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J Philip McCoy

Publications and source records attributed to J Philip McCoy.

At least 19 recordsLinked to original sources
PubMed2006

Age-dependent accumulation of mtDNA mutations in murine hematopoietic stem cells is modulated by the nuclear genetic background.

Alterations in mitochondrial DNA (mtDNA) and consequent loss of mitochondrial function underlie the mitochondrial theory of aging. In this study, we systematically analyzed the mtDNA control region somatic mutation pattern in 2864 single hematopoietic stem cells (HSCs) and progenitors, isolated by flow cytometry sorting on Lin(-)Kit(+)CD34(-) parameters from young and old C57BL/6 (B6) and BALB/cBy (BALB) mice, to test the hypothesis that the accumulated mtDNA mutations in HSCs were strain-correlated and associated with HSC functional senescence during aging. An increased level of mtDNA mutations in single HSCs was observed in old B6 when compared with young B6 mice (P=0.003); in contrast, no significant age-dependent accumulation of mutations was observed in BALB mice (old versus young, P=0.202) and the level of mutations in both young and old BALB mice was close to that of old B6 mice (P>0.280). Cellular reactive oxygen species (ROS) in mouse HSCs could not be correlated with the level of mtDNA mutations in these cells, although B6 mice had a higher proportion of ROS(-) cells when compared with the BALB mice. Propagation assays of single HSCs showed B6 cells form larger colonies compared with cells from BALB mice, irrespective of age and mtDNA mutation load. We infer from our data that age-related mtDNA somatic mutation accumulation in mouse HSCs is influenced by the nuclear genetic background and that these mutations may not obviously correlate to either cellular ROS content or HSC senescence.

Aging↗
PubMed2006

Reduction of GVHD and enhanced antitumor effects after adoptive infusion of alloreactive Ly49-mismatched NK cells from MHC-matched donors.

We investigated if an infusion of alloreactive natural killer (NK) cells would reduce GVHD and mediate antitumor effects in mice undergoing MHC-matched allogeneic stem cell transplantation (SCT). Balb/c mice bearing RENCA tumors underwent an allogeneic SCT from MHC-matched B10.d2 donors and were given a single infusion of either Ly49 ligand-matched, ligand-mismatched, or no donor NK cells. Recipients of Ly49 ligand-mismatched NK cells had a reduced incidence of graft-versus-host disease (GVHD; 39% vs 100%; P < .01), and prolonged survival (median 84 days vs 39 days; P < .01) compared with SCT recipients not receiving NK cells. Recipients of Ly49 ligand-matched NK cells had the same incidence of GVHD and similar survival compared with controls not receiving NK cells. Pulmonary tumor burden was significantly (P < .01) lower in recipients that received Ly49-mismatched or Ly49-matched NK cells compared with recipients not receiving NK cells. These data provide in vivo evidence that a single infusion of alloreactive donor NK cells reduces GVHD and mediates antitumor effects following MHC-matched allogeneic transplantation.

Animals↗
PubMed2006

Targeted disruption of Kaposi's sarcoma-associated herpesvirus ORF57 in the viral genome is detrimental for the expression of ORF59, K8alpha, and K8.1 and the production of infectious virus.

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 regulates viral gene expression at the posttranscriptional level during viral lytic infection. To study its function in the context of the viral genome, we disrupted KSHV ORF57 in the KSHV genome by transposon-based mutagenesis. The insertion of the transposon into the ORF57 exon 2 region also interrupted the 3' untranslated region of KSHV ORF56, which overlaps with the ORF57 coding region. The disrupted viral genome, Bac36-Delta57, did not express ORF57, ORF59, K8alpha, K8.1, or a higher level of polyadenylated nuclear RNA after butyrate induction and could not be induced to produce infectious viruses in the presence of valproic acid, a histone deacetylase inhibitor and a novel KSHV lytic cycle inducer. The ectopic expression of ORF57 partially complemented the replication deficiency of the disrupted KSHV genome and the expression of the lytic gene ORF59. The induced production of infectious virus particles from the disrupted KSHV genome was also substantially restored by the simultaneous expression of both ORF57 and ORF56; complementation by ORF57 alone only partially restored the production of virus, and expression of ORF56 alone showed no effect. Altogether, our data indicate that in the context of the viral genome, KSHV ORF57 is essential for ORF59, K8alpha, and K8.1 expression and infectious virus production.

Cell Line↗
PubMed2006

Mitochondrial DNA sequence variation in single cells from leukemia patients.

A high frequency of mtDNA somatic mutation has been observed in many tumors as well as in aging tissues. In this study, we analyzed the mtDNA control region sequence variation in 3534 single normal cells and individual blasts from 18 patients with leukemia and 10 healthy donors, to address the mutation process in leukemic cells. We found significant differences in mtDNA sequence, as represented by the number of haplotypes and the mean number of cells with each nonaggregate haplotype in a population of cells, in patients compared to controls. Patients with similar clinical leukemia types, particularly acute myeloid leukemia (AML), did not show a uniform pattern of sequence variation in single blasts. Some patients at relapse presented a complex shift of major haplotypes in single cells. Four patients showed high frequencies of cells containing mutations 189, 260, 16150, and 16488, respectively, as a result of clonal expansion and could be considered as potential markers for their respective disease progression. To our knowledge, this is the first large-scale study of mtDNA variation in single malignant cells. Our results suggest that the somatic mutation process in leukemia is complex, leading to diverse levels of genetic alterations due to either intrinsic aspects of leukemia pathophysiology or chemotherapy effects.

Adolescent↗
PubMed2006

The mammalian target of rapamycin (mTOR) pathway regulates mitochondrial oxygen consumption and oxidative capacity.

Metabolic rate and the subsequent production of reactive oxygen species are thought to contribute to the rate of aging in a wide range of species. The target of rapamycin (TOR) is a well conserved serine/threonine kinase that regulates cell growth in response to nutrient status. Here we demonstrate that in mammalian cells the mammalian TOR (mTOR) pathway plays a significant role in determining both resting oxygen consumption and oxidative capacity. In particular, we demonstrate that the level of complex formation between mTOR and one of its known protein partners, raptor, correlated with overall mitochondrial activity. Disruption of this complex following treatment with the mTOR pharmacological inhibitor rapamycin lowered mitochondrial membrane potential, oxygen consumption, and ATP synthetic capacity. Subcellular fractionation revealed that mTOR as well as mTOR-raptor complexes can be purified in the mitochondrial fraction. Using two-dimensional difference gel electrophoresis, we further demonstrated that inhibiting mTOR with rapamycin resulted in a dramatic alteration in the mitochondrial phosphoproteome. RNA interference-mediated knockdown of TSC2, p70 S6 kinase (S6K1), raptor, or rictor demonstrates that mTOR regulates mitochondrial activity independently of its previously identified cellular targets. Finally we demonstrate that mTOR activity may play an important role in determining the relative balance between mitochondrial and non-mitochondrial sources of ATP generation. These results may provide insight into recent observations linking the TOR pathway to life span regulation of lower organisms.

Adenosine Triphosphate↗
PubMed2006

Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA.

The measurement of soluble cytokines and other analytes in serum and plasma is becoming increasingly important in the study and management of many diseases. As a result, there is a growing demand for rapid, precise, and cost-effective measurement of such analytes in both clinical and research laboratories. Multiplex bead array assays provide quantitative measurement of large numbers of analytes using an automated 96-well plate format. Enzyme-linked immunosorbent assay (ELISAs) have long been the standard for quantitative analysis of cytokines and other biomarkers, but are not well suited for high throughput multiplex analyses. However, prior to replacement of ELISA assays with multiplex bead array assays, there is a need to know how comparable these two methods are for quantitative analyses. A number of published studies have compared these two methods and it is apparent that certain elements of these assays, such as the clones of monoclonal antibodies used for detection and reporting, are pivotal in obtaining similar results from both assays. By careful consideration of these variables, it should be possible to utilize multiplex bead array assays in lieu of ELISAs for studies requiring high throughput analysis of numerous analytes.

Antibodies, Monoclonal↗
PubMed2006

Multilineage involvement of the fusion gene in patients with FIP1L1/PDGFRA-positive hypereosinophilic syndrome.

Myeloproliferative hypereosinophilic syndrome (MHES) is a disorder characterised by male predominance, marked eosinophilia, splenomegaly, tissue fibrosis, elevated serum tryptase and the presence of the FIP1L1/PDGFRA fusion gene in peripheral blood mononuclear cells. The characteristic hypercellular bone marrow with dysplastic eosinophils and spindle-shaped mast cells suggest that multiple lineages may be involved in the clonal process. To determine which haematopoietic lineages are involved in MHES, we purified cells of specific lineages from patients with MHES and used nested reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR and fluorescence in situ hybridisation to analyse the purified cell populations for the presence of the fusion gene. The fusion gene was detected in eosinophils, neutrophils, mast cells, T cells, B cells and monocytes. These results suggest that the mutation arises in a pluripotential haematopoietic progenitor cell capable of giving rise to multiple lineages. The basis for the preferential expansion of eosinophils and mast cells remains unclear.

Antigens, CD↗
PubMed2006

Overcoming graft rejection in heavily transfused and allo-immunised patients with bone marrow failure syndromes using fludarabine-based haematopoietic cell transplantation.

Allogeneic haematopoietic cell transplantation (HCT) can cure a variety of non-malignant haematological disorders. Although transplant outcomes for selected patients with severe aplastic anaemia (SAA) and paroxysmal nocturnal haemoglobinuria (PNH) have improved, older age, allo-immunisation from transfusions, prior immunosuppressive therapy and a prolonged time from diagnosis to transplantation are associated with worse outcome. Because of its potent immunosuppressive effects, we investigated a fludarabine-based non-myeloablative conditioning regimen in patients with transfusion-dependent non-malignant haematological disorders at increased risk for graft rejection with conventional transplant conditioning. Twenty-six patients with transfusion dependent/anti-thymocyte globulin (ATG)-refractory SAA, PNH or pure red cell aplasia underwent HCT from a human leucocyte antigen (HLA)-compatible relative. Transplant conditioning consisted of cyclophosphamide (120 mg/kg) and fludarabine (125 mg/m2) with or without ATG. Ciclosporine, alone or combined with mycophenolate mofetil or methotrexate, was used as graft-versus-host disease (GVHD) prophylaxis. All patients achieved durable engraftment and transfusion-independence. Twenty-four of 26 patients are alive at a median of 21 months following transplantation. Although a high cumulative incidence of acute (65% grades II-IV, 54% grades III-IV) and chronic GVHD (56%) was observed, only one patient died from transplant-related causes (cumulative incidence 7%). These data show that HCT following fludarabine-based non-myeloablative conditioning results in durable engraftment and excellent survival in SAA and PNH patients at high risk for graft rejection.

Adolescent↗
PubMed2006

The E7 oncoprotein is translated from spliced E6*I transcripts in high-risk human papillomavirus type 16- or type 18-positive cervical cancer cell lines via translation reinitiation.

High-risk human papillomaviruses (HPVs) encode two viral oncoproteins, E6 and E7, from a single bicistronic pre-mRNA containing three exons and two introns. Retention of intron 1 in the E6 coding region is essential for production of the full-length E6 oncoprotein. However, splicing of intron 1 is extremely efficient in cervical cancer cells, leading to the production of a spliced transcript, E6*I, of E6. Here, we investigated whether this splicing of intron 1 might benefit E7 production. Using RNA interference as a tool, we targeted the intron 1 region using small interfering RNAs (siRNAs) in HPV-positive cell lines. At an effective low dose, the siRNAs specifically suppressed E6 expression but not E7 expression, as demonstrated by the stabilization of p53. However, at high doses the HPV18 intron 1-specific siRNA substantially and specifically reduced the level of the 18E6*I mRNA lacking the intron region in HeLa cells, implying its nuclear silencing on the pre-mRNA before RNA splicing. Two other siRNAs targeting the exon 2 regions of HPV16 and -18, which encode the E7 oncoprotein, reduced the E6*I mRNAs to a remarkable extent and preferentially suppressed expression of E7, leading to accumulation of hypophosphorylated p105Rb and cell cycle arrest, indicating that the majority of E7 proteins are the translational products of E6*I mRNAs. This was confirmed by transient transfection in 293 cells: E7 could be translated only from the E7 open reading frame (ORF) on E6*I mRNA in a distance-dependent matter of upstream E6*I ORF by translation reinitiation. The data thus provide direct evidence that the E6*I mRNAs of high-risk HPVs are responsible for E7 production.

Cell Line↗
PubMed2005

cGMP-independent nitric oxide signaling and regulation of the cell cycle.

BACKGROUND: Regulatory functions of nitric oxide (NO*) that bypass the second messenger cGMP are incompletely understood. Here, cGMP-independent effects of NO* on gene expression were globally examined in U937 cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. Differentiated U937 cells (>80% in G0/G1) were exposed to S-nitrosoglutathione, a NO* donor, or glutathione alone (control) for 6 h without or with dibutyryl-cAMP (Bt2cAMP), and then harvested to extract total RNA for microarray analysis. Bt2cAMP was used to block signaling attributable to NO*-induced decreases in cAMP. RESULTS: NO* regulated 110 transcripts that annotated disproportionately to the cell cycle and cell proliferation (47/110, 43%) and more frequently than expected contained AU-rich, post-transcriptional regulatory elements (ARE). Bt2cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO*, Bt2cAMP was associated with ARE-containing transcripts. A comparison of NO* and Bt2cAMP effects showed that NO* regulation of cell cycle genes was independent of its ability to interfere with cAMP signaling. Cell cycle genes induced by NO* annotated to G1/S (7/8) and included E2F1 and p21/Waf1/Cip1; 6 of these 7 were E2F target genes involved in G1/S transition. Repressed genes were G2/M associated (24/27); 8 of 27 were known targets of p21. E2F1 mRNA and protein were increased by NO*, as was E2F1 binding to E2F promoter elements. NO* activated p38 MAPK, stabilizing p21 mRNA (an ARE-containing transcript) and increasing p21 protein; this increased protein binding to CDE/CHR promoter sites of p21 target genes, repressing key G2/M phase genes, and increasing the proportion of cells in G2/M. CONCLUSION: NO* coordinates a highly integrated program of cell cycle arrest that regulates a large number of genes, but does not require signaling through cGMP. In humans, antiproliferative effects of NO* may rely substantially on cGMP-independent mechanisms. Stress kinase signaling and alterations in mRNA stability appear to be major pathways by which NO* regulates the transcriptome.

Blotting, Western↗
PubMed2005

Hematopoietic-specific microRNA expression in human cells.

We examined expression profiles of hematopoietic tissue-specific microRNAs (miRNAs; miR-142, miR-155, miR-181 and miR-223) in 17 commercially available malignant hematopoietic cell lines and compared to those in highly purified normal human B, T, monocytic and granulocytic lineages. Although malignant cell lines examined showed miRNA expression patterns similar to normal human hematopoietic lineages, the levels of miRNA expression among cell lines and normal cell lineages were considerably different, indicating the significance of miRNAs in human hematopoietic diseases. Further our results showed differences in miRNA expression between mouse and human hematopoietic cells, suggesting important regulatory roles of miRNAs in human hematopoiesis and oncogenesis.

Animals↗
PubMed2005

Uncoupling proteins 2 and 3 function in concert to augment tolerance to cardiac ischemia.

Transient cardiac ischemia activates cell survival signaling, conferring subsequent ischemia tolerance to the heart. This biological phenomenon, termed ischemic preconditioning, results in improved clinical outcome and attenuated infarct size following myocardial infarction. To explore genomic modifications underpinning this ischemia tolerance, we delineated the regulation and function of the cardiac enriched mitochondrial uncoupling proteins 2 and 3 during delayed ischemic preconditioning in the rat. Cardiac transcripts of genes encoding uncoupling proteins 2 and 3 are up-regulated in parallel with infarct size reduction in preconditioned hearts. Mitochondria isolated from preconditioned hearts exhibit an augmented inducible proton leak. In parallel, following anoxia-reoxygenation these mitochondria generate less hydrogen peroxide compared with non-preconditioned mitochondria. Preconditioning in rat cardiac derived myoblasts is abolished following uncoupling protein-2 depletion by RNA-interference. RNAi of uncoupling protein-3 partially attenuates the capacity to precondition these cells. Functional characterization of anoxia and reoxygenation tolerance following uncoupling protein 2 or 3 and combined 2 and 3 RNAi shows the largest reduction in viability follows depletion of both homologues. Uncoupling protein-2 depletion alone significantly attenuates anoxia-reoxygenation tolerance but uncoupling protein-3 depletion does not reduce anoxia tolerance. In parallel combined uncoupling protein depletion and isolated uncoupling protein-2 depletion augments ROS production in viable cardiomyocytes following anoxia-reoxygenation. Concurrent anti-oxidant administration ameliorates the uncoupling protein-depleted anoxia-susceptible phenotype. In conclusion, mitochondrial uncoupling proteins are necessary components of ischemia tolerance and function as components of the cellular antioxidant defense program. In the cytoprotective hierarchy, uncoupling protein-2 appears to play a greater role than uncoupling protein-3 in modulating ischemia/anoxia tolerance in heart-derived cells.

Animals↗
PubMed2005

Mitochondrial DNA spectra of single human CD34+ cells, T cells, B cells, and granulocytes.

Previously, we described the age-dependent accumulation of mitochondrial DNA (mtDNA) mutations, leading to a high degree of mtDNA heterogeneity among normal marrow and blood CD34+ clones and in granulocytes. We established a method for sequence analysis of single cells. We show marked, distinct mtDNA heterogeneity from corresponding aggregate sequences in isolated cells of 5 healthy adult donors-37.9% +/- 3.6% heterogeneity in circulating CD34+ cells, 36.4% +/- 14.1% in T cells, 36.0% +/- 10.7% in B cells, and 47.7% +/- 7.4% in granulocytes. Most heterogeneity was caused by poly-C tract variability; however, base substitutions were also prevalent, as follows: 14.7% +/- 5.7% in CD34+ cells, 15.2% +/- 9.0% in T cells, 15.4% +/- 6.7% in B cells, and 32.3% +/- 2.4% in granulocytes. Many poly-C tract length differences and specific point mutations seen in these same donors but assayed 2 years earlier were still present in the new CD34+ samples. Additionally, specific poly-C tract differences and point mutations were frequently shared among cells of the lymphoid and myeloid lineages. Secular stability and lineage sharing of mtDNA sequence variability suggest that mutations arise in the lymphohematopoietic stem cell compartment and that these changes may be used as a natural genetic marker to estimate the number of active stem cells.

Adult↗
PubMed2005

Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry.

The finding of angiogenic and vasculogenic cells in the peripheral circulation may have profound effects on the course of a variety of diseases ranging from cancer to cardiovascular disease. These cells are ascribed to be endothelial in nature and are generally referred to as circulating endothelial cells if mature or as endothelial progenitor cells if immature. Different approaches have been used to detect these cells, including in vitro culture, magnetic bead isolation, and flow cytometry. We review flow cytometric methods for the detection and enumeration of these cells and provide technical suggestions to promote the accurate enumeration of circulating endothelial cells and endothelial progenitor cells.

Antigens, CD↗
PubMed2005

c-Kit expression and stem cell factor-induced hematopoietic cell proliferation are up-regulated in aged B6D2F1 mice.

Expression of c-Kit (CD117) and stem cell factor/c-Kit-mediated cell proliferation were tested in vitro in young and old B6D2F1 mice to study the role of c-Kit signaling in hematopoietic stem cell (HSC) senescence. Increasing age is associated with a significant increase in bone marrow (BM) cells without affecting mature blood cells. The number of c-Kit-expressing BM cells increased significantly in old mice when compared to young controls, to 201% in total BM cells, 261% in Lin(-) cells, 517% in Lin(-)CD34(+)Sca1(+) progenitor cells, and 1272% in Lin(-)CD34(-)Sca1(+) HSCs. Sorted Lin(-)Sca1(+)CD117(+) BM cells from an old mouse expanded 5-fold when cultured in vitro for 72 hours with stem cell factor at 25 ng/ml, which was significantly higher than a 2.5-fold expansion of the same cells from a young donor. HSCs and progenitor cells from B6D2F1 mice maintain extremely high proliferative potentials and do not reach proliferative arrest at old age during a normal life span.

Aging↗
PubMed2004

Molecular and genetic analysis of disseminated neoplastic cells in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a multisystem disorder of women, characterized by cystic degeneration of the lungs, renal angiomyolipomas (AML), and lymphatic abnormalities. LAM lesions result from the proliferation of benign-appearing, smooth muscle-like LAM cells, which are characterized by loss of heterozygosity (LOH) of one of the tuberous sclerosis complex (TSC) genes. LAM cells are believed to migrate among the involved organs. Because of the apparently metastatic behavior of LAM, we tried to isolate LAM cells from body fluids. A cell fraction separated by density gradient centrifugation from blood had TSC2 LOH in 33 of 60 (55%) LAM patients. Cells with TSC2 LOH were also found in urine from 11 of 14 (79%) patients with AML and in chylous fluid from 1 of 3 (33%) patients. Identification of LAM cells with TSC2 LOH in body fluids was not correlated with severity of lung disease or extrapulmonary involvement and was found in one patient after double lung transplantation. These studies are compatible with a multisite origin for LAM cells. They establish the existence of disseminated, potentially metastatic LAM cells through a relatively simple, noninvasive procedure that should be valuable for molecular and genetic studies of somatic mutations in LAM and perhaps other metastatic diseases.

Body Fluids↗
PubMed2004

Granulocyte colony-stimulating factor mobilizes functional endothelial progenitor cells in patients with coronary artery disease.

OBJECTIVE: Endothelial progenitor cells (EPCs) that may repair vascular injury are reduced in patients with coronary artery disease (CAD). We reasoned that EPC number and function may be increased by granulocyte colony-stimulating factor (G-CSF) used to mobilize hematopoietic progenitor cells in healthy donors. METHODS AND RESULTS: Sixteen CAD patients had reduced CD34(+)/CD133(+) (0.0224+/-0.0063% versus 0.121+/-0.038% mononuclear cells [MNCs], P<0.01) and CD133(+)/VEGFR-2(+) cells, consistent with EPC phenotype (0.00033+/-0.00015% versus 0.0017+/-0.0006% MNCs, P<0.01), compared with 7 healthy controls. Patients also had fewer clusters of cells in culture, with out-growth consistent with mature endothelial phenotype (2+/-1/well) compared with 16 healthy subjects at high risk (13+/-4/well, P<0.05) or 14 at low risk (22+/-3/well, P<0.001) for CAD. G-CSF 10 microg/kg per day for 5 days increased CD34(+)/CD133(+) cells from 0.5+/-0.2/microL to 59.5+/-10.6/microL and CD133(+)/ VEGFR-2(+) cells from 0.007+/-0.004/microL to 1.9+/-0.6/microL (both P<0.001). Also increased were CD133(+) cells that coexpressed the homing receptor CXCR4 (30.4+/-8.3/microL, P<0.05). Endothelial cell-forming clusters in 10 patients increased to 27+/-9/well after treatment (P<0.05), with a decline to 9+/-4/well at 2 weeks (P=0.06). CONCLUSIONS: Despite reduced EPCs compared with healthy controls, patients with CAD respond to G-CSF with increases in EPC number and homing receptor expression in the circulation and endothelial out-growth in culture. Endothelial progenitor cells (EPCs) are reduced in coronary artery disease. Granulocyte colony-stimulating factor (CSF) administered to patients increased: (1) CD133+/VEGFR-2+ cells consistent with EPC phenotype; (2) CD133+ cells coexpressing the chemokine receptor CXCR4, important for homing of EPCs to ischemic tissue; and (3) endothelial cell-forming clusters in culture. Whether EPCs mobilized into the circulation will be useful for the purpose of initiating vascular growth and myocyte repair in coronary artery disease patients must be tested in clinical trials.

AC133 Antigen↗