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Selected AbstractsRANK Expression as a Cell Surface Marker of Human Osteoclast Precursors in Peripheral Blood, Bone Marrow, and Giant Cell Tumors of BoneJOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2006Gerald J Atkins Abstract RANK expression in vivo on hematopoietic subsets including pre-osteoclasts, identified by monoclonal antibodies, has not been described. We describe the lineages that express RANK in bone marrow, peripheral blood, and GCTs. We show that CD14+RANKhigh cells constitute a circulating pre-osteoclast pool. Introduction: The expression of RANK by subsets of hematopoietic cells has not been adequately studied in humans. While attributed to the monocytoid lineage, the phenotype of the pre-osteoclast (pre-OC) with respect to RANK expression in vivo remains unclear. We tested monoclonal antibodies (MAbs) raised against the extracellular domain of recombinant human RANK for reactivity with normal peripheral blood (PB) and bone marrow (BM) mononuclear cells (PBMNCs and BMMNCs, respectively). We also tested reactivity with giant cell tumor cells (GCT), a confirmed source of pre-OC and mature OCs. Materials and Methods: Human PBMNCs, BMMNCs, and GCT cells were analyzed for reactivity with anti-RANK MAbs by flow cytometry in combination with hematopoietic lineage restricted markers. GCTs were also analyzed by immunofluorescence. CD14+ monocytoid cells were sorted by fluorescence-activated cell sorting (FACS) based on their relative RANK expression and cultured under OC-forming conditions. Results: RANK+ cells were detected similarly by three independent anti-RANK MAbs. One MAb (80736) immunoprecipitated RANK,RANKL complexes from surface-biotinylated GCT lysates. Using dual-color flow cytometry, RANK was detected on CD14+ (monocytoid), CD19+ (B-lymphoid), CD56+ (NK cell), and glycophorin A+ erythroid progenitors. Minor populations of both CD3+ T lymphocytes and BM CD34+ hematopoietic progenitors also expressed cell surface RANK. In GCTs, RANK expression was identified on mononuclear CD45+CD14+,V,3+c-Fms+ cells, likely to be committed pre-OC, and on multinucleated CD45+,V,3+TRACP+ OCs. Importantly, sorted CD14+RANKhigh PBMNCs treated with recombinant RANKL and macrophage-colony stimulating factor (M-CSF) gave rise to approximately twice the number of osteoclasts than RANKmid or RANKlow cells. Conclusions: These results suggest that committed monocytoid RANK+ pre-OCs are represented in the marrow and circulate in the periphery, forming a pool of cells capable of responding rapidly to RANKL. The ability to reliably detect committed pre-OC in peripheral blood could have important clinical applications in the management of diseases characterized by abnormal osteoclastic activity. [source] Diagnostic value of GLUT-1 immunoreactivity to distinguish benign from malignant cystic squamous lesions of the head and neck in fine-needle aspiration biopsy materialDIAGNOSTIC CYTOPATHOLOGY, Issue 5 2004Michael F. Weiner M.D. Abstract The distinction of cystic squamous-cell carcinoma (SCC) from benign cystic squamous lesions (BCSLs) of the head and neck can be problematic on fine-needle aspiration biopsy (FNAB) material, particularly when BCSLs display epithelial reactive atypia or when SCC is well differentiated. Glucose transporter 1 (GLUT-1), a facilitative cell surface glucose transport protein, is aberrantly expressed in many cancers including oral and hypopharyngeal SCC. We evaluated the expression of GLUT-1 by immunochemistry on FNAB material to determine its value in distinguishing cystic SCC from BCSL of the head and neck. A 5-yr retrospective review of all head and neck cystic squamous lesions having FNAB specimens with cell block material, radiological studies, and histological confirmation was performed at our institution. Cell block material from 24 cystic squamous lesions, including 8 (33%) BCSL (7 branchial cleft cysts and 1 thyroglossal duct cyst[TDC]) and 16 (67%) metastatic SCCs with cystic/liquefactive degeneration, was retrieved and immunostained with anti-GLUT-1. GLUT-1 expression was considered positive when at least 10% of squamous cells exhibited distinct cell membrane reactivity. Positive GLUT-1 immunostaining was detected in all 16 SCCs and in none of the 8 BCSLs. In the carcinoma cases, the majority of malignant cells exhibited GLUT-1 reactivity; only a minor population of well-differentiated SCC cells displaying keratinization and arranged as squamous pearls did not express GLUT-1. GLUT-1 expression in cell block material can help to distinguish cystic SCCs from BCSLs of the head and neck. In conjunction with clinical and radiological correlation, GLUT-1 immunoreactivity can be an important diagnostic aid when the cytological findings are ambiguous. Diagn. Cytopathol. 2004;31:294,299. © 2004 Wiley-Liss, Inc. [source] Functional characterization of human nucleosome assembly protein 1-like proteins as histone chaperonesGENES TO CELLS, Issue 1 2010Mitsuru Okuwaki Nucleosome Assembly Protein 1 (NAP1) is a highly conserved histone chaperone protein suspected to be involved in the dynamical regulation of the histone H2A-H2B hetero-dimer. However, the exact mechanism by which NAP1-like proteins act is currently unknown. In this work, we characterized the biochemical properties of two human NAP1-like proteins, hNAP1L1 and hNAP1L4, including a previously uncharacterized subtype, with the aim of determining their exact mechanistic role. Both hNAP1L1 and hNAP1L4 were found to be localized mainly to the cytoplasm and a minor population of them was suggested to be in the nucleus. Biochemical analyses demonstrated that both hNAP1L1 and hNAP1L4 mediated nucleosome formation. In addition, hNAP1L1 was shown to possess a significantly greater nucleosome disassembly activity than hNAP1L4, suggesting that hNAP1L1 and hNAP1L4 may play distinct roles in the regulation of histone dynamics. Building upon this initial discovery we also found that histone H2A-H2B and various histone H2A variants-H2B dimers were found to associate with both hNAP1L1 and hNAP1L4 in cell extracts. These results suggest that human NAP1-like proteins play overlapping roles in transport and deposition of histone H2A-H2B or H2A variants-H2B dimers on chromatin and nonoverlapping roles in nucleosome disassembly. [source] Natural Killer Cell Receptor+ T-Lymphocytes in Normal and Helicobacter pylori -Infected Human Gastric MucosaHELICOBACTER, Issue 6 2008Joan O'Keeffe Abstract Background:,Helicobacter pylori infection is associated with development of chronic inflammation and infiltration of immune cells into the gastric mucosa. As unconventional T-lymphocytes expressing natural killer cell receptors are considered to play central roles in the immune response against infection, a study investigating their frequencies in normal and H. pylori -infected gastric mucosa was undertaken. Materials and Methods:, Flow cytometry was used to quantify T-cells expressing the natural killer cell markers CD161, CD56, and CD94 in freshly isolated lymphocytes from the epithelial and lamina propria layers of gastric mucosa. Thirteen H. pylori -positive and 24 H. pylori -negative individuals were studied. Results:, CD94+ T-cells were the most abundant (up to 40%) natural killer receptor-positive T-cell population in epithelial and lamina propria layers of H. pylori -negative gastric mucosa. CD161+ T-cells accounted for about one-third of all T-cells in both compartments, but the lowest proportion were of CD56+ T-cells. Compared with H. pylori -negative mucosa, in H. pylori -infected mucosa the numbers of CD161+ T-cells were significantly greater (p = .04) in the epithelium, whereas the numbers of CD56+ T-cells were lower (p = .01) in the lamina propria. A minor population (< 2%) of T-cells in both mucosal layers of H. pylori -negative subjects were natural killer T-cells, and whose proportions were not significantly different (p > .05) to those in H. pylori -infected individuals. Conclusions:, The predominance, heterogeneity, and distribution of natural killer cell receptor-positive T-cells at different locations within the gastric mucosa reflects a potential functional role during H. pylori infection and warrants further investigation. [source] CD4+ CD5+ regulatory T cells render naive CD4+ CD25, T cells anergic and suppressiveIMMUNOLOGY, Issue 4 2007Miao Qiao Summary CD4+ CD25+ Foxp3+ naturally occurring regulatory T cells (nTreg) are potent inhibitors of almost all immune responses. However, it is unclear how this minor population of cells is capable of exerting its powerful suppressor effects. To determine whether nTreg mediate part of their suppressor function by rendering naive T cells anergic or by converting them to the suppressor phenotype, we cocultured mouse nTreg with naive CD4+ CD25, T cells from T-cell receptor (TCR) transgenic mice on a RAG deficient (RAG,/,) background in the presence of anti-CD3 and interleukin-4 (IL-4) to promote cell viability. Two distinct responder cell populations could be recovered from the cocultures. One population remained undivided in the coculture and was non-responsive to restimulation with anti-CD3 or exogenous IL-2, and could not up-regulate IL-2 mRNA or CD25 expression upon TCR restimulation. Those responder cells that had divided in the coculture were anergic to restimulation with anti-CD3 but responded to restimulation with IL-2. The undivided population was capable of suppressing the response of fresh CD4+ CD25, T cells and CD8+ T cells, while the divided population was only marginally suppressive. Although cell contact between the induced regulatory T cell (iTreg) and the responders was required for suppression to be observed, anti-transforming growth factor-, partially abrogated their suppressive function. The iTreg did not express Foxp3. Therefore nTreg are not only able to suppress immune responses by inhibiting cytokine production by CD4+ CD25, responder cells, but also appear to modulate the responder cells to render them both anergic and suppressive. [source] Expression of CD8, identifies a distinct subset of effector memory CD4+ T lymphocytesIMMUNOLOGY, Issue 2 2006Iole Macchia Summary Circulating CD4+ CD8+ T lymphocytes have been described in the peripheral blood of humans and several animal species. However, the origin and functional properties of these cells remain poorly understood. In the present study, we evaluated the frequency, phenotype and function of peripheral CD4+ CD8+ T cells in rhesus macaques. Two distinct populations of CD4+ CD8+ T cells were identified: the dominant one was CD4hi CD8lo and expressed the CD8,, homodimer, while the minor population was CD4lo CD8hi and expressed the CD8,, heterodimer. The majority of CD4hi CD8,lo T cells exhibited an activated effector/memory phenotype (CCR5lo CD7, CD28, HLA-DR+) and expressed relatively high levels of granzyme B. Intracellular cytokine staining assays demonstrated that the frequency of cytomegalovirus-specific T cells was enriched five-fold in CD4hi CD8,lo T cells compared to single-positive CD4+ T cells, whereas no consistent enrichment was observed for simian immunodeficiency virus (SIV)-specific T cells. Cross-sectional studies of SIV-infected animals demonstrated that the frequency of CD4hi CD8,lo T cells was lower in wild-type SIV-infected animals compared to uninfected controls, although prospective studies of SIV-infected animals demonstrated depletion of CD4hi CD8,lo lymphocytes only in a subset of animals. Taken together, these data suggest that CD4+ T cells expressing CD8, represent an effector/memory subset of CD4+ T cells and that this cell population can be depleted during the course of SIV infection. [source] Laser-capture microdissection: Applications in routine molecular dermatopathologyJOURNAL OF CUTANEOUS PATHOLOGY, Issue 7 2004Amir S. Yazdi Applications for PCR-based diagnostics are particularly helpful for the determination of clonality in cutaneous lymphocytic infiltrates and for detection of infectious agents, such as herpes simplex virus (HSV), varicella zoster virus (VZV), Borrelia burgdorferi, Mycobacteria, Leishmania, and Treponema pallidum. As biopsies are always composed of different cells, the cells of interest are often only a minor population. As a consequence, their specific DNA is diluted by the majority of contaminating cells. Another problem is the time- and labor-intensive DNA extraction, because usually only formalin-fixed, paraffin-embedded tissue is available, which makes molecular diagnostics a time and labor consuming, and consequently a cost-intensive procedure. To overcome these shortcomings and to eventually shorten the time to generate a result, we introduce a laser-capture microdissection (LCM)-based method for the detection of infectious agents and clonality. Only the cells of interest for the particular indication are microdissected (e.g. epidermal cells for HSV and VZV and lymphocytes for clonality analysis) and subjected to PCR amplification. Due to an accelerated DNA-extraction procedure which generates DNA in 5 h (compared to 3,4 days using conventional DNA extraction), we are able to generate a result within one working day. [source] P2X2, P2X2,2 and P2X5 receptor subunit expression and function in rat thoracolumbar sympathetic neuronsJOURNAL OF NEUROCHEMISTRY, Issue 5 2001H. Schädlich The present study investigated the pharmacological properties of excitatory P2X receptors and P2X2 and P2X5 receptor subunit expression in rat-cultured thoracolumbar sympathetic neurons. In patch-clamp recordings, ATP (3,1000 µm; applied for 1 s) induced inward currents in a concentration-dependent manner. Pyridoxal-phosphate-6-azophenyl-2,,4,-disulfonate (PPADS; 30 µm) counteracted the ATP response. In contrast to ATP, ,,,-meATP (30 µm; for 1 s) was virtually ineffective. Prolonged application of ATP (100 µm; 10 s) induced receptor desensitization in a significant proportion of sympathetic neurons in a manner typical for P2X2,2 splice variant-mediated responses. Using single-cell RT-PCR, P2X2, P2X2,2 and P2X5 mRNA expression was detectable in individual tyrosine hydroxylase-positive neurons; coexpression of both P2X2 isoforms was not observed. Laser scanning microscopy revealed both P2X2 and P2X5 immunoreactivity in virtually every TH-positive neuron. P2X2 immunoreactivity was largely distributed over the cell body, whereas P2X5 immunoreactivity was most distinctly located close to the nucleus. In summary, the present study demonstrates the expression of P2X2, P2X2,2 and P2X5 receptor subunits in rat thoracolumbar neurons. The functional data in conjunction with a preferential membranous localization of P2X2/P2X2,2 compared with P2X5 suggest that the excitatory P2X responses are mediated by P2X2 and P2X2,2 receptors. Apparently there exist two types of P2X2 receptor-bearing sympathetic neurons: one major population expressing the unspliced isoform and another minor population expressing the P2X2,2 splice variant. [source] Glutamate Receptor Subunit ,2 Is Highly Expressed in a Novel Population of Glial-Like Cells in Rat Pineal Glands in CultureJOURNAL OF NEUROCHEMISTRY, Issue 3 2000Shouki Yatsushiro Abstract: The mammalian pineal gland uses L-glutamate as an intercellular chemical transmitter to regulate negatively melatonin synthesis. To receive glutamate signals, pinealocytes express at least three kinds of glutamate receptors: metabotropic receptor types 3 and 5 and an ionotropic receptor, GluR1. In this study, we examined whether or not the fourth class of ionotropic receptor, ,, which is known for its nondefinitive molecular function and its unique expression pattern in brain, is expressed in pineal gland. RT-PCR analyses with specific probes indicated the expression of mRNA of ,2 but not that of ,1 in pineal gland and cultured pineal cells. Western blotting analysis with polyclonal antibodies specific to the carboxyl-terminal region of the ,2 receptor recognized a single 110-kDa polypeptide of cerebellar membranes and specifically immunostained Purkinje cells. The ,2 antibodies recognized a 110-kDa polypeptide of pineal membranes and specifically immunostained huge glial-like cells with the occasional presence of several long, branching processes in a pineal cell culture. ,2 is not uniformly distributed throughout the cells and is relatively abundant at the periphery of the cell bodies and long processes, where the terminals of synaptophysin-positive processes of pinealocytes, a site for glutamate secretion, are frequently present. The ,2-positive cells constitute a very minor population among total pineal cells (,0.03%). Double immunolabeling with ,2 antibodies and antibodies against marker proteins for pineal interstitial cells clearly distinguishes ,2-positive pineal cells and other known interstitial cells, including glial fibrillary acidic protein- or vimentin-positive glial-like cells. These results indicated that the ,2 glutamate receptor is expressed in a novel subpopulation of pineal glial-like cells in culture and suggest the presence of a glutamate-mediated intercellular signal transduction mechanism between pinealocytes and ,2-expressing cells. The pineal cells may provide a good experimental system for studies on the function of glutamate receptor ,2. [source] Hypoxia and low-nutrition double stress induces aggressiveness in a murine model of melanomaCANCER SCIENCE, Issue 5 2009Tsuyoshi Osawa Antiangiogenic therapy is a potent cancer treatment, however, the possibility of recurrence and resistance to this approach remains. Here we show that hypoxia and low-nutrition double-deprivation stress induces reversible tumor aggressiveness. In a stress-cycle-dependent manner, murine melanoma cells showed morphological changes, up-regulated phospho-Akt, and abnormal regulation of multiple genes including fibroblast growth factor-21, a metabolic regulator, resulting in increased cell proliferation in vitro, and increased tumorigenesis and invasive potential in vivo. In this system, altered cellular metabolism participates in the adaptation of tumor to the double-deprivation stress. Our results suggest the targeting of a minor population of cancer cells resistant to both hypoxia and low nutrition to be an effective new antitumor strategy in combination with antiangiogenic therapy. (Cancer Sci 2009; 100: 844,851) [source] Diagnosing PNH with FLAER and multiparameter flow cytometryCYTOMETRY, Issue 3 2007D. Robert Sutherland Abstract Background: PNH is an acquired hematopoietic stem cell disorder leading to a partial or absolute deficiency of all glycophosphatidyl-inositol (GPI)-linked proteins. The classical approach to diagnosis of PNH by cytometry involves the loss of at least two GPI-linked antigens on RBCs and neutrophils. While flow assays are more sensitive and specific than complement-mediated lysis or the Hams test, they suffer from several drawbacks. Bacterial aerolysin binds to the GPI moiety of cell surface GPI-linked molecules and causes lysis of normal but not GPI-deficient PNH cells. FLAER is an Alexa488-labeled inactive variant of aerolysin that does not cause lysis of cells. Our goals were to develop a FLAER-based assay to diagnose and monitor patients with PNH and to improve detection of minor populations of PNH clones in other hematologic disorders. Methods: In a single tube assay, we combined FLAER with CD45, CD33, and CD14 allowing the simultaneous analysis of FLAER and the GPI-linked CD14 structure on neutrophil and monocyte lineages. Results: Comparison to standard CD55 and CD59 analysis showed excellent agreement. Because of the higher signal to noise ratio, the method shows increased sensitivity in our hands over single (CD55 or CD59) parameter analysis. Using this assay, we were able to detect as few as 1% PNH monocytes and neutrophils in aplastic anemia, that were otherwise undetectable using CD55 and CD59 on RBC's. We also observed abnormal FLAER staining of blast populations in acute leukemia. In these cases, the neutrophils stained normally with FLAER, while the gated CD33bright cells failed to express normal levels of CD14 and additionally showed aberrant CD45 staining and bound lower levels of FLAER. Conclusion: FLAER combined with multiparameter flow cytometry offers an improved assay for diagnosis and monitoring of PNH clones and may have utility in detection of unsuspected myeloproliferative disorders. © 2007 Clinical Cytometry Society [source] Characterization of a chemokine receptor CCR5-negative T cell line and its use in determining human immunodeficiency virus type 1 phenotypeJOURNAL OF MEDICAL VIROLOGY, Issue 2 2008Dorothea Binninger-Schinzel Abstract A human CD4-positive T cell line from a donor homozygous negative for the chemokine receptor CCR5 was established, characterized, and used for determining the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) isolates. Clones of this IL-2 dependent human T-cell lymphotropic virus type 1 (HTLV-I) immortalized cell line, named IsnoR5 clones 1 and 2, are susceptible to infection by HIV-1 isolates that use CXCR4 as a coreceptor but resistant to infection by CCR5 tropic HIV-1 viruses. HIV-1 isolates whose replication is inhibited in IsnoR5 cells in the presence of the bicyclam AMD 3100, a CXCR4 specific inhibitor, utilize a coreceptor distinct from CCR5 and CXCR4. Using a panel of primary HIV-1 isolates we have shown that a single T cell line is sufficient to discriminate between use of CCR5, CXCR4 or an alternative coreceptor. As IsnoR5 clone 1 cells revealed the existence of even minor populations of CXCR4-using virus variants, they could be useful for the early identification of changes in coreceptor usage in HIV infected individuals facilitating the timely introduction of appropriate clinical treatments. J. Med. Virol. 80:192,200, 2008. © 2007 Wiley-Liss, Inc. [source] | |||||||||||||