Biomedical subjects

K Muirhead

Publications and source records attributed to K Muirhead.

10 recordsLinked to original sources

PubMed2000

Response of chemosensitive and chemoresistant leukemic cell lines to drug therapy: simultaneous assessment of proliferation, apoptosis, and necrosis.

BACKGROUND: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively. METHODS: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance. RESULTS: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug. CONCLUSIONS: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Optimized fluorescent probe combinations for evaluation of proliferation and necrosis in anthracycline-treated leukaemic cell lines.

Proliferation and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukaemia (AML). Anthracyclines such as daunorubicin (DNR) are typically used to treat AML and can induce drug resistance. The goal of the studies described here was to select a combination of fluorescent probes that could be used in combination with flow cytometry to monitor cell proliferation vs. cell death/necrosis as a function of anthracycline uptake. Propidium iodide (PI), the most commonly used marker of membrane integrity, cannot be used to evaluate necrosis in DNR-containing cells because of spectral overlap. A membrane integrity probe compatible with the use of a dye dilution method using PKH67 to study cell proliferation was also selected. The results show that DAPI and Cascade Blue (CB), like PI, were able to detect necrotic cells when no DNR was present, although CB gave less resolution between viable and necrotic cells than PI or DAPI. In the presence of DNR, DAPI cannot be used owing to the fluorescence quenching by DNR. However, it was found that a combination of DNR, CB, and PKH67 allows simultaneous identification of chemoresistant cells, based on reduced DNR accumulation, necrotic cells based on CB incorporation, and proliferating cells based on partitioning of PKH67 fluorescence between daughter cells. It was also found that unless a marker of necrosis is used in combination with the dye dilution assay, a moderate decrease of fluorescence as a result of necrosis may be incorrectly interpreted as proliferation.

Antibiotics, Antineoplastic↗

PubMed1998

Indicators of T-cell activation: correlation between quantitative CD38 expression and soluble CD8 levels in asymptomatic HIV+ individuals and healthy controls.

Increased activation of CD8+ T cells, particularly increased expression of CD38 antigen, has been shown to strongly correlate with progression of human immunodeficiency virus-positive (HIV+) individuals to acquired immunodeficiency syndrome (AIDS) and death. As part of a study evaluating responses to a recombinant gp160 vaccine, we have used quantitative three-color flow cytometry (QFCM) to further investigate the relationships among several measures of lymphocyte activation/immunological status. Parameters evaluated included 1) absolute circulating counts for the major lymphocyte phenotypes (T, B, NK) and selected activated/regulatory subsets believed to have clinical value in the monitoring of patients with HIV infection; 2) level of CD38 expression (antibody-binding capacity [ABC]) on the lymphocyte subsets defined by CD8, CD38, and HLA-DR; and 3) serum levels of soluble CD8. CD8+DR+CD38+ counts were found to be markedly increased (approximately 10-fold) in HIV+ individuals, whereas CD4+CD45RA+ counts were markedly decreased (approximately 5-fold). We confirmed previous reports that CD38 expression on CD8 T cells (here reported as CD38 ABC) are increased in asymptomatic HIV+ individuals as compared with healthy controls, and further found that CD38 ABC was elevated approximately 2-fold on CD8+DR+ cells as compared with CD8+DR- cells in healthy controls, and almost 2-fold further elevated on CD8+DR+ cells in HIV+ individuals compared with CD8+DR+ cells in healthy controls. In agreement with previous studies, we found increased serum CD8 levels (sCD8) and increased CD8+DR+ counts in asymptomatic HIV+ individuals. However, when sCD8 was expressed relative to CD8+DR+ cell counts (RsCD8), this index was found to be significantly decreased in HIV+ individuals. Although CD38 ABC on CD8+DR+ cells showed no correlation with sCD8, it was significantly correlated with RsCD8 in both HIV+ and HIV- individuals. Absolute lymphocyte counts were strongly correlated with both CD38 ABC and RsCD8 in HIV+ individuals. However, CD4 counts were correlated with CD38 ABC (but not RsCD8) in HIV+ patients and with RsCD8 (but not CD38 ABC) in HIV-controls. Our results suggest that QFCM is significant in understanding the role of CD8+DR+CD38+ cells in processes such as lymphocyte homeostasis and HIV-induced CD4-cell depletion.

ADP-ribosyl Cyclase↗

PubMed1997

Incorporation of nisin in micro-particles of calcium alginate.

Nisin was successfully incorporated into a matrix of calcium alginate and ground into micro-particles smaller than 150 microns. Formation of micro-particles and incorporation of nisin was verified by scanning electron microscopy and by the reduction in the inactivation of nisin activity with proteolytic enzymes. Incorporation efficiency was 87-93% and the nisin in the alginate-incorporated form was 100% active against an indicator culture of Lactobacillus curvatus both in MRS broth and reconstituted skim milk.

Alginates↗

PubMed1996

Successful in vitro antigen-dependent activation of 24-hour-old peripheral blood lymphocytes.

We describe a simple, rapid and reproducible in vitro culture system in which human peripheral blood lymphocytes (PBLs), donated 24 h prior to initiation of culture can be stimulated to produce antigen-specific antibodies. Peripheral blood lymphocytes purified by Ficoll-Hypaque centrifugation were passed over a G10 Sephadex column and then activated in vitro in the presence of 0.003% staphylococcus Cowan A, 2.8 x 10(-6) M indomethacin and appropriate concentrations of tetanus toxoid antigen. After the first 24 h in culture, a five-fold concentrated supernatant from an allogeneic mixed lymphocyte culture was added. The cell surface phenotypes of the PBLs were analyzed by flow cytometry at the initiation and termination of culture, in order to provide a comprehensive characterization of the cellular composition of a successful in vitro stimulation system. Our results clearly show that the majority of peripheral blood B cells can be induced to an activated stage (blast transformation) and interleukin 2 (IL-2) receptor expression, following very simple manipulations of the lymphoid population. Tetanus toxoid-specific antibody production can be readily generated in this cell population. In contrast, T cells were not activated to express IL-2 receptors and reach blast transformation, and did not show appreciable proliferation. Our system provides a population of B cells producing antibodies of desired specificity which could be utilized for the generation of human hybridomas or could serve as a donor population for antibody engineering via the combinatorial library approach. Careful light scattering and cell surface phenotypic analyses of the cells entering, proliferating and differentiating in these cultures enabled several novel observations to be made.

Antibodies, Bacterial↗

PubMed1995

Partial characterisation of pediocin PO2 and comparison with nisin for biopreservation of meat products.

A plasmid associated bacteriocin (pediocin PO2) was isolated by ammonium sulphate precipitation from cell-free growth media and subsequent studies showed that the partially purified pediocin PO2 was most likely identical (molecular mass approximately 3200 daltons in size by SDS-PAGE, stable to low pH and heat at 121 degrees C for 15 min, inactivated by various proteolytic enzymes and resistant to treatment with a range of solvents, except 10% formaldehyde) to other pediocins (PA-1 and AcH) previously reported. The antagonistic spectrum of activity of pediocin PO2 was compared with nisin and showed a narrower host-range, but a much greater activity against Listeria species including strains of Listeria monocytogences, than did nisin. A rapid method of reflectance colorimetry was used to quantitate growth and acid production (as determined by the colour change in bromcresol purple) of Lactobacillus curvatus, added to a meat product model system. The combined effects of refrigeration temperature, microbial load and bacteriocin concentration were determined in the model over 15 days storage. Both nisin and pediocin demonstrated inhibitory activity against Lactobacillus curvatus in the model system. However, when bacteriocins were incorporated into a manufactured cooked meat product only low nisin activity and no pediocin activity was detected, after challenge of vacuum packaged slices of product with Lactobacillus curvatus, over a 21 day storage trial under refrigeration temperatures.

Bacteriocins↗

PubMed1985

Normal human blood density gradient lymphocyte subset analysis: I. An interlaboratory flow cytometric comparison of 85 normal adults.

An interlaboratory flow cytometric comparison of several commercially available human lymphocyte subset reagents was undertaken in three different laboratories. Fresh Hypaque-Ficoll purified blood mononuclear cells were stained at 4 degrees C or 22 degrees C. Direct or indirect surface immunofluorescence was carried out at all sites using an EPICS V flow cytometer. Fullbright, 10-micron fluorescent polystyrene microspheres were used for optical alignment and standardization. A log integral fluorescent histogram gated on forward and right angle scatter was collected on 1-2 X 10(4) cells for each reagent and the proportion, of positive cell determined for each reagent. With the exception of one reagent, anti-B1, which showed an approximately twofold variation, all three laboratories showed remarkable agreement. Thus there was no significant difference noted for the following reagents: OKT4, CCT4, Leu 3a, Leu 2a, OKT8, or CCT8. We attribute these findings to the availability of quality reagents, precision instrumentation, and a standard lymphocyte preparation.

Adult↗

PubMed1985

Binding of histamine and histamine analogs to lymphocyte subsets analyzed by flow cytometry.

The binding of histamine, 4-methylhistamine (a histamine type 2 receptor agonist), cimetidine (a histamine type 2 receptor antagonist), and telemethylhistamine (an inactive analog) to human peripheral blood mononuclear cell subsets was investigated by flow cytometry by using conjugates of these ligands coupled to fluorescein-labeled human serum albumin. Our results indicate that binding of fluorescent protein conjugates of histamine and its analogs does not selectively identify a lymphocyte subset(s) that mediates the immunomodulatory effects of histaminergic ligands. Conjugates with both low (2.5 to 2.8:1) and high (28 to 57:1) ligand to protein coupling ratios were used. No binding above background could be detected for the low mole ratio reagents. The high mole ratio reagents were bound by 95 to 99% of all lymphocytes when used at ligand concentrations of 50 microM or greater. At lower ligand concentrations, the number of lymphocytes exceeding a set fluorescence threshold was decreased, but fluorescence distributions remained unimodal at all concentrations used (1 to 500 microM). Monocytes also bound the high mole ratio reagents and gave rise to a second high-intensity peak in the fluorescence distribution unless they were excluded by other means. Levels of conjugate binding detected by flow cytometry did not parallel ligand potencies at classical histamine type 2 receptors; at equivalent ligand concentrations, approximately equal amounts of histamine or 4-methylhistamine conjugate were bound per lymphocyte, and only 30% less telemethylhistamine conjugate was bound. Competition with free ligands (10(2)- to 10(4)-fold excess histamine, 4-methylhistamine, cimetidine, or telemethylhistamine) did not significantly decrease the level of binding observed for the high mole ratio reagents at bound ligand concentrations of 1 to 25 microM. Dual staining with fluorescein-labeled conjugate and phycoerythrin-labeled monoclonal antibodies Leu-3ab (anti-helper T), Leu-2a (anti-suppressor T), Leu-M3 (anti-monocyte), or anti-HLA-DR (B cells and monocytes) was also carried out. The extent of conjugate binding to helper and suppressor cells was identical for each of the ligands used, but higher levels of conjugate binding were seen for monocytes and B cells than for T cells in every case. Our data do not exclude the possibility of enhanced conjugate binding to small numbers of activated (HLA-DR positive) T cells that might be involved in mediation of histamine effects.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗

PubMed1985

Evaluation of the in vivo antitumor activity and in vitro cytotoxic properties of auranofin, a coordinated gold compound, in murine tumor models.

The coordinated gold compound, 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S-triethyl phosphine gold (auranofin; Ridaura), was evaluated for antitumor activity in a variety of mouse tumor models. Of the 15 tumor models evaluated, auranofin was found to be active only against i.p. P388 leukemia. A number of dose schedules was used to measure activity against P388 with optimal activity observed at 12 mg/kg given daily, i.p., on Days 1 to 5. Auranofin was active against i.p. P388 leukemia only when administered i.p.; the drug was completely inactive when administered i.v., s.c., or p.o. on Days 1 to 5. Evaluation of the effects of auranofin in vitro demonstrated that survival curves for B16 melanoma cells as measured by the clongenic and dye exclusion assays were exponential and monophasic; cell cycle distribution was not altered, and auranofin displayed no preferential cytotoxicity to logarithmic or plateau growth phase cell populations; auranofin inhibited DNA, RNA, and protein synthesis at cytotoxic concentrations but showed no selective effect; the cytotoxic activity and cellular association of gold from auranofin were dose, time, and temperature dependent; and binding of auranofin gold to serum proteins markedly decreased cellular uptake of gold and cytotoxicity of auranofin in vitro.

Animals↗

PubMed1984

Identification and characterization of a monoclonal antibody to an antigen expressed on activated macrophages.

A hybridoma clone secreting a monoclonal antibody, designated MA158.2, that reacts with an antigen expressed on lymphokine-treated macrophages was produced by fusion of mouse myeloma cells with rat spleen cells immunized against C57BL/6 peritoneal macrophages rendered tumoricidal in vitro by incubation with the lymphokine macrophage-activating factor. The specificity of the antibody for activated macrophages and lack of reactivity with histologically diverse cell types was determined by radioimmune indirect binding and flow cytometry. MA158.2 antibody binds to mouse peritoneal macrophages elicited by nonspecific inflammatory agents and to tumoricidal macrophages elicited with Corynebacterium parvum. Resident peritoneal, splenic, and alveolar macrophages were only weakly positive. Several macrophage cell lines (P388D1, WEH1-231, J774, RAW 264.7), murine fibroblasts, and neutrophils did not bind detectable amounts of MA158.2. Radioimmune indirect binding analysis demonstrated that cell suspensions prepared from C57BL/6 mouse spleen, thymus, and lymph node as well as polymorphonuclear leukocytes, lymphocytes, and T- and B-cell murine lymphomas were MA158.2 negative. Expression of the reactive antigen on the macrophage cell surface was enhanced 3-fold following in vitro activation of elicited macrophages with macrophage-activating factor and the kinetics of activation to the tumoricidal state paralleled the increased expression of the antigen recognized by MA158.2. MA158.2 is a rat IgG2a antibody containing a single specific heavy and light chain that does not detect a polymorphic determinant. This monoclonal antibody will be a useful tool for monitoring the efficacy of agents in activating murine macrophages to the tumoricidal state and in analyzing the sequence of biochemical events that culminate in macrophage activation.

Animals↗