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Biomedical subjects

D H Gillespie

Publications and source records attributed to D H Gillespie.

At least 19 recordsLinked to original sources
PubMed1995

IgE-dependent expression of interleukin-5 mRNA and protein in human lung: modulation by dexamethasone.

mRNA and protein expression of the Th2 cytokines IL-4 and IL-5 from human lung were examined during the first 4 hr following IgE-mediated triggering, a time representative of the evolving late-phase reaction (LPR). Lung explants were incubated for 16 hr at 37 degrees C in culture media alone or with added dexamethasone (10(-6) M), washed, and then challenged with buffer or anti-IgE (3 micrograms/ml). Using RNase protection assays, in 16/16 individual lungs IL-5 mRNA expression was observed at 4 hr following anti-IgE and at no points following buffer challenge. Fragments released 1129 +/- 499 ng of IL-5/g wet wt over a 24-hr period (mean +/- SEM, n = 5). Neither IL-4 transcripts nor protein were detected in any anti-IgE challenges. Both the IgE-mediated IL-5 mRNA and protein responses were below the limits of detection following dexamethasone preincubation, suggesting a mechanism for the potent inhibitory effects of these agents observed in the LPR.

Dexamethasone↗
PubMed1992

Interleukin-1 alpha mediates an alternative pathway for the antiproliferative action of poly(I.C) on human endothelial cells.

The antiproliferative effect of double-stranded RNA (dsRNA) on human tumor and normal cells has been well established. However, the genes involved in the dsRNA-induced antiproliferative response and the molecular mechanisms by which this occurs remain less well defined. We have studied the ability of synthetic dsRNA, polyinosinic:polycytidylic acid (poly(I.C)) to modify human umbilical vein endothelial cell (HUVEC) growth and report that poly(I.C) induces a dose-dependent inhibition of HUVEC proliferation in vitro. In addition, the mRNA levels for the cytokines interleukin-1 alpha (IL-1 alpha) and interferon-beta 1 are induced in poly(I.C)-treated cells. Moreover, the growth inhibitory effects of poly(I.C) are relieved when cells are grown in the presence of an IL-1 alpha antisense oligonucleotide to the human IL-1 alpha transcript. Thus, the effects of poly(I.C) appear to be mediated, in part, through the function of IL-1 alpha, suggesting an alternative pathway for dsRNA-mediated inhibition of human endothelial cell growth.

Base Sequence↗
PubMed1992

Cellular and enzymatic activities of a synthetic heteropolymer double-stranded RNA of defined size.

We have synthesized a novel heteropolymer double-stranded RNA (dsRNA) molecule of defined length and strandedness (dsRNA309) and evaluated its ability to induce cytokine gene expression, activate dsRNA-dependent enzymes, and inhibit both tumor cell growth and virus replication. Unlike the conventionally studied synthetic homopolymer dsRNAs, polyinosinic acid:polycytidylic acid (poly(I-C)) and its mismatched analogue polyinosinic:polycytidylic, uridylic acid (poly(I-C12,U), dsRNA309 possessed restricted biological activity. dsRNA309 was unable to inhibit tumor cell growth or efficiently induce cytokine (i.e. interferon-beta and interleukin-1 alpha) gene expression. However, dsRNA309 was able to inhibit virus replication and activate dsRNA-dependent intracellular enzymes, 2'-5' oligoadenylate synthetase (2'-5' A synthetase) and the dsRNA-activated inhibitor kinase in in vitro assay systems. Overall, dsRNA309 provided a means for examining the mechanisms governing the dsRNA-regulated antiviral and antiproliferative responses, and studies with dsRNA309 demonstrated that the ability of a synthetic dsRNA to activate dsRNA-dependent intracellular enzymes does not necessarily predict the same gene inducing capacity.

2',5'-Oligoadenylate Synthetase↗
PubMed1992

RNA abundance measured by a lysate RNase protection assay.

We describe a sensitive ribonuclease protection assay that we have used to measure the amount of interferon-beta RNA directly in lysates of human cells. Cell lysates were prepared in concentrated guanidine thiocyanate. Molecular hybridization with RNA probes was then performed directly in crude cell lysate, and native RNase-resistant duplexes were characterized by polyacrylamide gel electrophoresis. Comparison of interferon-beta RNA abundance by quantitative solution hybridization and lysate RNase protection showed that lysate RNase protection was highly quantitative. A high degree of reproducibility of the method was determined with a glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene probe. Sensitivity of lysate RNase protection was determined using both induced interferon-beta RNA and synthetic human endogenous reverse transcriptase RNA as target. The lysate RNase protection method was able to measure as few as 10(4)-10(5) RNA molecules.

Genetic Techniques↗
PubMed1991

Cellular response to double-stranded RNA.

The study of double-stranded RNA (dsRNA) encompasses a variety of fields. Basic research in this area has contributed to a greater mechanistic understanding of gene induction, tumor cell growth arrest, the establishment of antiviral states, and immunomodulation. Because of the possible clinical value of these molecules, physicians are now exploring the use of synthetic dsRNA to treat patients with cancer, HIV-1 disease, and immune dysfunction. Continued studies of the mechanisms of action of dsRNA are likely to suggest an even wider scope of clinical applications.

Animals↗
PubMed1991

Mismatched double-stranded RNA, Ampligen (poly(I): poly(C12U), demonstrates antiviral and immunostimulatory activities in HIV disease.

Mismatched double-stranded RNA (Ampligen) has broad spectrum antiviral and immunomodulatory activities. These activities generate stabilization or improvement in three important surrogate markers of HIV disease progression. Patients with HIV disease treated with Ampligen do not become positive for p24 antigen, in contrast to patients treated with AZT or placebo. Viral burden can also be decreased in patients receiving Ampligen/AZT therapy. In vitro studies indicate that both AZT sensitive and AZT resistant viruses can be inhibited by Ampligen alone and are synergistically inhibited by Ampligen in combination with AZT. The immunomodulatory effects of Ampligen are manifested as a stabilization of CD4 counts. When Ampligen is combined with AZT, an increase in CD4 count is seen. Furthermore, a return or increase in delayed type hypersensitivity to mumps, Candida, and trichophyton was seen in approximately 70% of patients treated with Ampligen. The activity of Ampligen in HIV disease is due to its multifunctional activity as an antiviral and immune stimulating agent. The antiviral effect directly inhibits HIV-infection and other viruses which have been implicated in HIV disease acceleration and progression. The immunomodulatory activity can stabilize, increase, or restore immune function. This enhanced immune function can also lead to the further inhibition of additional infections associated with disease progression. Thus, Ampligen has multiple mechanisms of action against HIV disease.

Adjuvants, Immunologic↗
PubMed1990

Detection of poly(I):poly(C12U), mismatched double-stranded RNA, by rapid solution hybridization: blood values after intravenous infusion.

A rapid solution hybridization technique has been developed for estimating blood concentrations of poly(I):poly(C12U), a high molecular weight bioactive double-stranded RNA. Samples were prepared by mixing 100 microL of blood with, 165 microL of 6 M guanidine thiocyanate (GuSCN) and 0.16 M EDTA pH 8.0, and freezing. Hybridizations were carried out with a [3H]poly(C) probe in 3 M GuSCN for 10 min at 37 degrees C. Ribonuclease-resistant hybrids were collected by precipitation with trichloroacetic acid and filtration. Validation studies demonstrated minor interassay variance; the assay was accurate in the range 0.1 to 10 ng poly(I):poly(C12U). Thirty-one blood samples from 15 patients were collected and prepared before and immediately after an average 35 min intravenous infusion of 40-500 mg poly(I):poly(C12U). Postadministration values averaged 48% (s.d. 23%) of the theoretical maximum (range 20-102%). These results confirm previous observations of rapid elimination kinetics of poly(I):poly(C12U) in patients.

Adult↗
PubMed1989

Inhibition of HIV-1 proviral DNA synthesis and RNA accumulation by mismatched dsRNA.

The antiviral activity of mismatched dsRNA of the form poly(I):poly(C12-U)n (Ampligen) against the human immunodeficiency virus type 1 (HIV-1) was investigated by RNA-RNA and RNA-DNA hybridizations. Mismatched dsRNA delayed the appearance of newly transcribed HIV-1 RNA as detected by liquid dot-blot hybridization in cultures of H9 T-lymphoblastoid cells following virus challenge. The appearance of proviral DNA as detected by Southern hybridization following virus challenge in H9 cells was also delayed. Mismatched dsRNA had no effect in syncytium inhibition assays performed by fusing MT-2 cells with H9/HTLV-IIIB cells. These results suggest that the in vitro anti-HIV-1 activity of mismatched dsRNA occurs, at least in part, at an early stage in the viral replication cycle following initial gp120-CD4 binding.

Antiviral Agents↗
PubMed1989

Complement-mediated, antibody-dependent enhancement of HIV-1 infection in vitro is characterized by increased protein and RNA syntheses and infectious virus release.

Antibody-dependent enhancement (ADE) of human immunodeficiency virus type 1 (HIV-1) infection in vitro has been described recently and was shown to occur by two mechanisms: either participation of the alternative pathway of complement or to involve an Fc receptor-mediated, complement-independent mechanism. Complement-mediated ADE results in an accelerated cytopathic effect in target cells that can abrogate the protective properties of neutralizing antibodies. This study characterizes the surface antigens of MT-2 cells using flow cytometric analysis and shows that these cells express high levels of both CD4 and complement receptor type 2 (CR2) while several CD4+ cell lines that do not demonstrate complement-mediated ADE lack high levels of complement receptors. Further, utilizing MT-2 cell cultures, it is demonstrated that complement-mediated ADE of HIV-1 infection is conferred by the sera from more than 80% of HIV-1 antibody-positive individuals (N = 85). Complement-mediated ADE of HIV-1 infection causes an acceleration of several parameters indicative of HIV-1 infection in vitro including increased HIV-1 antigen synthesis as detected by indirect immunofluorescence, RNA accumulation as measured by a solution hybridization protocol, reverse transcriptase release, and progeny virus production.

Cell Line↗
PubMed1987

Implications of retroviral and oncogene activity in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a stem cell disease which, on a clinical level, progresses from the release from growth control of normally differentiated cells (a preleukemic state) to an acute leukemia. On a molecular level, the evolution of CML to acute leukemia is a multistep process. We propose that an early step, at the stem cell level, is acquisition of the ability for gene movement, which allows subsequent submicroscopic and chromosomal rearrangements that cause changes in the growth characteristics and regulation of the stem cell. A specific platelet DNA polymerase (PDP - reverse transcriptase) may play a role in gene movement. The characteristic reciprocal translocation of chromosomes #9 and #22, causing the activation of the c-abl oncogene, appears to be responsible for the uncontrolled cellular growth. Yet, other growth factors (e.g., platelet derived growth factor) and activated oncogenes (e.g., c-sis) must be responsible for the stimulation, progression, and variability seen during the course of the disease. Because CML is a progressive disease with clinically definable stages, CML appears to be a model system for the study of the molecular basis of the progression of preleukemia to leukemia specifically, and preneoplasia to aggressive neoplasia in general.

Blood Platelets↗
PubMed1987

Synergistic antiproliferative effect of recombinant alpha-interferons with recombinant gamma-interferon.

Two human tumor cell lines were studied for their response to the antiproliferative effect of recombinant human interferons (IFNs) alpha 2, alpha 4, a hybrid alpha (delta 4 alpha 2 Bgl II alpha 1), and gamma, individually and in combination. Natural human alpha-IFN was used as a reference point for all experiments. RT4 (bladder carcinoma) cells were overall more sensitive to the antiproliferative effects of the IFNs than A2182 (lung adenocarcinoma) cells. Three-way analysis of variance indicated that the relative effectiveness of the alpha-IFNs was alpha 2 less than alpha 4 less than hybrid alpha less than natural alpha-IFN. On an international reference unit per milliliter basis, gamma-IFN was 50- and 75-fold more effective than natural alpha-IFN and hybrid alpha-IFN in RT4 cells and 5.6-, 12.1-, and 14.9-fold more effective than alpha 4-, hybrid alpha-, and natural alpha-IFN in A2182 cells. In contrast, when recalculated on a nanogram per milliliter basis, gamma-IFN was only threefold more effective than the hybrid alpha-IFN in RT4 and approximately twofold less effective than alpha 4 and the hybrid alpha in A2182. Combinations of alpha-IFNs gave additive or antagonistic effects. When any of the alpha-IFNs were combined with the gamma-IFN, however, a synergistic antiproliferative effect was seen. The magnitude of the synergy was dependent upon the concentration of gamma-IFN used and the type of alpha-IFN in the combination. Antagonistic effects were seen at the lowest gamma-IFN concentration studied (0.2 IRU/ml). Synergy also varied according to the potency of the alpha-IFN used.

Carcinoma↗
PubMed1985

Independent evolution of antiviral and growth-modulating activities of interferon.

Interferon (IFN) may be an extraordinarily ancient antiviral defense used by animals and plants. In animals, the antiviral properties of IFN may have been rendered redundant in part by the evolution of cell-mediated immunity. Comparative analysis of gene and protein structures, combined with examinations of biological specificities, suggest that IFN-beta and IFN-gamma have evolved new functions that include regulation of hematopoietic cell differentiation. An inspection of the events required for growth control by IFN suggests that this evolution consisted of the addition of an "antiproliferative domain" to a primitive IFN molecule that was previously capable of eliciting only an antiviral response. These evolutionary considerations lead to the speculation that some molecular species of IFN-alpha retain specific and potent biological activities, whereas other species may be losing both target cell specificity and biological potency over time. Important clinical problems, as well as opportunities, are raised by these hypotheses, particularly the problem of toxicity to normal organs resulting from the chronic use of certain cloned interferons at the relatively high doses that may be necessary to compensate for evolutionary alterations in their structure and function.

Amino Acid Sequence↗
PubMed1984

Platelet DNA polymerase levels in CML: implications for oncogenesis.

Partially purified platelet DNA polymerase (PDP) was able to synthesize DNA transcripts of platelet polyadenylated RNA. PDP was elevated in the earliest stages of CML and PV. In PV, successful chemotherapy resulted in rapid return of PDP to normal levels while in CML this was not the case. An hypothesis is presented proposing that PDP contributes to oncogenesis by regulating the expression of oncogenes.

Blood Platelets↗
PubMed1982

Quantitation of RNA-dependent platelet DNA polymerase in patients with myeloproliferative disorders.

Platelets from 28 patients with the myeloproliferative diseases (MPD) polycythaemia vera (9), essential thrombocythaemia (6), myelofibrosis with myeloid metaplasia (5) and chronic myelogenous leukaemia (8) were examined for an RNA-dependent DNA polymerase activity using standardized conditions permitting highly reproducible quantitation. Low levels of activity were detected in platelets of normal individuals, but platelets of nearly all MPD patients (25/28) possessed higher levels. The polymerase activity correlated with diagnosis (P = 0.001) and did not correlate with platelet counts (P greater than 0.2). Quantitation of this RNA-dependent DNA polymerase activity may be a useful parameter in the diagnosis of myeloproliferative disorders.

Blood Platelets↗