Biomedical subjects

D Ou

Publications and source records attributed to D Ou.

50 records · Page 3Linked to original sources

PubMed1989

Rabies virus-specific T cell hybridomas: identification of class II MHC-restricted T-cell epitopes using synthetic peptides.

Rabies virus-specific T-cell hybridomas were produced from immune mice by somatic cell fusion. Cloned T-cell hybridomas were studied for antigen specificity using purified virus, a recombinant vaccinia virus expressing rabies glycoprotein and synthetic peptides containing amino acid sequences of rabies viral antigens. Two closely situated T-cell epitopes of rabies glycoprotein, and one of rabies nucleoprotein were identified using synthetic peptides corresponding to amino acid sequences of these proteins. The major histocompatibility gene complex elements that determine the recognition of antigen by these T-cell hybrids were determined using mouse fibroblasts (L cells) transfected with and expressing the I-Ad and I-Ed genes. Some of the T cell hybridomas exhibited significant cytotoxic activity against target cells expressing surface rabies antigens. This T cell mediated cytotoxicity requires cell-to-cell contact between target and effector cells since no by-stander cytotoxicity was observed. The results are discussed in the context of their significance for the design of newer subunit vaccines to prevent rabies infection.

Animals↗

PubMed1988

Recognition of hepatitis B surface antigen by human T lymphocytes. Proliferative and cytotoxic responses to a major antigenic determinant defined by synthetic peptides.

The antigenic sites for human T lymphocytes on hepatitis B surface Ag (HBsAg) were studied by using synthetic oligopeptides. T cell lines of the helper/inducer class, which were isolated from hepatitis B vaccine recipients, were found to react strongly and in an Ag-specific way with peptides corresponding to a sequence of 10 to 30 amino acids near the amino terminus of the HBsAg molecule. Cells with surface expression of the antigenic determinant contained in these synthetic peptides induced both proliferative and cytotoxic responses in the hepatitis B-specific T cells. The results indicate that amino acid residues 24-27 of HBsAg could be directly involved in this T cell determinant. Inhibition studies with mAb to MHC class II Ag and target cells from various HLA-typed individuals suggest that some T cell responses to this determinant of HBsAg might be restricted by the DPw4 molecule. However, the possibility exists that more than one of the MHC class II molecules could be involved as restricting elements of T cell responses to this synthetic peptide. In vivo experiments with synthetic peptides such as those described here are needed to demonstrate the possibility of enhancing HBsAg immune responses in some individuals.

Amino Acid Sequence↗

PubMed1988

Recognition of rabies and rabies-related viruses by T cells derived from human vaccine recipients.

Human peripheral blood mononuclear cells and T-cell lines and clones from individuals immunized with rabies PM vaccine were tested for the ability to recognize antigenic determinants in rabies and rabies-related viruses in an antigen-induced proliferation assay. Some, but not all, of the T cells from these individuals cross-reacted with various laboratory strains of rabies virus with rabies-related viruses such as Duvenhage and Mokola. In addition, these T cells were shown to react with epitopes of either the ribonucleoprotein or the viral glycoprotein. Rabies-specific cytotoxic T-cell responses by a CD4+ T-cell line were evident against antigenic determinants of the ribonucleoprotein and glycoprotein.

Antibodies, Viral↗

PubMed1982

Alpha chain disease in the Thai man.

A 61-year-old Thai man presented with a history of chronic diarrhoea of 1-2 years duration. Stool examination revealed a heavy parasitic infection. Several anthelminthics were given without benefit, despite disappearance of the intestinal parasites. Serum protein studies revealed abnormal alpha heavy chain. Two courses of cyclophosphamide, vincristine and prednisolone were followed by a brief remission in his symptoms. A 21 day course of tetracycline was then given, resulting in an improvement in his condition. He is still symptom free nine months after confirmation of the diagnosis, without any further treatment. Serum protein studies should be included in laboratory investigations of a patient with chronic diarrhoea.

Cyclophosphamide↗

PubMed1980

Erythrocyte gamma-glutamylcysteine synthetase from normal and low-glutathione sheep.

gamma-Glutamylcysteine synthetase (L-glutamate:L-cysteine gamma-ligase (ADP-forming), EC 6.3.2.2) was purified from the erythrocytes of normal and low-glutathione sheep. The molecular weight (78 000), pH optimum (pH 7), substrate specificity, inhibition constant for glutathione (0.44-0.50 mM), electrophoretic mobility, and heat stability were similar for the purified enzyme from both sources. Using immunological techniques, the specific activity of gamma-glutamylcysteine synthetase from low-glutathione sheep was lower than that from normal sheep.

Animals↗

PubMed1976

Oxygen-hemoglobin equilibrium of normal and glutathione-deficient sheep.

The affinity of hemoglobin for oxygen did not differ significantly between normal and glutathione-deficient sheep. Although phlebotomy in sheep of both types did not significantly alter the partial pressure values at which hemoglobin is 50% saturated with oxygen, it did increase significantly the erythrocyte glutathione and 2,3-diphosphoglycerate (GSH) and (DPG) concentrations. Because DPG does not bind to sheep hemoglobin, its increase in hypoxic sheep must mean DPG is metabolically controlled in the absence of physiologic effect.

Animals↗

PubMed1975

Effect of storage on oxygen dissociation of canine blood.

Oxygen dissociation curve and adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) contents in red blood cells (RBC) were determined in canine blood stored in acid citrate dextrose (ACD) and citrate phosphate dextrose (CPD) solutions. The oxygen-unloading ability decreased, as shown by the left shift of oxygen dissociation curve during storage, and the shift correlated with decreasing DPG but not decreasing ATP concentrations. After 2 weeks of storage in ACD solution, oxygen dissociation curves were shifted significantly to the left. For blood stored in CPD solution, 4 weeks was required before the shift was significant. It was concluded that canine blood collected and stored in CPD solution is more efficient than that stored in ACD solution in delivering oxygen to the tissues.

Adenosine Triphosphate↗

PubMed1974

Biological and physical properties of the suckling mouse cataract agent grown in chicken embryos.

Experiments were carried out with the suckling mouse cataract agent (SMCA) in an attempt to better characterize the nature of this agent. Despite the fact that high titers of SMCA were found in the chorioallantoic fluid of inoculated chicken embryos, the agent did not replicate in chicken embryo epithelial cell cultures. SMCA did not interfere with Sindbis virus plaque formation and was not inhibited by chicken interferon or polyinosinic-polycytidylic acid. The agent was relatively resistant to high pH and osmotic shock but was inactivated by low pH. Electron micrographs of negatively stained particles concentrated and semipurified from infected chicken embryo chorioallantoic fluids revealed pleomorphic particles ranging from approximately 70 to 400 nm in diameter. SMCA was resistant to kanamycin, novobiocin, and sodium aurothiomalate.

Animals↗

PubMedDate not supplied

Recognition of contiguous allele-specific peptide elements in the rubella virus E1 envelope protein.

Peptides which bind to human HLA-DRB1 class II molecules in an allele-specific fashion were derived from the immunodominant E1 envelope protein of rubella virus. Two nonoverlapping E1 peptide epitopes were recognized by rubella virus-specific T cells in the context of independent HLA alleles when presented either separately or as a contiguous polypeptide containing both epitopes. Direct binding analysis of potential peptide epitopes to distinct HLA molecules provides a direct approach for selecting antigenic peptides useful for epitope-based vaccine targeted to multiple HLA types.

Alleles↗

PubMedDate not supplied

Altered oncogene, tumor suppressor and cell-cycle gene expression in PANC-1 cells cultured with the pleiotrophic 5-lipoxygenase inhibitor, MK886, assessed with a gene chip.

We describe a genomic response of mRNAs associated with a subset of oncogenes, tumor suppressor and cell cycle-related genes in proliferating human Panc-1 pancreatic cancer cells after 24 hours of culture with MK886, a pleotrophic 5-lipoxygenase inhibitor. Ninety-eight of these cDNAs are represented in one of the sub-arrays included in the Clontech Human cDNA Expression Array. In this initial analysis, control cells exhibited apparent widespread low levels of disparate mRNA synthesis. In cells cultured with 40 microM MK886 for 24 hr, while most expressed genes, including a number of specific proliferation-enhancing genes such as c-myc were inhibited, 19 other ones including some countervailing genes including tyrosine SRC protein kinase, cyclins B1 and D1, CDC25B phosphatase and 40s ribosomal S19, amounting to 19 percent of the cDNAs resident on the chip were up-regulated at > 1.10 experimental/control values. Therapy-induced activation of compensatory proliferative genomic responses provides an additional explanation why malignant cells can fail therapy. Among their many future uses, gene chips clearly will be an extremely powerful tool for identifying relationships between the hierarchical linear and non-linear control and implementation-related cellular events and for identifying potential molecular targets tor cancer therapy.

Blotting, Western↗

PubMedDate not supplied

A response of Panc-1 cells to cis-platinum, assessed with a cDNA array.

BACKGROUND: The problem posed by the lack of response of cells in most solid cancers to current chemotherapy generally remains intractable. MATERIALS AND METHODS: The use of cDNA arrays represents one global approach to identifying reasons for this failure. A messenger RNA response of pancreatic cancer (Panc-1) cells after culture for 24 hours with 12 microM cis-platinum was analyzed with a commercial cDNA array. RESULTS: Major drug-induced events included inhibition of messenger RNAs associated with cell proliferation and up-regulation of generally countervailing DNA repair, cellular stress, heat shock protein, glutathione stress-related and multiple drug resistance enzyme messenger RNAs, accompanied by a limited programmed cell death response. CONCLUSION: Induction of widespread normal stress-induced countervailing mRNAs by comparatively non-selective agents such as cis-platinum strongly biases against a successful therapeutic outcome. This paradoxical result of a therapeutic intent provides a further compelling argument for the use of specifically-targeted therapy such as growth factor receptor, tyrosine kinase and other discretely focused agents, probably employed in combinations based on expression of their targets in an individual patient's cancer, as identified by cDNA or proteonomic arrays.

Antineoplastic Agents↗

PubMedDate not supplied

An in vivo inhibitor of 5-lipoxygenase, MK886, at micromolar concentration induces apoptosis in U937 and CML cells.

MK886 (Merck Frosst) is a selective in vivo inhibitor of 5-lipoxygenase, active at nanomolar concentrations. At micromolar concentrations, it inhibited the proliferation of U937 monoblastoid cells and of cultured malignant cells from patients with chronic myelogenous leukemia. These cells became morphologically apoptotic, a form of physiologic cell death. U937 cell apoptosis was assessed by flow cytometry, ultrastructure, DNA laddering and immuno-histology for free 3'OH-DNA. MK886-induced apoptosis developed over time as cells were recruited in concert with reduction in their numbers. Some CML cells exhibited cytoplasmic changes of apoptosis without typical nuclear changes. Under conditions used for measuring Ca2+ with Fura 2, 10 micromolar MK886 increased U937 intracellular Ca2+ 4-fold or more over the 8 minute period of measurement. Since MK886 inhibits the association of arachidonic acid with the 5-lipoxygenase activating protein, altered arachidonic acid metabolism may have contributed to these results.

Apoptosis↗

PubMedDate not supplied

Five-lipoxygenase inhibitors reduce Panc-1 survival: the mode of cell death and synergism of MK886 with gamma linolenic acid.

The 5-lipoxygenase inhibitors ETYA, SC41661A and MK886 reduced the proliferation and viability of Panc-1 human pancreatic cancer cells. The extent of inhibition depended upon drug concentration, and with continued culture, cells detached and stained with trypan blue. Although results from flow cytometry were those associated with programmed cell death, despite repeated attempts, no DNA laddering consistent with its later stages was detected, and studies with the TUNEL assay were negative. Light and electron microscopy of cells cultured with SC41661A provided morphologic evidence of a population of "dark" cells and of an incompletely expressed type 1 programmed cell death including margination of chromatin at the nuclear membrane and by consolidation and degeneration of cytoplasmic organelles, along with extensive vacuolization. Cells cultured with MK886 exhibited compact "dark" cells and an unusual cytoplasmic mode of cell death characterized by vacuolization and widely separated smooth internal membranes without diagnostic nuclear changes. This is in marked contrast to the extensive type 1 PCD induced by 5-lipoxygenase inhibitors cultured with human U937 monoblastoid cells. On balance, the response of Panc-1 cells to MK886 suggests expression of a variant type 2 (autophagic) cellular suicide, although some contribution from components of a "cytoplasmic" (type 3?) form of non-necrotic cell death may also be considered. In a European clinical trial, gamma linolenic acid, a polyunsaturated fatty acid that generates free radicals has been combined with 5-fluorouracil as chemotherapy for pancreatic cancer. Panc-1 cell proliferation was insensitive to inhibition by several chemotherapeutic agents employed clinically, including 5-fluorouracil, cisplatin or gemcitabine and only somewhat sensitive to GLA. When gamma linolenic acid was combined with MK886, the more effective of the two 5-lipoxygenase inhibitors, a synergistic reduction in Panc-1 cell number and viability occurred.

Apoptosis↗

PubMedDate not supplied

NTBN, a free radical spin trap induces programmed cell death in human pancreatic cancer (PANC-1) cells.

N-tertiary butyl-a-phenylnitrone, a free radical spin trap at > or = 10 mM concentration, inhibited proliferation and reduced the viability of human pancreatic cancer (Panc-1) cells. The drug concentration determined the extent of inhibition, and with continued culture a proportion of the cells detached, most of which stained with trypan blue. Although hypodiploid cells were detected by flow cytometry of cells cultured with 20 mM NTBN, DNA laddering was absent and the TUNEL reaction negative. "Dark" cells present in samples cultured with 10 mM NTBN exhibited decreased cytoplasmic volume and increased staining with methylene blue and azure II, but lacked characteristic nuclear changes of type 1 programmed cell death. Cells cultured with > 10 mM of the spin trap exhibited nuclear and cytoplasmic changes more consistent with a non-type 1, type 2 variant of PCD with extensive cytoplasmic vacuolization. Careful analysis revealed evidence of marked pinocytosis in some cells. In view of the spin-trap associated pinocytosis, augmented uptake of chemotherapy in affected cells might be anticipated, but additive, synergistic or antagonistic interactions between NTBN and 5-fluorouracil were not observed.

Antimetabolites, Antineoplastic↗