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Cell Pool (cell + pool)
Kinds of Cell Pool Selected AbstractsFast set-up of doxycycline-inducible protein expression in human cell lines with a single plasmid based on Epstein,Barr virus replication and the simple tetracycline repressorFEBS JOURNAL, Issue 3 2007Markus Bach We have developed a novel plasmid vector, pEBTetD, for full establishment of doxycycline-inducible protein expression by just a single transfection. pEBTetD contains an Epstein,Barr virus origin of replication for stable and efficient episomal propagation in human cell lines, a cassette for continuous expression of the simple tetracycline repressor, and a cytomegalovirus-type 2 tetracycline operator (tetO2)-tetO2 promoter. As there is no integration of vector into the genome, clonal isolation of transfected cells is not necessary. Cells are thus ready for use 1 week after transfection; this contrasts with 3,12 weeks for other systems. Adequate regulation of protein expression was accomplished by abrogation of mRNA polyadenylation. In northern analysis of seven cDNAs coding for transport proteins, pools of transfected human embryonic kidney 293 cells showed on/off mRNA ratios in the order of 100 : 1. Cell pools were also analyzed for regulation of protein function. With two transport proteins of the plasma membrane, the on/off activity ratios were 24 : 1 and 34 : 1, respectively. With enhanced green fluorescent protein, a 23 : 1 ratio was observed based on fluorescence intensity data from flow cytometry. The unique advantage of our system rests on the unmodified tetracycline repressor, which is less likely, by relocation upon binding of doxycycline, to cause cellular disturbances than chimera of tetracycline repressor and eukaryotic transactivation domains. Thus, in a comprehensive comparison of on- and off-states, a steady cellular background is provided. Finally, in contrast to a system based on Flp recombinase, the set-up of our system is inherently reliable. [source] Fine-tuning CD4+ central memory T cell heterogeneity by strength of stimulationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2008Vandana Kalia Abstract The memory T cell pool serves as a relatively long-lived heterogeneous repository of antigen-experienced T cells that "remember" previous encounters with antigen. While heterogeneity in the memory T cell pool is now well established, signals regulating the generation of this memory T cell heterogeneity are not fully understood. Two articles in this issue of the European Journal of Immunology highlight the importance of the strength of antigenic stimulation in regulating the generation of phenotypically and functionally distinct CD4+ T cell memory subsets. New insights are also provided into key molecular players that likely mediate differences in homeostatic and secondary expansion between the memory subsets. See accompanying articles: http://dx.doi.org/10.1002/eji200737611 and http://dx.doi.org/10.1002/eji200737852 [source] Splenic function and IgM-memory B cells in Crohn's disease patients treated with infliximabINFLAMMATORY BOWEL DISEASES, Issue 5 2008Antonio Di Sabatino MD Abstract Background: Under experimental chronic inflammation, tumor necrosis factor (TNF)-, plays a role in damaging spleen marginal zone. This latter has a crucial function in mounting B cell-dependent immune responses against infections by encapsulated bacteria. In Crohn's disease (CD), a chronic inflammatory disorder where TNF-, is centrally involved, impaired splenic function may increase the susceptibility to bacterial infections. On this basis, we aimed to investigate the influence of anti-TNF therapy on splenic function in CD patients. Methods: Peripheral blood samples were obtained from 15 CD patients before and after treatment with infliximab administered at weeks 0, 2, and 6 at a dose of 5 mg/kg. Counting of erythrocytes with membrane abnormalities (pitted red cells) was used as an indicator of splenic function. Multicolor flow cytometry was performed to analyze circulating B cells. Results: A substantial clinical improvement in 10 of the 15 CD patients was associated with a significant reduction of pitted red cells (from median 6.0% to 3.6%; P < 0.01) after 10 weeks of treatment. In responder patients the improvement of splenic function was accompanied by a parallel increase of circulating IgM-memory B cells (from median 6.9% to 13.3%; P < 0.005). Splenic function was not ameliorated in nonresponder patients. Conclusions: Splenic function improved in CD patients who responded to infliximab and was accompanied by a concomitant restoration of the IgM-memory B cell pool responsible for the protection against encapsulated bacteria. Restoration of splenic function after infliximab treatment is intriguing and requires further investigation. (Inflamm Bowel Dis 2008) [source] CD4+CD25+ cell depletion from the normal CD4+ T cell pool prevents tolerance toward the intestinal flora and leads to chronic colitis in immunodeficient miceINFLAMMATORY BOWEL DISEASES, Issue 6 2006Claudia Veltkamp MD Abstract Background: CD4+CD25+ regulatory T cells have been shown to prevent immune-mediated colitis in mice; however, it is unclear whether the absence of CD4+CD25+ in the normal CD4+ T cell pool is responsible for the development of chronic colitis. Using the T cell-deficient Tg,26 mouse model, we show that CD4+CD25, cells but not CD4+CD25+ cells induce a severe intestinal inflammation. Transfer of CD4+CD25+ cells, together with CD4+CD25, cells, ameliorated intestinal inflammation, and reconstitution with the whole mesenteric lymph node cell pool did not induce colitis in recipients. Transferred CD4+CD25, cells were found mainly in the mesenteric lymph nodes, where they showed an activated TH1-like phenotype. In the absence of regulatory CD4+CD25+ T cells, recipient CD4+ cells secreted IFN-, in response to stimulation with intestinal bacterial antigen that was prevented in vivo and in vitro by regulatory CD4+CD25+ cells. These studies suggest that CD4+CD25, cells have a strong colitogenic effect in the Tg,26 colitis model and that CD4+CD25+ cells may be the main regulators that prevent or downregulate the proinflammatory effect of colitogenic T cells in the Tg,26 mouse model. [source] Prospective evaluation of intestinal homing memory T cells in ulcerative colitisINFLAMMATORY BOWEL DISEASES, Issue 5 2004A. L. Hart Abstract Background: Intestinal homing (,7+) memory T cells reflect the mucosal environment in which they were primed. We hypothesized that prospective assessment of cytokine production by intestinal homing (,7+) memory T cells in ulcerative colitis patients followed from remission to early relapse may elucidate shifts in cytokine production relevant to the mucosal environment associated with the early phase of inflammation. Methods: Twelve patients with frequently relapsing ulcerative colitis (,2 relapses in the previous 12 months) were recruited in remission and followed prospectively until relapse. Antibody labeling of whole blood and flow cytometry were used to identify ,7+ cells and ,7, populations within CD3+CD45RA, leukocytes. Production of cytokines (IFN-,, TNF-,, IL-2, IL-10, TGF-,, and IL-4) was determined by intracellular labeling. Results: Early relapse of ulcerative colitis was associated with a shift of T cells from the naive to the memory T cell pool, and further the ratio of ,7+:,7, memory T cells was significantly reduced at relapse (p < 0.01). A greater proportion of intestinal homing ,7+ memory T cells produced IL-4 (p < 0.02) and TNF-, (p < 0.05) at disease relapse compared with remission. Non-intestinal homing ,7,memory T cells also showed a tendency toward an increased production of TH1 and TH2 cytokines. Conclusions: The earliest phase of intestinal inflammation in ulcerative colitis patients is associated with an increase in both TH1 (TNF-, and TH2 (IL-4) cytokines by intestinal homing ,7+ memory T cells. These data support the principles of targeting lymphocyte trafficking as therapies in ulcerative colitis. [source] New model for the formation and function of sagittocysts: Symsagittifera corsicae n. sp. (Acoela)INVERTEBRATE BIOLOGY, Issue 2 2002Robert Gschwentner Abstract. This study is focused on the formation and function of sagittocysts, which are secretions typical of members of the acoel family Sagittiferidae. The needle-shaped sagittocysts are produced in specialized gland cells (sagittocytes) whose distal necks are often surrounded by muscle mantles. Contraction of the muscle mantle ejects the sagittocyst. We establish a model for the development of sagittocytes and muscle mantles out of the stem cell pool of the new acoel species Symsagittifera corsicae. We used various techniques, especially interference and phase-contrast microscopy of living specimens as well as labeling of the body-wall musculature, for species characterization. In addition to the morphological features, we provide the third complete sequence of the 18S rDNA gene in the family Sagittiferidae. [source] Regulation of Human Skeletal Stem Cells Differentiation by Dlk1/Pref-1JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2004Basem M Abdallah Abstract Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. Introduction: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. Materials and Methods: As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. Results: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPAR,2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor ,1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected. Conclusion: During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes. [source] Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2004Genevieve M. Boland Abstract Multipotential adult mesenchymal stem cells (MSCs) are able to differentiate along several known lineages, and lineage commitment is tightly regulated through specific cellular mediators and interactions. Recent observations of a low/high bone-mass phenotype in patients expressing a loss-/gain-of-function mutation in LRP5, a coreceptor of the Wnt family of signaling molecules, suggest the importance of Wnt signaling in bone formation, possibly involving MSCs. To analyze the role of Wnt signaling in mesenchymal osteogenesis, we have profiled the expression of WNTs and their receptors, FRIZZLEDs (FZDs), and several secreted Wnt inhibitors, such as SFRPs, and examined the effect of Wnt 3a, as a representative canonical Wnt member, during MSC osteogenesis in vitro. WNT11, FZD6, SFRP2, and SFRP3 are upregulated during MSC osteogenesis, while WNT9A and FZD7 are downregulated. MSCs also respond to exogenous Wnt 3a, based on increased ,-catenin nuclearization and activation of a Wnt-responsive promoter, and the magnitude of this response depends on the MSC differentiation state. Wnt 3a exposure inhibits MSC osteogenic differentiation, with decreased matrix mineralization and reduced alkaline phosphatase mRNA and activity. Wnt 3a treatment of fully osteogenically differentiated MSCs also suppresses osteoblastic marker gene expression. The Wnt 3a effect is accompanied by increased cell number, resulting from both increased proliferation and decreased apoptosis, particularly during expansion of undifferentiated MSCs. The osteo-suppressive effects of Wnt 3a are fully reversible, i.e., treatment prior to osteogenic induction does not compromise subsequent MSC osteogenesis. The results also showed that sFRP3 treatment attenuates some of the observed Wnt 3a effects on MSCs, and that inhibition of canonical Wnt signaling using a dominant negative TCF1 enhances MSC osteogenesis. Interestingly, expression of Wnt 5a, a non-canonical Wnt member, appeared to promote osteogenesis. Taken together, these findings suggest that canonical Wnt signaling functions in maintaining an undifferentiated, proliferating progenitor MSC population, whereas non-canonical Wnts facilitate osteogenic differentiation. Release from canonical Wnt regulation is a prerequisite for MSC differentiation. Thus, loss-/gain-of-function mutations of LRP5 would perturb Wnt signaling and depress/promote bone formation by affecting the progenitor cell pool. Elucidating Wnt regulation of MSC differentiation is important for their potential application in tissue regeneration. Published 2004 Wiley-Liss, Inc. [source] Dietary restriction enhances germline stem cell maintenanceAGING CELL, Issue 5 2010William Mair Summary Dietary restriction (DR) increases lifespan in species ranging from yeast to primates, maintaining tissues in a youthful state and delaying reproductive senescence. However, little is known about the mechanisms by which this occurs. Here we demonstrate that, concurrent with extending lifespan, DR attenuates the age-related decline in male germline stem cell (GSC) number in Drosophila. These data support a model whereby DR enhances maintenance of GSCs to extend the reproductive period of animals subjected to adverse nutritional conditions. This represents the first example of DR maintaining an adult stem cell pool and suggests a potential mechanism by which DR might delay aging in the tissues of higher organisms. [source] Depletion of the neural precursor cell pool by glucocorticoidsANNALS OF NEUROLOGY, Issue 1 2010Shuang Yu MD Objective Glucocorticoids (GCs) are indicated for a number of conditions in obstetrics and perinatal medicine; however, the neurodevelopmental and long-term neurological consequences of early-life GC exposure are still largely unknown. Preclinical studies have demonstrated that GCs have a major influence on hippocampal cell turnover by inhibiting neurogenesis and stimulating apoptosis of mature neurons. Here we examined the fate of the limited pool of neural progenitor cells (NPCs) after GC administration during neonatal development; the impact of this treatment on hippocampal structure was also studied. Methods Phenotype-specific genetic and antigenic markers were used to identify cultured NPCs at various developmental stages; the survival of these cells was monitored after exposure to the synthetic glucocorticoid dexamethasone (DEX). In addition, the effects of neonatal DEX treatment on the neurogenic potential of the rat hippocampus were examined by monitoring the incorporation of bromodeoxyuridine and expression of Ki67 antigen at various postnatal ages. Results Multipotent nestin-expressing NPCs and T,1-tubulin,expressing immature neurons succumb to GC-induced apoptosis in primary hippocampal cultures. Neonatal GC treatment results in marked apoptosis among the proliferating population of cells in the dentate gyrus, depletes the NPC pool, and leads to significant and sustained reductions in the volume of the dentate gyrus. Interpretation Both NPCs and immature neurons in the hippocampus are sensitive to the proapoptotic actions of GCs. Depletion of the limited NPC pool during early life retards hippocampal growth, thus allowing predictions about the potential neurological and psychiatric consequences of neonatal GC exposure. ANN NEUROL 2010;67:21,30 [source] Monoclonal anti-CD8 therapy induces disease amelioration in the K/BxN mouse model of spontaneous chronic polyarthritisARTHRITIS & RHEUMATISM, Issue 10 2010Bruno R. Raposo Objective CD8+ T cells are part of the T cell pool infiltrating the synovium in rheumatoid arthritis (RA). However, their role in the pathogenesis of RA has not been fully delineated. Using the K/BxN mouse model of spontaneous chronic arthritis, which shares many similarities with RA, we studied the potential of CD8+ T cell depletion with monoclonal antibodies (mAb) to stop and reverse the progression of experimental arthritis. Methods CD8+ T cells from the blood and articular infiltrate of K/BxN mice were characterized for cell surface phenotypic markers and for cytokine production. Additionally, mice were treated with specific anti-CD8 mAb (YTS105 and YTS169.4), with and without thymectomy. Results CD8+ T cells from the peripheral blood and joints of K/BxN mice were mainly CD69+ and CD62L,CD27+ T cells expressing proinflammatory cytokines (interferon-, [IFN,], tumor necrosis factor , [TNF,], interleukin-17a [IL-17A], and IL-4), and granzyme B. In mice receiving anti-CD8 mAb, the arthritis score improved 5 days after treatment. Recovery of the CD8+ T cells was associated with a new increase in the arthritis score after 20 days. In thymectomized and anti-CD8 mAb,treated mice, the arthritis score improved permanently. Histologic analysis showed an absence of inflammatory infiltrate in the anti-CD8 mAb,treated mice. In anti-CD8 mAb,treated mice, the serologic levels of TNF,, IFN,, IL-6, and IL-5 normalized. The levels of the disease-related anti,glucose-6-phosphate isomerase antibodies did not change. Conclusion These results indicate that synovial activated effector CD8+ T cells locally synthesize proinflammatory cytokines (IFN,, TNF,, IL-17, IL-6) and granzyme B in the arthritic joint, thus playing a pivotal role in maintaining chronic synovitis in the K/BxN mouse model of arthritis. [source] Continuous occurrence of both insufficient neovascularization and elevated vascular permeability in rabbit proximal femur during inadequate repair of steroid-associated osteonecrotic lesionsARTHRITIS & RHEUMATISM, Issue 10 2009Ge Zhang Objective To examine the features of the intraosseous vasculature, the size of the marrow stem cell pool (MSCP), and expression of vascular endothelial growth factor A (VEGF) during inadequate repair of steroid-associated osteonecrotic lesions in rabbits. Methods Steroid-associated osteonecrosis was induced in male rabbits. At 0, 1, 2, 4, and 6 weeks postinduction, vascularization and permeability indices were quantified by dynamic magnetic resonance imaging (MRI). In addition, the size of the MSCP in the hematopoietic and mesenchymal compartments was determined, and marrow mononuclear cells expressing specific surface markers for endothelial progenitor cells or periendothelial mural precursor cells were counted. At various time points after the rabbits were killed, the proximal femora were dissected to examine the intraosseous vasculature by angiography, histomorphometry, and ultramorphology. In addition, osteonecrotic lesion repair and marrow VEGF expression were evaluated. Results Lesion formation without repair was observed at 2 weeks after induction of steroid-associated osteonecrosis. Rabbits displaying destructive repair (DR+) and those displaying reparative osteogenesis (DR,) from 4 weeks to 6 weeks postinduction were identified. From week 2 to week 6, the vascularization index was significantly lower in DR+ rabbits compared with DR, rabbits, whereas the permeability index was significantly higher in DR+ rabbits compared with DR, rabbits. The features of the intraosseous vasculature determined by angiography, histomorphometry, and ultramorphology were consistent with those determined by dynamic MRI. The MSCP size and number of marrow mononuclear cells expressing specific surface markers were all significantly lower in DR+ rabbits than in DR, rabbits from week 1 to week 6. The increased VEGF expression at 2 weeks was maintained through week 6 in DR+ rabbits, whereas VEGF expression decreased in DR, rabbits from week 2 to week 6. Conclusion Continuous occurrence of both insufficient neovascularization and elevated vascular permeability is accompanied by a continuously low- level MSCP and uncontrolled VEGF expression during inadequate repair of steroid-associated osteonecrotic lesions. [source] Controlling the stem cell niche: right time, right place, right strengthBIOESSAYS, Issue 1 2006Catherin Niemann Wnt signalling through ,-catenin plays a pivotal role during embryonic pattern formation, cell fate determination and tissue homeostasis in the adult organism. In the skin, as in many other tissues, Wnt/,-catenin signalling can control lineage determination and differentiation. However, it was not known whether Wnt/,-catenin signalling is an immediate regulator of the stem cell niche in skin tissue. A recent publication now provides evidence that Wnt/,-catenin signalling exerts a direct effect on the stem cell compartment by inducing quiescent stem cells to enter the cell cycle during early stages of hair follicle regeneration. In addition, the authors demonstrate that ,-catenin is required for maintenance of the stem cell pool in the tissue.1 The data suggest that a gradient in Wnt/,-catenin activity levels can induce different responses within distinct cell populations reflected by activation of distinct transcriptional profiles. BioEssays 28:1,5, 2006. © 2005 Wiley Periodicals, Inc. [source] Structure of an N-terminally truncated Nudix hydrolase DR2204 from Deinococcus radioduransACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009A. M. D. Gonçalves Nudix pyrophosphatases are a well represented protein family in the Deinococcus radiodurans genome. These hydrolases, which are known to be enzymatically active towards nucleoside diphosphate derivatives, play a role in cleansing the cell pool of potentially deleterious damage products. Here, the structure of DR2204, the only ADP-ribose pyrophosphatase in the D. radiodurans genome that is known to be active towards flavin adenosine dinucleotide (FAD), is presented at 2.0,Å resolution. [source] Stem cell separation: A bottleneck in stem cell therapyBIOTECHNOLOGY JOURNAL, Issue 1 2010Kornelia Schriebl Dr. Abstract The substantial progress in embryonic stem cell (ESC) research could lead to new possibilities in the treatment of various diseases. Currently, applications of ESC for cell therapy are impeded by the presence of potentially teratoma-forming undifferentiated ESC. Thus, a selective and quantitative removal of undifferentiated ESC from a pool of differentiated and undifferentiated cells is essential before cell therapy. We evaluated the highly selective magnetic activated cell sorting (MACS) method for the quantitative removal of undifferentiated ESC. We found that the clearance rates for undifferentiated ESC decreased with decreasing amount of undifferentiated ESC in the cell pool. Using a simplified model calculation we could predict that, assuming an initial purity of 60%, an estimated 31 steps are required to achieve less than 10,1 cell per 109 cells. Thus, a log clearance rate of 10, which would be necessary for a therapeutically application, is hard to achieve. Our work clearly indicates that the current MACS technology is insufficient to meet the purification needs for cell therapy. [source] Developmental consequences of abnormal folate transport during murine heart morphogenesis ,BIRTH DEFECTS RESEARCH, Issue 7 2004Louisa S. Tang Abstract BACKGROUND Folic acid is essential for the synthesis of nucleotides and methyl transfer reactions. Folic acid,binding protein one (Folbp1) is the primary mediator of folic acid transport into murine cells. Folbp1 knockout mouse embryos die in utero with multiple malformations, including severe congenital heart defects (CHDs). Although maternal folate supplementation is believed to prevent human conotruncal heart defects, its precise role during cardiac morphogenesis remains unclear. In this study, we examined the role of folic acid on the phenotypic expression of heart defects in Folbp1 mice, mindful of the importance of neural crest cells to the formation of the conotruncus. METHODS To determine if the Folbp1 gene participates in the commitment and differentiation of the cardiomyocytes, relative levels of dead and proliferating precursor cells in the heart were examined by flow cytometry, Western blot, and immunohistostaining. RESULTS Our studies revealed that impaired folic acid transport results in extensive apoptosis-mediated cell death, which concentrated in the interventricular septum and truncus arteriosus, thus being anatomically restricted to the two regions of congenital heart defects. Together with a reduced proliferative capacity of the cardiomyocytes, the limited size of the available precursor cell pool may contribute to the observed cardiac defects. Notably, there is a substantial reduction in Pax-3 expression in the region of the presumptive migrating cardiac neural crest, suggesting that this cell population may be the most severely affected by the massive cell death. CONCLUSIONS Our findings demonstrate for the first time a prominent role of the Folbp1 gene in mediating susceptibility to heart defects. Birth Defects Research (Part A), 2004. © 2004 Wiley-Liss, Inc. [source] B-cell chronic lymphocytic leukaemia cells show specific changes in membrane protein expression during different stages of cell cycleBRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2007Fiona Bennett Summary The proliferating component in chronic lymphocytic leukaemia (CLL) is usually small (<1%) and restricted to a specific micro-environmental niche. To characterize the proliferating component, CLL cells from bone marrow or lymph nodes of 23 patients were assessed for expression of up to 66 surface antigens in combination with nuclear Ki-67/MCM6. Ki-67 expression was associated with step-wise increases in CD23/CD95/CD86/CD39/CD27 and decreases in CD24/CD69/CXCR4/CXCR5. Ki-67+ cells showed increased CD38 expression, but with considerable inter-patient variability: in some cases Ki-67 expression was only detectable in CD38, CLL cells. The results suggest continuous re-entry into the cell cycle as no distinct stem cell pool was detectable. [source] Bone morphogenetic protein 4 modulates c-Kit expression and differentiation potential in murine embryonic aorta-gonad-mesonephros haematopoiesis in vitroBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2007Caroline J. Marshall Summary The transforming growth factor- , -related factor bone morphogenetic protein 4 (BMP4) is expressed in the human embryonic aorta-gonad-mesonephros (AGM) coincident with the emergence of haematopoietic cells and influences postnatal mammalian haematopoietic stem cells in vitro. To investigate the role of BMP4 in mammalian embryonic haematopoiesis, cells were isolated from murine AGM and two populations of CD34+ cells with different levels of c-Kit expression and multipotency were identified. CD34+/c-Kithigh cells express CD45 and are haematopoietic-restricted progenitors. In contrast, CD34+/c-Kitlow cells are Flk1+/CD45neg and generate adherent colonies in ex vivo culture that resemble haemangioblast colonies identified in other systems. The addition of BMP4 to AGM cells resulted in expansion of the CD34+/c-Kitlow cell pool within 48 h, via a combination of down modulation of the c-Kit receptor in CD34+/c-Kithigh cells and proliferation. In long-term culture, BMP4 increased the growth/survival of CD34+/c-Kithigh haematopoietic progenitors, effects that were blocked by BMP inhibitors. CD34+/c-Kithigh progenitors cultured with BMP4 also generated adherent colonies typical of c-Kitlow cells. These results suggest that BMP4 regulates c-Kit expression and differentiation potential in CD34+ AGM cells and supports a role for BMP signalling in the maintenance of multipotency during embryonic haematopoiesis, providing an insight into stem cell homeostasis within the mammalian haematopoietic niche. [source] Obesity predisposes to Th17 biasEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2009Shawn Winer Abstract Obesity is associated with numerous inflammatory conditions including atherosclerosis, autoimmune disease and cancer. Although the precise mechanisms are unknown, obesity-associated rises in TNF-,, IL-6 and TGF-, are believed to contribute. Here we demonstrate that obesity selectively promotes an expansion of the Th17 T-cell sublineage, a subset with prominent pro-inflammatory roles. T-cells from diet-induced obese mice expand Th17 cell pools and produce progressively more IL-17 than lean littermates in an IL-6-dependent process. The increased Th17 bias was associated with more pronounced autoimmune disease as confirmed in two disease models, EAE and trinitrobenzene sulfonic acid colitis. In both, diet-induced obese mice developed more severe early disease and histopathology with increased IL-17+ T-cell pools in target tissues. The well-described association of obesity with inflammatory and autoimmune disease is mechanistically linked to a Th17 bias. [source] Mathematical modelling of the impact of haematopoietic stem cell-delivered gene therapy for HIVTHE JOURNAL OF GENE MEDICINE, Issue 12 2009John M. Murray Abstract Background Gene therapy represents a new treatment paradigm for HIV that is potentially delivered by a safe, once-only therapeutic intervention. Methods Using mathematical modelling, we assessed the possible impact of autologous haematopoietic stem cell (HSC) delivered, anti-HIV gene therapy. The therapy comprises a ribozyme construct (OZ1) directed to a conserved region of HIV-1 delivered by transduced HSC (OZ1+HSC). OZ1+HSC contributes to the CD4+ T lymphocyte and monocyte/macrophage cell pools that preferentially expand under the selective pressure of HIV infection. The model was used to predict the efficacy of OZ1 in a highly active antiretroviral therapy (HAART) naïve individual and a HAART-experienced individual undergoing two structured treatment operations. In the standard scenario, OZ1+HSC was taken as 20% of total body HSC. Results For a HAART-naïve individual, modelling predicts a reduction of HIV RNA at 1 and 2 years post-OZ1 therapy of 0.5 log10 and 1 log10, respectively. Eight years after OZ1 therapy, the CD4+ T-lymphocyte count was 271 cells/mm3 compared to 96 cells/mm3 for an untreated individual. In a HAART-experienced individual HIV RNA was reduced by 0.34 log10 and 0.86 log10 at 1 and 2 years. The OZ1 effect was maximal when both CD4+ T lymphocytes and monocytes/macrophages were protected from successful, productive infection by OZ1. Conclusions The modelling indicates a single infusion of HSC cell-delivered gene therapy can impact on HIV viral load and CD4 T-lymphocyte count. Given that gene therapy avoids the complications associated with HAART, there is significant potential for this approach in the treatment of HIV. Copyright © 2009 John Wiley & Sons, Ltd. [source] A retrovirus-based system to stably silence GDF-8 expression and enhance myogenic differentiation in human rhabdomyosarcoma cellsTHE JOURNAL OF GENE MEDICINE, Issue 8 2008Zhuo Yang Abstract Background Myostatin, also called GDF-8, a secreted growth and differentiating factor that belongs to the transforming growth factor-, superfamily, is a known negative regulator of myogenesis in vivo. Overexpression of GDF-8 contributes to the lack of differentiation in human rhabdomyosarcoma (RMS) cells. We investigated whether a retrovirus-based RNA interference (RNAi) system against GDF-8 expression in human RMS cells would enhance myogenic differentiation. Methods A retrovirus-based RNAi system was developed that utilized the U6-RNA polymerase III promoter to drive efficient expression and deliver the GDF8-specific short hairpin RNAs (shRNAs) in human RMS cell A204. In this system, the retrovirus vector was integrated into the host cell genome and allowed stable expression of shRNAs. GDF-8 expression was determined by real-time polymerase chain reaction and western blotting analysis. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cell proliferation. Myogenic differentiation markers were monitored by western blotting analysis. Cell cycle and apoptosis was determined by propidium iodide staining and analysed in a flow cytometer. Results In the siGDF8 A204 cell pools, the levels of both GDF-8 mRNA and protein were dramatically reduced by this RNAi system. In differentiation conditions, inhibition of myostatin synthesis led to enhanced cell cycle withdrawal, consequently stimulated myogenic differentiation and increased the rate of tumor cell apoptosis. Conclusions The results demonstrate that deactivation of myostatin by using retrovirus-based RNAi thus may be useful for therapy in rhabdomyosarcomas. Copyright © 2008 John Wiley & Sons, Ltd. [source] On the Structure of the Adrenal Gland of the Common Seal (Phoca vitulina vitulina)ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 5 2004H. Bragulla Summary The adrenal gland is a vitally important endocrine gland that occupies a central role in the regulatory mechanisms of the body metabolism. Environmental stress factors lead to permanent strain and overload of the body resulting in structural alterations of the adrenals that in turn are followed by hormonal imbalances. This leads to an increased susceptibility to bacterial and viral diseases. The recurrence of numerous fatalities in the different seal populations of the North Sea (during the years 1988, 1989 and 2002), of the Baikal Lake and Caspian Sea (during the years 2000 and 2001) were the motive for a morphological investigation of the species-specific structure of the adrenal gland of the common seal in order to differentiate environmental stress-induced pathological alterations from the physiological structure of this organ. The study was based on adrenals of 112 common seals (Phoca vitulina vitulina) using light microscopic and transmission and scanning electron microscopic methods. The phocine adrenal gland displays several structural characteristics. Originating from the connective tissue organ capsule, narrow and broad septa intersperse the adrenal cortex. These septa contain blastemata as a reserve for the regeneration of hormone-producing cortical cells. Such blastemata are also occurring in the form of an intermediate zone in between the zona glomerulosa and zona fasciculata in the phocine adrenal cortex. Another species-specific characteristic is an inverse part of the adrenal cortex encircling the central vein of the organ. These structural features have to be considered in assessment and definition of pathological alterations of the adrenals as observed in the form of exhausted blastema cell pools in the adrenocortex of seals perished in the mentioned phocine mass mortalities. [source] Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodiesBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2010Laetitia Malphettes Abstract IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily ,1,6-fucosylated, a modification that reduces antibody-dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a ,1,6-fucosyltransferase. FUT8,/, CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein-production cell lines has prevented the widespread adoption of FUT8,/, cells as hosts for antibody production. We have created zinc-finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8,/, CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN-treated cells can be directly selected to give FUT8,/, cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN-derived FUT8,/, cell lines were as transfectable as wild-type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild-type and ZFN-generated FUT8,/, cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN-generated FUT8,/, cell lines was fourfold higher than in the best-producing clones of FUT8,/, cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN-mediated transfer of the Fut8, phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies. Biotechnol. Bioeng. 2010;106: 774,783. © 2010 Wiley Periodicals, Inc. [source] Telomerase activity in disseminated prostate cancer cellsBJU INTERNATIONAL, Issue 6 2006JESCO PFITZENMAIER OBJECTIVE To analyse telomerase activity in disseminated prostate cancer cells isolated from bone marrow aspirates taken from men with localized prostate cancer before radical prostatectomy (RP). PATIENTS AND METHODS Disseminated epithelial prostate cancer cells were isolated from bone marrow aspirates from 69 men with localized prostate cancer before RP, by magnetic column-chromatography enrichment, followed by isolation of fluorescently labelled epithelial cells by micropipetting. We used pools of 10 non-epithelial bone marrow cells after tumour cell enrichment as control samples. These pure cell pools were tested for the presence of telomerase activity. RESULTS In all, 49 of the patient samples contained disseminated prostate cancer cells. Homogeneous pools of 10 cells were obtained from 35 of these; 49% of the 35 specimens showed telomerase activity, whereas all five control samples did not. Telomerase activity in the 35 samples was not significantly associated with Gleason score, preoperative prostate-specific antigen level, tumour stage, or surgical margin status. Follow-up is continuing to assess an association with disease recurrence. CONCLUSION This work shows the feasibility of isolating disseminated cancer cells for analysing individual or pooled cells. Compared to tissue staining, where telomerase is detected in 80,90% of samples, we found lower rates of telomerase activity in the disseminated tumour cells (49%). Telomerase-negative cells might provide information about cell dormancy, as telomerase is a marker of cell proliferation in immortal and cancer cells. Telomerase-positive cells might predict early disease recurrence, but a longer follow-up is needed to test this possibility. [source] Differential distribution of haematopoietic and nonhaematopoietic progenitor cells in intralesional and extralesional keloid: do keloid scars provide a niche for nonhaematopoietic mesenchymal stem cells?BRITISH JOURNAL OF DERMATOLOGY, Issue 6 2010S.A. Iqbal Summary Background, Keloid disease is a benign, quasineoplastic disease with a high recurrence rate. Mesenchymal-like stem cells (MLSC) have previously been demonstrated in keloid scars and may be involved in keloid pathobiology. However, as these cells have only been examined by single colour fluorescence activated cell sorting (FACS) alone, they need to be more comprehensively characterized so that the key cellular contributors to keloid scars can be better understood. Objectives, To identify and characterize MLSC in intralesional and extralesional keloid, and to distinguish haematopoietic stem cells (HSC) from mesenchymal stem cells (MSC). Methods and patients, Punch biopsies from intralesional (top, middle and margin) and extralesional keloid scar sites were obtained from 17 patients with a keloid. Multicolour FACS analysis using antibodies specific for HSC markers CD34 and CD117 and MSC markers CD13, CD29, CD44 and CD90 was performed on freshly isolated keloid scar cells and on passage 0 and 1 cells. This was complemented by real-time quantitative polymerase chain reaction (PCR) and immunohistological in situ analyses. Results, Keloid scars contain distinct subpopulations of MLSCs. Cells positive for CD13, CD29, CD44 and CD90 were found to be significantly (P < 0·05) higher in the top and middle compartments of keloid scars compared with extralesional skin, where cells positive for CD34, CD90 and CD117 (representing HSCs) predominated. A unique population of CD34+ cells (cells positive for CD13, CD29, CD34, CD44 and CD90) were found in keloid scars and in extralesional skin. FACS and quantitative PCR analysis showed that many of the MSC markers were progressively downregulated and all HSC markers were lost during extended keloid fibroblast culture up to passage 1. Conclusion, We have found distinct subpopulations of haematopoietic and nonhaematopoietic MSC in keloid scars, whereby HSC accumulate extralesionally, while keloids seem to provide a niche environment for nonhaematopoietic MSC. Future therapy of keloids may have to target differentially both stem cell populations in order to deprive these tumours of their regenerative cell pools. [source] | ||||||||||||||||