Biomedical subjects

Jonni S Moore

Publications and source records attributed to Jonni S Moore.

8 recordsLinked to original sources

PubMed2006

Flow cytometric measurement of circulating endothelial cells: the effect of age and peripheral arterial disease on baseline levels of mature and progenitor populations.

BACKGROUND: Age and cardiovascular disease status appear to alter numbers and function of circulating endothelial progenitor cells (EPCs). Despite no universal phenotypic definition, numerous studies have implicated progenitors with apparent endothelial potential in local responses to vascular injury and with cardiovascular disease in general. To further define the role of this lineage in peripheral artery disease (PAD), we developed a multiparameter flow cytometry assay to analyze multiple phenotypic definitions of progenitor cells (PCs), EPCs, and mature endothelial cells (ECs) and evaluate effects of age and PAD on baseline levels of each subset. METHODS: Blood was collected from young healthy subjects (N = 9, mean age 33 +/- 8 years), older healthy subjects (N = 13, mean age 66 +/- 8 years), and older subjects with PAD (N = 15, mean age 69 +/- 8 years). After ammonium chloride lysis, cells were stained and analyzed on a Becton-Dickinson LSR II with a 5-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3,-CD19,-CD33 (lineage panel), PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion, and only viable, low to medium side scatter lineage-negative singlets were analyzed. In some studies, cells were sorted for morphological studies. Subsets were defined as indicated later. RESULTS: Our results, using a comprehensive flow cytometric panel, indicate that CD133+, CD34+, and CD133+/CD34+ PCs are elevated in younger healthy individuals compared to older individuals, both healthy and with PAD. However, the number of EPCs and mature ECs did not significantly differ among the three groups. Assessment of endothelial colony forming units and dual acLDL-lectin staining supported the flow cytometric findings. CONCLUSIONS: We describe a comprehensive flow cytometric method to detect circulating mature and progenitor endothelial populations confirmed by conventional morphological and functional assays. Our findings suggest that aging may influence circulating levels of PCs, but not EPCs or ECs; PAD had no effect on baseline levels of any populations investigated. This study provides the basis for evaluating the potential effects of acute stress and therapeutic intervention on circulating progenitor and endothelial populations as a biomarker for cardiovascular status.

AC133 Antigen↗

PubMed2006

An alternative spliced RNASEL variant in peripheral blood leukocytes.

2-5A-Dependent RNase L is an endoribonuclease that catalyzes RNA degradation and promotes apoptosis during the innate antiviral response in mammalian cells. Prior studies showed that RNASEL is widely expressed and suggested the presence of mRNA species of different sizes but lacked a characterization of these variants. Using RT-PCR, we show that RNASEL is expressed in all human tissues examined, whereas an alternatively generated spliced variant lacking the third exon (RNASEL del_Ex3) is solely expressed in peripheral blood leukocytes (PBL). Quantitative RT-PCR measurements of RNA from different PBL cell types separated by fluorescence activated cell sorting (FACS) showed that complete RNASEL mRNA levels were significantly elevated in granulocytes compared with all other PBL cell types, whereas expression was lowest in CD8(+) T cells. The alternatively spliced RNASEL del_Ex3 transcript was present in all PBL cell types examined but at lower levels than the full-length RNASEL mRNA. The presence of high levels of RNase L protein in granulocytes was confirmed by immunohistochemistry. Our findings are the first to demonstrate the presence of an alternatively spliced RNASEL mRNA and to demonstrate the variable expression of RNase L in different leukocytes. Our results suggest that RNase L plays an important role in granulocytes as an innate immunity enzyme that controls viral infections.

Alternative Splicing↗

PubMed2005

Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species.

Acute cessation of flow (ischemia) leads to depolarization of the endothelial cell (EC) membrane mediated by K(ATP) channels and followed by production of reactive oxygen species (ROS) from NADPH oxidase. We postulated that ROS are a signal for initiating EC proliferation associated with the loss of shear stress. Flow cytometry was used to identify proliferating CD31-positive pulmonary microvascular endothelial cells (mPMVECs) from wild-type, Kir6.2-/-, and gp91phox-/- mice. mPMVECs were labeled with PKH26 and cultured in artificial capillaries for 72 h at 5 dyn/cm2 (flow adaptation), followed by 24 h of stop flow or continued flow. ROS production during the first hour of ischemia was markedly diminished compared with wild-type mice in both types of gene-targeted mPMVECs. Cell proliferation was defined as the proliferation index (PI). After 72 h of flow, >98% of PKH26-labeled wild-type mPMVECs were at a single peak (PI 1.0) and the proportion of cells in the S+G2/M phases were at 5.8% on the basis of cell cycle analysis. With ischemia (24 h), PI increased to 2.5 and the ratio of cells in S+G2/M phases were at 35%. Catalase, diphenyleneiodonium, and cromakalim markedly inhibited ROS production and cell proliferation in flow-adapted wild-type mPMVECs. Significant effects of ischemia were not observed in Kir6.2-/- and gp91phox-/- cells. ANG II activation of NADPH oxidase was unaffected by KATP gene deletion. Thus loss of shear stress in flow-adapted mPMVECs results in cell division associated with ROS generated by NADPH oxidase. This effect requires a functioning cell membrane KATP channel.

Angiotensin II↗

PubMed2004

Flow cytometric test for beryllium sensitivity.

BACKGROUND: Chronic beryllium disease (CBD) is an occupational granulomatous disorder characterized by hypersensitivity to beryllium, mediated by CD4+ T lymphocytes, and predominantly affects the lungs. In this disorder, lymphocyte proliferative responses to beryllium, measured by 3H thymidine incorporation, are used for diagnosis of CBD, for screening asymptomatic workers or former workers to detect unrecognized disease, and for surveillance as a bioassay to detect abnormal exposures. Problems with test variability and the use of radioactivity have recently led to the search for alternative methods. METHODS: We applied a 5,6-carboxyfluorescein diacetate succinimidyl ester flow cytometric technique for measurement of mitogen- and antigen-induced T-lymphocyte proliferation to a group of beryllium-exposed sensitized individuals and beryllium-unexposed controls. RESULTS: We detected mitogen and antigen proliferative responses in CD3+, CD4+, and CD8+ subpopulations. Phytohemagglutinin and Candida stimulated CD4+ and CD8+ T-cell responses, but beryllium appeared to stimulate only CD3+/CD4+ responses. CONCLUSIONS: This technique may provide a sensitive, nonradioactive alternative to the traditional proliferation tests that measure beryllium sensitivity. It offers the added specificity of enabling phenotypic description of the responding cell type and may prove to be easier to standardize for clinical use.

Air Pollutants, Occupational↗

PubMed2004

Endothelial cell proliferation associated with abrupt reduction in shear stress is dependent on reactive oxygen species.

We have shown previously that flow-adapted endothelial cells respond to cessation of flow with cell membrane depolarization and increased production of reactive oxygen species, resulting in activation of transcription factors and increased DNA synthesis. This study utilized flow cytometry to evaluate cellular proliferation with ischemia and to determine the role of reactive oxygen species and apoptosis. PKH26-labeled rat pulmonary microvascular endothelial cells were seeded in an artificial capillary system and subjected to flow at 5 dynes/cm(2) for 96 h or for 72 h followed by 24 h of simulated "ischemia." Ischemia resulted in a 2.5-fold increase in the cellular proliferation index. Cell-cycle analysis showed G0/G1 arrest and decreased S plus G2/M during flow adaptation, whereas ischemia resulted in a three-fold increase of cells in S plus G2/M phases. Apoptotic cells as detected by TUNEL and annexin V binding assays were ~5% of total cells with no differences between static, flow-adapted, and "ischemic" groups. Reactive oxygen species production during a 1-h period following onset of ischemia was confirmed by oxidation of the fluorophore, dichlorofluorescein, and was inhibited by cromakalim, a K(ATP) channel agonist, or diphenyleneiodonium, a flavoprotein inhibitor. Cromakalim and diphenyleneiodonium also markedly inhibited cell proliferation in the flow-adapted ischemic cells, but had no effect on subconfluent cells cultured under static conditions. These results indicate reactive oxygen species-dependent endothelial cell proliferation in flow-adapted microvascular endothelial cells as a response to ischemia and indicate that this response is not a consequence of apoptosis.

Animals↗

PubMed2002

The role of passenger leukocyte genotype in rejection and acceptance of rat liver allografts.

BACKGROUND: Although graft-resident passenger leukocytes are known to mediate acute rejection by triggering direct allorecognition, they may also act in an immunomodulatory fashion and play an important role in tolerance induction. Our purpose in the current study was to utilize rat bone marrow chimeras to evaluate the role of the genotype of passenger leukocytes in both acute rejection and tolerance of liver allografts. METHODS: The fate of livers bearing donor-type, recipient-type, and third-party passenger leukocytes was evaluated in the MHC class I and II mismatched rejector combination ACI-->LEW and the acceptor combination PVG-->DA. RESULTS: We report that although treatment of ACI liver donors with lethal irradiation does not lead to prolongation of graft survival in the ACI-->LEW strain combination, ACI livers bearing recipient-type (LEW) or third-party passenger leukocytes (BN) are rejected at a significantly slower rate. We confirm that lethal irradiation of PVG donor animals leads to abrogation of tolerance induction with acute rejection of their livers by DA recipients. However, the majority of PVG livers carrying donor-type (PVG), recipient-type (DA), or third-party (LEW) passenger leukocytes are accepted for >100 days. These DA recipients develop immune tolerance to the donor parenchyma (PVG). CONCLUSIONS: Our findings demonstrate that long-term acceptance of liver allografts and tolerance induction is not dependent on the presence of donor-type passenger leukocytes and can be achieved with organs carrying donor-type, recipient-type, or third-party passenger leukocytes. The importance of the MHC framework on the surface of passenger leukocytes as a critical regulator of the immune response after transplantation of chimeric organs is substantiated by the delayed tempo of rejection of ACI livers bearing recipient-type or third-party passenger leukocytes in the ACI-->LEW strain combination.

Animals↗

PubMed2002

CD3 monitoring of antithymocyte globulin therapy in thoracic organ transplantation.

BACKGROUND: Antithymocyte globulin is frequently used as a component of induction therapy in thoracic organ transplantation. This study evaluates the utility of monitoring peripheral CD3 lymphocytes to rationally adjust antithymocyte globulin therapy in this patient population. METHODS: A total of 17 heart and 19 lung transplant recipients received antithymocyte globulin (ATGAM or thymoglobulin) as induction therapy or to treat steroid-resistant acute or chronic rejection. Absolute CD3 counts were maintained between 50 and 100 cells/microl. RESULTS: With CD3 monitoring, the doses of antithymocyte globulin were reduced from 10-15 mg/kg to 1-5 mg/kg during the course of therapy. The total amount of antithymocyte globulin given to each CD3 monitored patient was reduced by 48%. Dose reduction did not alter the number of acute rejection or infectious episodes, and hematological side effects were infrequent. CONCLUSION: CD3 monitoring of antithymocyte globulin therapy in thoracic organ recipients reduced the amount of drug received by each patient, while maintaining CD3 counts less than 100 cells/microl.

Acute Disease↗

PubMed2002

Diminished expression of the type II receptor for TGFbeta (TGFbetaRII) in T lymphocytes from patients with Sezary syndrome is not due to mutations in the receptor's poly-A tract: limitations of the standard RT-PCR in cDNA sequence analysis of homopolymeric base stretches.

Peripheral blood lymphocytes from patients with Sezary syndrome (SzS) frequently demonstrate decreased surface expression of transforming growth factor beta receptor II (TGFbetaRII). The mechanism of this low TGFbetaRII expression remains unknown. Because mutations within the poly-A tract of the TGFbetaRII sequence (nucleotides 709-718) were shown to result in diminished TGFbetaRII expression in other types of malignant tumors, we examined the sequence of the TGFbetaRII poly-A tract in two SzS-derived cell lines and in peripheral blood SzS cells from 17 SzS patients and 4 control, healthy individuals using DNA sequencing and single-stranded conformation polymorphism (SSCP) analysis. A standard bidirectional, automated sequence analysis of the RT-PCR-generated cDNA TGFbetaRII fragment showed a heterogenous population of the normal length, 10-, with admixed, shortened, 9-base poly-A stretches. Surprisingly, this mixture was present not only in the cells from 5 SzS patients and 2 SzS cell lines, but also in cells from 2 healthy control individuals. Importantly, the proportion of the shortened, 9-base fragments was markedly reduced or practically eliminated when the procedure was modified by usage of high-fidelity DNA polymerase, labeled primers and/or cloned RT-PCR products, which indicates that the presence of the shortened, 9-base fragments represented a procedural phenomenon rather than a true deletional mutation within an allele of the TGFbetaRII gene. Accordingly, SSCP analysis of genomic DNA did not reveal any mutations within the poly-A tract-containing region. These results indicate that a mechanism different from mutations in the polyadenine tract underlies the diminished TGFbetaRII expression in SzS cells and that the results of an unmodified, direct sequence analysis of homopolymeric base streaches in RT-PCR-derived cDNA should be interpreted with caution.

Base Sequence↗