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D Darling

Publications and source records attributed to D Darling.

At least 19 recordsLinked to original sources
PubMed2001

Streptavidin paramagnetic particles provide a choice of three affinity-based capture and magnetic concentration strategies for retroviral vectors.

Three strategies have been designed to concentrate infectious retroviral vectors from the supernatants of human- (HT1080) and murine- (NIH 3T3) based packaging cells. Streptavidin-conjugated paramagnetic particles in conjunction with (i) antibodies directed against murine fibronectin, (ii) biotinylated lectins, or (iii) biotin-modified packaging cell-surface proteins allow affinity-mediated magnetic concentration of retroviral vectors. Retroviral titers (assayed by colony formation of human myeloid K562 cells) are increased by 1-4 x 10(3)-fold after volume reductions of only 125-fold. Using these procedures, preparations of 5 x 10(8) cfu/ml are routinely made from relatively low-titer (2-5 x 10(5) cfu/ml) starting material. High-titer (paramagnetic) retroviral vector preparations can be used for magnetic field-dependent retroviral infection in vitro. Magnetic field-dependent localization such as this may enable the in vivo administration of formulations that concentrate retroviral infection to the required target tissues and organs.

3T3 Cells↗
PubMed2001

Protein transduction: an alternative to genetic intervention?

Protein transduction, an emerging technology with potential applications in gene therapy, can best be described as the internalisation of proteins into the cell, from the external environment. This process relies on the inherent property of a small number of proteins and peptides of being able to penetrate the cell membrane. The transducing property of these molecules can be conferred upon proteins which are expressed as fusions with them and thus offers an alternative to gene therapy for the delivery of therapeutic proteins into target cells. This review describes the three most commonly used protein transduction vehicles; the antennapedia peptide, the herpes simplex virus VP22 protein and HIV TAT protein transduction domain. The future prospects for the application of this technology in gene therapy are also discussed.

Antennapedia Homeodomain Protein↗
PubMed2000

Protein transduction: a new tool for the study of cellular ageing and senescence.

Protein transduction can be described as the direct uptake by the cell of exogenous proteins/peptides or protein/peptide:chemical complexes, as a result of a specific property of the protein/peptide component. In this review, the three most widely studied protein transducing activities are described, with particular emphasis on the TAT protein transduction domain. Current progress in protein transduction technology suggests the potential development of a variety of molecular and cell biology tools that will enable researchers to by-pass conventional genetic routes for modulating the cells' biological activity, thus negating many of the problems associated with genetic intervention. The potential application of this class of molecule in the development of tools for the study of senescent populations is discussed.

Animals↗
PubMed2000

Local versus systemic interleukin-2: tumor formation by wild-type and B7-1-positive murine melanoma cells.

Modification of murine K1735 melanoma cells to express the immune costimulator B7-1 had no effect on tumor formation in syngeneic mice. In contrast, <40% of mice inoculated with K1735 cells modified to secrete murine interleukin-2 (IL-2) formed tumors, and no tumors formed when the K1735 cells coexpressed both murine IL-2 and B7-1. However, administration of systemic recombinant human IL-2 had no detectable effect on the formation of tumors by the B7-1-expressing K1735 cells. By contrast, admixtures of IL-2-secreting and B7-1-expressing K1735 cells formed fewer tumors than either cell type alone. Murine IL-2 was effective only when secreted locally, because the IL-2-secreting cells inoculated into the right flank did not affect the growth of the B7-1-expressing cells inoculated into the opposite flank.

Animals↗
PubMed2000

Low-speed centrifugation of retroviral vectors absorbed to a particulate substrate: a highly effective means of enhancing retroviral titre.

For many gene therapy applications the effective titre of retroviral vectors is a limiting factor both in vitro and in vivo. Purification and concentration of retrovirus from packaging cell supernatant can overcome this problem. To this end we have investigated a novel procedure which involves complexing retrovirus to a dense and particulate substrate followed by a short low-speed centrifugation. The study reported here uses heat-killed, formaldehyde fixed Staphylococcus aureus (Pansorbin) absorbed to PG13 derived retrovirus. This complex was then used to harvest retrovirus from packaging cell supernatant: centrifugation and washing of this complex allows the retrovirus to be both purified and concentrated. This procedure increases the effective titre of retrovirus by up to 7500-fold after an only 200-fold reduction in volume. The affinity of Pansorbin for retrovirus allows concentration regardless of its encoded genes and makes this protocol applicable to other popular packaging cells and envelope proteins. Possible explanations for the marked increase in titre of concentrated virus and the mechanism governing the complexing of retrovirus to Pansorbin are discussed.

Animals↗
PubMed2000

Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron.

Transduction of cells with multiple genes, allowing their stable and co-ordinated expression, is difficult with the available methodologies. A method has been developed for expression of multiple gene products, as fusion proteins, from a single cistron. The encoded proteins are post-synthetically cleaved and processed into each of their constituent proteins as individual, biologically active factors. Specifically, linkers encoding cleavage sites for the Golgi expressed endoprotease, furin, have been incorporated between in-frame cDNA sequences encoding different secreted or membrane bound proteins. With this strategy we have developed expression vectors encoding multiple proteins (IL-2 and B7.1, IL-4 and B7.1, IL-4 and IL-2, IL-12 p40 and p35, and IL-12 p40, p35 and IL-2 ). Transduction and analysis of over 100 individual clones, derived from murine and human tumour cell lines, demonstrate the efficient expression and biological activity of each of the encoded proteins. Fusagene vectors enable the co-ordinated expression of multiple gene products from a single, monocistronic, expression cassette.

Animals↗
PubMed1999

Effect of costimulation and the microenvironment on antigen presentation by leukemic cells.

Costimulatory signals supplied by genetically modified tumor cells can enable T-cell recognition of tumor-associated antigens that were previously silent when presented by unmodified tumor cells. Although the mechanism of the CD80/CD28 costimulation has been studied extensively in the normal T-cell/antigen-presenting cell (APC) interactions, it is unclear how expression of CD80 by tumor cells mediates its effect. We demonstrate here that optimal CD80 expression on a leukemic cell enhances T-cell recognition of alloantigen primarily by lowering the level of T-cell receptor (TCR) stimulation required for activation. CD80 expression by leukemic cells leads to increased survival of activated T cells by inducing upregulation of the antiapoptotic protein BCL-2, but not BCL-X(L). The cytokine microenvironment in which T cells are activated is crucial in determining their differentiation and consequently the nature of the immune response generated. Many tumor cells produce immunosuppressive cytokines that may not favor the induction of cell-mediated immunity. In this study, the presence of CD80 on leukemic cells increased T-cell activation in vitro, but this did not result in the production of Th1 cytokines. We show that this is due to a leukemia-derived soluble factor that inhibits the production of Th1 cytokines. Optimal expression of a costimulatory molecule, therefore, enhances the ability of leukemic cells to present antigen by amplifying TCR signals, but the microenvironment generated by leukemic cells may suppress the immune response required for their eradication. Thus, strategies aimed at inducing antileukemic immunity by providing leukemic cells with costimulatory functions must ensure the presence of an appropriate microenvironment.

Antigen Presentation↗
PubMed1999

Expression of a variant of CD28 on a subpopulation of human NK cells: implications for B7-mediated stimulation of NK cells.

The ability of NK cells to kill tumor cells is controlled by a balance between activating and inhibitory signals transduced by distinct receptors. In murine tumor models, the costimulatory molecule B7.1 not only acts as a positive trigger for NK-mediated cytotoxicity but can also overcome negative signaling transduced by MHC class I molecules. In this study, we have evaluated the potential of human B7.1-CD28 interaction as an activating trigger for human blood NK cells. Using multiparameter flow cytometric analysis and a panel of different CD28 mAbs, we show that human peripheral blood NK cells (defined by CD56+, CD16+, and CD3- surface expression) express the CD28 costimulatory receptor, with its detection totally dependent on the mAb used. In addition, the level of CD28 varies among individuals and on different NK cell lines, irrespective of CD28 steady-state mRNA levels. By performing Ab binding studies on T cells, our data strongly suggest that binding of two of the anti-CD28 Abs (clones 9.3 and CD28.2) is to a different epitope to that recognized by clones L293 and YTH913.12, which is perhaps modified in the CD28 molecule expressed by the NK cells. We also show that B7.1 enhances the NK-mediated lysis of NK-sensitive but not of NK-resistant tumor cells and that this increased lysis is dependent on CD28-B7 interactions as shown by the ability of Abs to block this lysis. Coculture of the B7.1-positive NK-sensitive cells also led to the activation of the NK cells, as determined by the expression of CD69, CD25, and HLA class II.

Adult↗
PubMed1997

Enhanced immune costimulatory activity of primary acute myeloid leukaemia blasts after retrovirus-mediated gene transfer of B7.1.

Gene modification of malignant cells to express immune stimulators (cytokines and immune costimulators) has provided the basis for a novel form of immunotherapy. Using a MPSV-based retroviral vector with hygromycin resistance gene as a selectable marker, we have studied retrovirus-mediated gene transfer of an immune costimulator, B7.1, into primary human acute myeloid leukaemia (AML) cells and the subsequent induction of immune costimulatory function. AML blasts from 10 patients were transduced by co-culture for 48 h with or without haemopoietic growth factors (HGFs). In the absence of HGFs, transduction efficiency (TE), as judged by % B7.1 expressing cells, was low, varying from 0.3 to 8.2% (median 1.5%). Addition of HGFs increased the median TE 1.8-fold with stem cell factor alone and 2.6-fold with SCF, interleukin-3 and GM-CSF. Effects on cell cycling alone could not explain this difference, suggesting other factors such as virus binding and promoter activity, are also involved. CFU-AL assays indicated a higher transduction efficiency of clonogenic cells, which was not improved by growth factors. Limited duration of cell growth prevented significant expansion of transduced populations by culture in the presence of hygromycin. Although not increasing transduction efficiency, CD34 enrichment enhanced drug selection, by targeting cells with the greatest self-renewal capacity. Immunoselection of B7.1 expressing cells produced transduced populations with 30-60% expressing B7.1. In an allogeneic mixed leukaemic cell/T lymphocyte reaction (MLLR) transduced AML cells enriched by immunoselection were able to stimulate allogeneic T cells (CD4 and CD8 positive), which could be inhibited by a solubilised B7 receptor, CTLA4.Ig. Our results demonstrate that using a replication incompetent retroviral vector, it is possible to introduce the immune costimulator B7.1 into primary AML-blasts and by immunoselection, enrich the transduced cells, which may be used for subsequent administration as an autologous cellular vaccine.

B7-1 Antigen↗
PubMed1997

In vitro immune modulation by antibodies coupled to tumour cells.

Modification of autologous tumour cells to express the immune costimulator B7.1 is a potential strategy for immunotherapy of cancer. Previously, this has involved introduction of genetic material into cells, in vitro culture, and confirmation of the protein product on the cell surface. This is possible only if sufficient tumour is obtainable and efficiently modified in a short time. Whilst progress has been made on ex vivo tumour cell culture and transfection/infection procedures there are still tumour types for which the present means of gene transfer are not efficient enough. We describe a highly efficient in vitro procedure for the modification of over 99% of the cells in a population, allowing the expression of cell surface proteins with potential immune modulatory activities. This procedure, which can be completed in as little as 24 h with no upper limit on cell number, utilizes succinimide esters to label cell surface proteins with biotin covalently. Biotinylated cell membrane proteins then anchor an avidin bridge for immobilizing protein G'-biotin. This can serve to bind immunoglobulin (Ig) molecules via their Fc region such that the variable region of the antibody is freely and functionally available. In the present study the binding of a stimulatory mouse anti-human CD28 monoclonal antibody to the surface of tumour cells is used to show that the modified cells are capable of co-stimulating T cells in vitro. The simplicity of the method, and the use of common reagents, represents a further step towards a realistic, truly 'off-the-shelf', nongene immunotherapy protocol.

Animals↗
PubMed1997

Serial measures of sinoatrial and atrioventricular nodal function in ambulatory patients.

We hypothesized that the outpatient assessment of SA and AV nodal (SAN, AVN) function could be a useful tool to determine the effectiveness of drugs and other treatments. We sought to examine the reproducibility, safety and ease of acquiring serial measurements of these parameters. Ten patients with permanent pacemakers underwent low current chest wall stimulation while their device was programmed to unipolar atrial triggered mode. Measurements at multiple conditioning drive train frequencies were obtained for: sinus nodal recovery time (SNRT); corrected sinus nodal recovery time (CSNRT); SA conduction time (SACT); AVN block cycle length (AVNBCL); and AVN effective refractory period (AVNERP). AVN function curves were also constructed. All studies were repeated after 2 weeks. Measures of sinus nodal and AVN function did not show significant differences between the two studies. The following co-efficients of correlation were obtained: SNRT800, r = 0.79; CSNRT800, r = 0.71; SNRT600, r = 0.71; CSNRT600, r = 0.44; SACT, r = 0.75; AVNBCL, r = 0.98; AVNERP800, r = 0.55; and AVNERP600, r = 0.99. AVN function curves did not significantly differ between week 1 versus week 2 at conditioning drive trains of either 800 ms or 600 ms. These data suggest that serial noninvasive electrophysiological measures of AVN and SAN function are reproducible over 2 weeks. Using data in this study, estimates of the sample size necessary for the evaluation of the effects of investigational drugs on the SAN and AVN in future studies are possible.

Arrhythmia, Sinus↗
PubMed1996

The effect of combined expression of interleukin 2 and interleukin 4 on the tumorigenicity and treatment of B16F10 melanoma.

The recent use of interleukin 2 (IL-2) and interleukin 4 (IL-4) single cytokine modified tumour cells in rodent models has demonstrated a potential use of these cytokines to produce autologous cancer cell vaccines. Here we compare the potential therapeutic benefit of transduction with IL-2 or IL-4 alone, and combined IL-2 + IL-4 in B16F10 cells, a murine malignant melanoma of poor immunogenicity. Transduction of B16F10 cells (MHC class I and II negative) to express either IL-2 or IL-4 alone delays the formation of tumours, IL-4 being more effective than IL-2. However, combined expression of IL-2 + IL-4 reduces tumorigenicity more than either cytokine alone. The eventual formation of tumours may result from loss of gene expression, and preliminary results suggest methylation of the retroviral long terminal repeat (LTR), rather than loss of the transduced DNA sequences. Histological examination of tumours expressing either IL-2 or IL-4 alone shows a non-specific inflammatory reaction with an increased tissue infiltrate of immune effectors (monocytes/macrophages, lymphocytes, granulocytes) localised around the tumour. In comparison, when cells expressing combined IL-2 + IL-4 were injected there were more granulocytes present, and perhaps more importantly, these were mainly localised within the tumour. The benefit of combined IL-2 + IL-4 expression results from a local rather than systemic effect as the growth of tumours from cells expressing IL-2 or IL-4 alone injected at distant sites was comparable with a single inoculation of cells expressing either cytokine alone. However, when cells expressing single cytokines IL-2 or IL-4 were mixed and injected at the same site, in comparison with the clonal population of cells expressing combined IL-2 + IL-4, tumour growth was characteristic of IL-4 alone rather than IL-2 + IL-4. Treatment of established tumours with a single injection of lethally irradiated tumour cells expressing IL-2 + IL-4 was sufficient to either reject tumours, or at least delay further tumour development. Furthermore, treatment stimulated an initial non-specific immune reaction that lead to a systemic immunity. Lethally irradiated wild-type cells were also successful in treating some established tumours, although this did not induce any systemic immunity. However, although successful in treatment studies, neither wild-type nor combined IL-2 + IL-4 expressing cells were able to vaccinate animals against a subsequent challenge with live wild-type tumour. These results indicate a potential therapeutic benefit with the use of combination IL-2 + IL-4 transduction of autologous cancer cells.

Animals↗
PubMed1996

Comparison of IL-2- and IL-4-transfected B16-F10 cells with a novel oil-microemulsion adjuvant for B16-F10 whole cell tumour vaccine.

The use of whole cell tumour vaccines in the treatment of malignant melanoma has given mixed results. Cytokine-transfected tumour cells as vaccine have shown efficacy in animal models but need to be compared with other means of enhancing a systemic anti-tumour immune response. A new generation of immunological adjuvants claimed to be more effective than the conventional adjuvants is now available for assessment. We have investigated the action of an oil-microemulsion adjuvant formulation (IDEC antigen formulation (IDEC-AF)) in the B16-F10 murine melanoma model. After standardisation of the whole cell tumour vaccination protocol we showed that mice vaccinated with whole irradiated cells combined with IDEC-AF produced a significant inhibition of tumour growth, following a challenge with live tumour cells, when compared with mice vaccinated with whole cell vaccine alone. IDEC-AF was superior to two conventional adjuvants, namely alum and incomplete Freund's adjuvant and a more reliable response was achieved with the oil-microemulsion adjuvant compared with IL-2-transfected cells. In addition, the adjuvant was comparable in efficacy to IL-4-transfected B16-F10 cells. Given the practical difficulty in using cytokine-transfected tumour cells and the limited therapeutic range of some cytokines, a cheap and easy to deliver adjuvant formulation proved equally or more effective than some of the currently clinically used transfected cytokines.

Alum Compounds↗
PubMed1994

The chicken retinoid-X-receptor-gamma gene gives rise to two distinct species of mRNA with different patterns of expression.

Retinoids are metabolites of vitamin A that can regulate gene expression in a range of embryonic and adult cell types. They do this by binding to nuclear receptors belonging to the steroid/thyroid hormone receptor superfamily of ligand-activated transcription factors. Vertebrates possess two classes of nuclear retinoid-receptor genes, each with three members. These are the RAR-alpha, RAR-beta and RAR-gamma genes and the RXR-alpha, RXR-beta and RXR-gamma genes. In this paper we show by cDNA cloning and ribonuclease protection that the chicken RXR-gamma gene gives rise to two mRNA species (RXR-gamma 1 and RXR-gamma 2) that differ at their 5' ends. The two mRNAs have different tissue distributions in the 10-day-old chick embryo. RXR-gamma 2 mRNA was present in the eye and dorsal root ganglia but was undetectable in the liver. In contrast, RXR-gamma 1 mRNA was present in liver, was undetectable in dorsal root ganglia and was just detectable in the eye, where it was much less abundant than RXR-gamma 2 mRNA. The predicted protein products of the RXR-gamma 1 and RXR-gamma 2 mRNAs differ at their N-termini, in a region thought to modulate transcriptional transactivation by the receptor. These results show that at least one of the retinoid-X-receptor (RXR) genes gives rise to more than one protein product, a principle previously established for the retinoic acid-receptor (RAR) genes. The existence of multiple RXR protein isoforms would increase the range of heterodimers formed between RXRs and other nuclear receptors, including RARs and the receptors for thyroid hormone, vitamin D and peroxisome proliferators. This could increase the diversity of transcriptional responses mediated by these molecules.

Amino Acid Sequence↗
PubMed1990

DMSO and retinoic acid induce HL-60 differentiation by different but converging pathways.

The expression of c-myc and two calcium-binding proteins, MRP8 and MRP14, has been analyzed in wild-type and differentiation-resistant HL-60 variants. In HL-R5 cells, resistant to the induction of differentiation by retinoic acid but not DMSO, the characteristic c-myc down-regulation which is associated with HL-60 differentiation, as well as increased levels of MRP8 and MRP14, is detectable only after DMSO treatment. By contrast HL-D4 cells, which were selected for resistance to the induction of differentiation by DMSO alone, are actually resistant to both DMSO and retinoic acid. However, treatment of HL-D4 cells with DMSO results in a transient c-myc down-regulation in the absence of either growth arrest or induction of differentiation. Neither agent can induce an increase in the level of either MRP8 or MRP14 in HL-D4. The resistance of HL-D4 cells to DMSO and retinoic acid, and the different effects of these agents on c-myc RNA levels, despite their common effect on the expression of MRP8 and MRP14, suggest that the two agents act through different pathways which coverage before the onset of myeloid differentiation in HL-60 cells.

Cell Differentiation↗
PubMed1989

DMSO induced modulation of c-myc steady-state RNA levels in a variety of different cell lines.

The nuclear proto-oncogene c-myc is believed to play a regulatory role in eukaryotic cellular growth and differentiation. Several studies have demonstrated rapid down-regulation of the steady-state levels of c-myc RNA during DMSO induced differentiation of the human 'promyelocytic' cell line, HL-60. However, little is known about the effect of DMSO on c-myc regulation in cells which are not induced to differentiate by DMSO. We have examined the effect of DMSO on c-myc RNA levels in a number of cell lines with different lesions in the c-myc gene, including those which do not differentiate in response to DMSO treatment. Here we demonstrate that DMSO induces a rapid, but transient, reduction in the steady-state level of c-myc RNA in human 'erythroid' K562 cells, Burkitt lymphoma lines Daudi and Raji, human T cell lymphoblastoid line CEM and mouse lymphoma line L1210. However, DMSO treatment does not produce a similar effect in the human colon adenocarcinoma cell line COLO 320 or HeLa epithelial cervical carcinoma cells. These observations demonstrate that the DMSO induced modulation of c-myc RNA levels is a more common phenomenon than previously recognised, and is not necessarily correlated with either the induction of cellular differentiation or growth arrest.

Cell Differentiation↗
PubMed1988

Binding of thyroid hormone receptor-associated factors to rGH gene 5' flanking sequences.

Binding of semi-purified rat liver triiodothyronine (T3) nuclear receptor (T3-nR) to sequences in the 5' flanking DNA of rat growth hormone (rGH) has been evaluated by the DNAse-footprinting technique. T3-nR was obtained by extraction in 0.3 M KCl and partially purified by column chromatography to 80 pmol/mg T3 maximal binding capacity (MBC). The T3-nR preparation protects the sequence -330 to -314 (5'-CAGTGAACAAACGATG-3') which may represent a repressor element, the sequence -224 to -192 (5'-CATTGCCCATAAACCTGAGC-3') which is thought to involve positive T3 regulation, the sequence -98 to -78 (5'-GCTCCAGCCATGAATAAATG-3') which may regulate basal expression, and the TATA box area. Although the DNAse footprints occur only with preparations containing functional T3-nR, it is uncertain whether each protected zone is due to binding of T3-nR or associated proteins. Protection is associated with enhanced DNAse digestion around the protected sites, involves both strands, and does not appear to require T3 occupancy of the receptor. Only the areas described are protected in the studies, and non-specific proteins such as albumin, core histones, and nuclear extracts without receptor activity, have no effect. A putative consensus enhancer sequence -CCAGAGCCAAGG- conveying T3 responsivity is suggested on the basis of comparison to sequences in four T3 responsive genes.

Animals↗