Biomedical subjects
Douglas P Olson
Publications and source records attributed to Douglas P Olson.
Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor.
During mammalian ontogeny, haematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, where haematopoiesis occurs throughout adulthood. Unique features of bone that contribute to a microenvironmental niche for stem cells might include the known high concentration of calcium ions at the HSC-enriched endosteal surface. Cells respond to extracellular ionic calcium concentrations through the seven-transmembrane-spanning calcium-sensing receptor (CaR), which we identified as being expressed on HSCs. Here we show that, through the CaR, the simple ionic mineral content of the niche may dictate the preferential localization of adult mammalian haematopoiesis in bone. Antenatal mice deficient in CaR had primitive haematopoietic cells in the circulation and spleen, whereas few were found in bone marrow. CaR-/- HSCs from fetal liver were normal in number, in proliferative and differentiative function, and in migration and homing to the bone marrow. Yet they were highly defective in localizing anatomically to the endosteal niche, behaviour that correlated with defective adhesion to the extracellular matrix protein, collagen I. CaR has a function in retaining HSCs in close physical proximity to the endosteal surface and the regulatory niche components associated with it.
The prognostic significance of P-glycoprotein, multidrug resistance-related protein 1 and lung resistance protein in pediatric acute lymphoblastic leukemia: a retrospective study of 295 newly diagnosed patients by the Children's Oncology Group.
Multidrug resistance (MDR) is a phenomenon by which cells become resistant to an array of structurally unrelated chemotherapeutic agents. The prognostic value that P-glycoprotein (Pgp), multidrug resistance-related protein 1 (MRP1), and lung resistance protein (LRP) have in the setting of pediatric acute lymphoblastic leukemia (ALL) is controversial. In a retrospective study, we analyzed samples obtained from 295 similarly treated pediatric ALL patients to assess whether the overexpression and/or function of these proteins at diagnosis affects outcome. Most patients (70%, 207/295) did not overexpress an MDR protein. A small number of patients expressed functional Pgp (1%, 3/295) and some overexpressed functional MRP1 (10%, 19/295), with a statistically significant number of the latter being of T-lineage as opposed to pre-B (P < 0.001). A small number of patients (2%, 6/295) also overexpressed both Pgp and MRP1. Additional patients expressed increased levels of LRP. Elevated levels of these proteins at diagnosis did not correlate with risk factors and did not predict an adverse prognosis. Life-table estimates and Kaplan-Meier plots did not show any significant differences between patients who overexpressed an MDR protein compared with those who did not, nor was any difference noted when the different MDR + groups were compared with one another. These data strongly support the conclusion that the overexpression of these functional drug efflux pumps at diagnosis does not contribute to treatment failure in pediatric ALL.
Health care delivery in the Texas prison system.
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Differential expression of cell surface antigens on subsets of CD4+ and CD8+ T cells.
BACKGROUND: The assessment of relative antigen density on T cell subsets is a feature of antigen expression that is infrequently characterized. Defining phenotypic differences is a first step in understanding associated differences in function. Additionally, a better understanding of T cell heterogeneity may aid in clinical diagnoses. MATERIAL/METHODS: To further elucidate phenotypic differences of T cell subsets, and begin to determine what information relative antigen density contributes to immunology, we analyzed normal human peripheral blood T cells for a variety of immunophenotypic (CD2, CD3, CD4, CD5, CD7, CD8, CD45RA, CD45RO, TCR alpha beta) and light scatter characteristics using 6 color flow cytometry. T-cell leukemia specimens were also analyzed. RESULTS: Our data show that statistically significant immunophenotypic differences exist between subsets of human CD4 and CD8 T cells. Normal T cells express different levels of relative antigen density for some antigens compared to malignant T cells. CONCLUSIONS: Significant differences are seen in relative antigen density for several cell surface markers between CD4+ and CD8+ T cells. Neither donor source nor flow cytometric calibration account for these differences. The data are applied to specimens from patients with T-lineage acute lymphoblastic leukemia to show how antigen density can be used clinically in aiding to diagnose disease. The data presented here can be used to further investigate these cell populations for functional differences.
Flowing cells through pulsed electric fields efficiently purges stem cell preparations of contaminating myeloma cells while preserving stem cell function.
Autologous stem cell transplantation, in the setting of hematologic malignancies such as lymphoma, improves disease-free survival if the graft has undergone tumor purging. Here we show that flowing hematopoietic cells through pulsed electric fields (PEFs) effectively purges myeloma cells without sacrificing functional stem cells. Electric fields can induce irreversible cell membrane pores in direct relation to cell diameter, an effect we exploit in a flowing system appropriate for clinical scale. Multiple myeloma (MM) cell lines admixed with human bone marrow (BM) or peripheral blood (PB) cells were passed through PEFs at 1.35 kV/cm to 1.4 kV/cm, resulting in 3- to 4-log tumor cell depletion by flow cytometry and 4.5- to 6-log depletion by tumor regrowth cultures. Samples from patients with MM gave similar results by cytometry. Stem cell engraftment into nonobese diabetic-severe combined immunodeficient (NOD/SCID)/beta2m-/- mice was unperturbed by PEFs. Flowing cells through PEFs is a promising technology for rapid tumor cell purging of clinical progenitor cell preparations.
Limited T cell receptor diversity of HCV-specific T cell responses is associated with CTL escape.
Escape mutations are believed to be important contributors to immune evasion by rapidly evolving viruses such as hepatitis C virus (HCV). We show that the majority of HCV-specific cytotoxic T lymphocyte (CTL) responses directed against viral epitopes that escaped immune recognition in HCV-infected chimpanzees displayed a reduced CDR3 amino acid diversity when compared with responses in which no CTL epitope variation was detected during chronic infection or with those associated with protective immunity. Decreased T cell receptor (TCR) CDR3 amino acid diversity in chronic infection could be detected long before the appearance of viral escape mutations in the plasma. In both chronic and resolved infection, identical T cell receptor clonotypes were present in liver and peripheral blood. These findings provide a deeper understanding of the evolution of CTL epitope variations in chronic viral infections and highlight the importance of the generation and maintenance of a diverse TCR repertoire directed against individual epitopes.
Monitoring caspase activity in living cells using fluorescent proteins and flow cytometry.
A molecular probe was developed to monitor caspase activity in living cells by flow cytometry. It consists of CFP and YFP with a peptide linker containing two caspase-cleavage sites (LEVD). Its expression resulted in intense fluorescence resonance energy transfer (FRET), whereas cleavage of this linker by caspases eliminated FRET because of physical separation of the CFP and YFP moieties. Using flow cytometry, cells expressing this probe exhibited two patterns, strong FRET and diminished or absent FRET. The appearance of diminished FRET was inhibited by a pan-caspase inhibitor z-VAD or D->A mutations in the LEVD sequence and was markedly increased by apoptosis-inducing agents, etoposide and camptothecin, or overexpression of a caspase 8-red fluorescent protein fusion protein. Importantly, this probe's ability to monitor caspase activity was comparable with results obtained with fluorogenic substrates or fluorochrome-labeled inhibitors of caspases. Specific caspase inhibitors indicated the probe was highly sensitive to cleavage by caspase 6 and 8, less sensitive to caspase 4, and resistant to other caspases. Activation of caspase 8 by Fas engagement markedly increased the probe's cleavage, whereas treatment of caspase 8-deficient cells with anti-Fas did not increase cleavage. However, staurosporine induced cleavage of the probe in caspase 8-deficient cells by a mechanism that was inhibited by overexpression of bcl-x. Taken together, the data indicate that this caspase-sensitive probe can be used to monitor the basal and apoptosis-related activities of caspases, including an initiator caspase, caspase 8, and effector caspases, such as caspase 6.
CD1d-restricted NKT cells express a chemokine receptor profile indicative of Th1-type inflammatory homing cells.
CD1d-restricted T cells (NKT cells) are innate memory cells activated by lipid Ags and play important roles in the initiation and regulation of the immune response. However, little is known about the trafficking patterns of these cells or the tissue compartment in which they exert their regulatory activity. In this study, we determined the chemokine receptor profile expressed by CD1d-restricted T cells found in the peripheral blood of healthy volunteers as well as CD1d-restricted T cell clones. CD1d-restricted T cells were identified by Abs recognizing the invariant Valpha24 TCR rearrangement or by binding to CD1d-Fc fusion tetramers loaded with alpha-GalCer. CD1d-restricted T cells in the peripheral blood and CD1d-restricted T cell clones expressed high levels of CXCR3, CCR5, and CCR6; intermediate levels of CXCR4 and CXCR6; and low levels of CXCR1, CCR1, CCR2, and CX(3)CR1, a receptor pattern often associated with tissue-infiltrating effector Th1 cells and CD8+ T cells. Very few of these cells expressed the lymphoid-homing receptors CCR7 or CXCR5. CCR4 was expressed predominantly on CD4+, but not on double-negative CD1d-restricted T cells, which may indicate differential trafficking patterns for these two functionally distinct subsets. CD1d-restricted T cell clones responded to chemokine ligands for CXCR1/2, CXCR3, CXCR4, CXCR6, CCR4, and CCR5 in calcium flux and/or chemotaxis assays. These data indicate that CD1d-restricted T cells express a chemokine receptor profile most similar to Th1 inflammatory homing cells and suggest that these cells perform their function in peripheral tissue sites rather than in secondary lymphoid organs.
P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrow hematopoietic stem cells.
Hematopoiesis in mammals undergoes a developmental shift in location from fetal liver to bone marrow accompanied by a gradual transition from highly proliferative to deeply quiescent stem cell populations. P2Y receptors are G-protein-coupled nucleotide receptors participating in vascular and immune responses to injury. We identified a P2Y-like receptor for UDP-conjugated sugars, GPR105 (P2Y14), with restricted expression on primitive cells in the hematopoietic lineage. Anti-GPR105 antibody selectively isolated a subset of hematopoietic cells within the fetal bone marrow, but not in the fetal liver, that was enriched for G0 cell cycle status and for in vitro stem-cell-like multipotential long-term culture capability. Conditioned media from bone marrow stroma induced receptor activation and chemotaxis that was sensitive to G alpha i and anti-receptor antibody inhibition. GPR105 is a G-protein-coupled receptor identifying a quiescent, primitive population of hematopoietic cells restricted to bone marrow. It mediates primitive cell responses to specific hematopoietic microenvironments and extends the known immune system functions of P2Y receptors to the stem cell level. These data suggest a new class of receptors participating in the regulation of the stem cell compartment.
A flow cytometric method to detect protein-protein interaction in living cells by directly visualizing donor fluorophore quenching during CFP-->YFP fluorescence resonance energy transfer (FRET).
BACKGROUND: Protein interactions at the molecular level can be measured by fluorescence resonance energy transfer (FRET) using a pair of fluorescent proteins, such as CFP and YFP, in which the emission spectrum of CFP significantly overlaps the excitation spectrum of YFP. The resulting energy given off from the donor CFP protein can directly excite the acceptor YFP protein when the proteins are closely approximated. During FRET, there is quenching of the emission of the donor CFP protein that is directly related to the efficiency of energy transfer and inversely proportional to the sixth power of the distance between the donor and acceptor proteins. In this study we describe a new approach to visualize donor CFP quenching during CFP-->YFP FRET and demonstrate how this parameter can be used to calculate FRET efficiency. METHODS: A novel flow cytometric method to detect protein-protein interactions in living cells was developed that utilized assessment of CFP donor quenching during CFP-->YFP FRET by comparing CFP intensity between FRET-positive and -negative populations. To accomplish this, we compared the CFP intensity in FRET-positive and FRET-negative cells within the same population transfected with a CFP/YFP fusion protein, in which the molar ratio of CFP:YFP was one. By using separate lasers to excite CFP and YFP, the detection of FRET was separated from that of YFP. Therefore, after direct excitation, the YFP emission spectrum remained constant in all transfected cells, whereas the emission spectrum of CFP varied with the extent of FRET in individual cells. Specific CFP/YFP fusion constructs were prepared to evaluate this approach. The first one consisted of CFP and YFP separated by two caspase cleavage sites (CFP-LEVD-YFP). A second construct consisted of CFP and YFP separated by a structurally restricted 232-amino acid (aa) spacer. No FRET was observed by transfectants expressing this construct. RESULTS: Transfection of CFP-LEVD-YFP into Hela cells resulted in a FRET-positive population and a FRET-negative one. The appearance of the FRET-negative population was inhibited by the caspase inhibitor z-VAD. Moreover, substituting D for A in the caspase cleavage sites of this probe abolished the FRET-negative population, demonstrating the probe's specificity for caspase activity. Comparison of the CFP emission in the FRET-positive and FRET-negative population was used to document the relationship of FRET to donor quenching and permit the calculation of FRET efficiency and relative molecular distance between CFP and YFP. Similar results were noted when cells transfected with the caspase-sensitive probe (in the presence of z-VAD) were mixed with cells expressing the CFP-YFP construct with the 232-aa spacer and therefore were FRET negative. This demonstrated the validity of calculating CFP donor quenching and FRET efficiency by comparing emission spectra of an unknown construct with that of a known positive control, both expressed by the same population of cells. Using this approach, we confirmed that members of the TNF receptor-associated factor (TRAF) family engaged in both homotypic and heterotypic interactions. CONCLUSIONS: We have established a novel flow cytometric approach to assess donor CFP quenching during CFP-->YFP FRET, which can be used for the calculation of FRET efficiency and relative biological molecular distance between CFP and YFP moieties. This method can be used not only to analyze cells that express a CFP and YFP fusion protein, but also independent CFP-coupled and YFP-coupled interacting proteins.
The protease inhibitor ritonavir inhibits the functional activity of the multidrug resistance related-protein 1 (MRP-1).
BACKGROUND: Efflux pumps situated on the plasma membrane, such as P-glycoprotein (Pgp) and the multidrug resistance related-protein 1 (MRP-1), have been shown to extrude HIV protease inhibitors from the cell. MRP-1 is present on many barrier sites throughout the body, such as the blood-brain and blood-testis interfaces and could reduce the concentration of protease inhibitors in these sanctuary sites for HIV-1 replication. Factors that modulate efflux pump function in vivo are poorly defined. OBJECTIVE: To analyze the inhibitory potential of the anti-retroviral drugs indinavir, amprenavir, ritonavir, lamivudine or zidovudine to modulate MRP-1 function. METHODS: Effect of anti-HIV drugs on the efflux pump activity of MRP-1 was evaluated in the presence of increasing concentrations of human plasma, using UMCC-1/VP cells which stably over-express MRP-1. MRP-1 activity was abrogated by probenecid. The potential of blocking MRP-1 function for an extended (3 day) time period, was also examined in MRP-1 over-expressing cells cultured with either probenecid or the anti-retroviral drugs and a cytotoxic compound (etoposide) that is transported by MRP-1. RESULTS: Ritonavir inhibited the functional activity of MRP-1 similarly to probenecid, as demonstrated by re-sensitization of MRP-1 over-expressing cells to cytotoxic effects of etoposide. Inhibition by ritonavir was inversely related to the concentration of human plasma added to the cells (r2 = 0.89). Other anti-HIV drugs didn't affect the MRP-1 mediated efflux of etoposide. CONCLUSIONS: These data may be exploitable to further improve sanctuary site concentrations of anti-HIV or anti-cancer drugs by using ritonavir as a lead compound to develop more potent MRP-1 inhibitors.
Receptor tyrosine kinase, EphB4 (HTK), accelerates differentiation of select human hematopoietic cells.
EphB4 (HTK) and its ligand, ephrinB2, are critical for angiogenesis and result in fatal abnormalities of capillary formation in null mice. EphB4 was originally identified in human bone marrow CD34(+) cells by us and has since been reported to be expressed in erythroid progenitors, whereas the ligand ephrinB2 is expressed in bone marrow stromal cells. Reasoning that the developmental relationship between angiogenesis and hematopoiesis implies common regulatory molecules, we assessed whether EphB4 signaling influences the function and phenotype of primitive human hematopoietic cells. Ectopically expressed EphB4 in cell lines of restricted differentiation potential promoted megakaryocytic differentiation, but not granulocytic or monocytic differentiation. Primary cord blood CD34(+) cells transduced with EphB4 resulted in the elevated expression of megakaryocytic and erythroid specific markers, consistent with EphB4 selectively enhancing some lineage-committed progenitors. In less mature cells, EphB4 depleted primitive cells, as measured by long-term culture-initiating cells or CD34(+)CD38(-) cell numbers, and increased progenitor cells of multiple cell types. Effects of ectopic EphB4 expression could be abrogated by either targeted mutations of select tyrosine residues or by the tyrosine kinase inhibitor, genistein. These data indicate that EphB4 accelerates the differentiation of primitive cells in a nonlineage-restricted manner but alters only select progenitor populations, influencing lineages linked by common ancestry with endothelial cells. EphB4 enforces preferential megakaryocytic and erythroid differentiation and may be a molecular bridge between angiogenesis and hematopoiesis.
Thymocyte emigration is mediated by active movement away from stroma-derived factors.
T cells leave the thymus at a specific time during differentiation and do not return despite elaboration of known T cell chemoattractants by thymic stroma. We observed differentiation stage-restricted egress of thymocytes from an artificial thymus in which vascular structures or hemodynamics could not have been playing a role. Hypothesizing that active movement of cells away from a thymic product may be responsible, we demonstrated selective reduction in emigration from primary thymus by inhibitors of active movement down a concentration gradient (chemofugetaxis). Immature intrathymic precursors were insensitive to an emigration signal, whereas mature thymocytes and peripheral blood T cells were sensitive. Thymic stroma was noted to elaborate at least two proteins capable of inducing emigration, one of which was stromal cell-derived factor-1. Thymic emigration is mediated, at least in part, by specific fugetaxis-inducing factors to which only mature cells respond.