells and anti-MAP1 against ganglion cells. Annexin-V-FITC/Propidium Iodide was used to identify apoptosis. Results Quantification of retinal neuronal cells was possible using flow cytometry. Photoreceptors had the 53.99%, the ganglion the 7%, the bipolars the 2%, the horizontal the 4% and the cholinergic amacrine cells the 1,5% in the hole mixed retinal population. Quantification of the apoptotic rate was also possible. The early apoptotic cells was 22.4% while in the control eye was 6.28% after retinal detachment. The experiments were repeated ten times and these measurements are the mean value. Conclusion Flow cytometry can be used to quantify the apoptotic neuronal cells as well as the healthy retinal neurons. It is quick and precise and it will be very useful in future in studies in neuroprotection and quantification of apoptosis during time. [source]


Timing and sequence of differentiation of embryonic rat hepatocytes along the biliary epithelial lineage

HEPATOLOGY, Issue 3 2003
Robbert G. E. Notenboom
To study the differentiation of hepatocytes along the biliary epithelial lineage in vivo, embryonic day 14 (E14) rat hepatocytes were isolated by differential centrifugation and transplanted as single-cell suspensions into the spleen of adult syngeneic rats. Hepatocytes and cholangiocytes were identified and their maturation characterized by the level of expression of ,-fetoprotein (AFP), glutamate dehydrogenase (GDH), and carbamoyl phosphate synthetase I (CPS); annexin IV, annexin V, cytokeratin 19 (CK-19), and cystic fibrosis transmembrane conductance regulator (CFTR); and electron microscopy. By correlating morphologic changes with the timing in the expression of these markers, we show that the organization of the transplanted E14 hepatocytes into lobular structures is accompanied by the formation and maturation of bile ducts around these developing lobules. Morphologic differentiation of the emerging bile ducts was accompanied by a gradual loss of hepatocyte markers and a gradual acquisition of cholangiocyte markers, with markers identifying a large-cholangiocyte phenotype appearing latest. Once fully differentiated, the intrasplenic liver lobules developed cholestatic features. The accompanying proliferation of bile ducts was due to cholangiocyte proliferation, but ductular transformation of hepatocytes was also observed. In conclusion, (1) bile duct formation at the interface between hepatocytes and connective tissue is an inherent component of liver development and (2) the susceptibility of developing hepatocytes to bile duct-inducing signals is highest in the fetal liver but that (3) this capacity is not irreversibly lost in otherwise mature hepatocytes. [source]


Phenotyping of epidermal dendritic cells allows the differentiation between extrinsic and intrinsic forms of atopic dermatitis

BRITISH JOURNAL OF DERMATOLOGY, Issue 6 2000
T. Oppel
Atopic dermatitis (AD) is a clinically characteristic, chronic inflammatory skin disease of unknown origin. IgE-mediated uptake and antigen focusing of environmental allergens by dendritic cells (DCs) is assumed to be a central immunopathogenetic event. A so-called intrinsic type of AD (IAD) has been delineated from the more common extrinsic AD (EAD) by normal serum IgE levels, negative RAST tests and negative immediate-type skin reactions towards environmental allergens. The recently characterized human autoantigen Hom S 1 has been proposed to play a part in the pathogenesis of IAD. Objectives,To compare clinical and laboratory data between patients with IAD and EAD, and to investigate potential differences in the inflammatory micromilieu of the epidermal compartment in IAD and EAD lesions. Methods,Epidermal DC phenotyping, a recently validated technique based on the three-colour flow cytometric analysis of Langerhans cells and the so-called inflammatory dendritic epidermal cells from epidermal single-cell suspensions, was performed on samples from 69 patients with AD (seven with IAD and 62 with EAD) and 94 controls. Results,Patients with EAD tended to have an earlier onset of disease but similar disease duration and family history of atopic diseases. Quantitative analysis of CD36 expression on DCs as a marker of inflammation, as well as the percentage of inflammatory dendritic epidermal cells in the CD1a+ epidermal DC pool, indicated a comparable disease activity in IAD and EAD. EAD was characterized by a significantly higher Fc,RI expression on the CD1a+ epidermal DCs than IAD. Using the Fc,RI/Fc,RII expression ratio as a disease marker for AD, values for IAD fell below the diagnostic cut-off level of 1·5 for this ratio. Conclusions,While IAD is clinically similar to EAD, the inflammatory microenvironment in this condition seems different from classical EAD and can be distinguished by phenotyping of epidermal DCs. [source]