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Biomedical subjects

M W Taylor

Publications and source records attributed to M W Taylor.

At least 19 recordsLinked to original sources
PubMed2001

Antitumor activity of recombinant adenoviral vectors expressing murine IFN-alpha in mice injected with metastatic IFN-resistant tumor cells.

Recent studies have shown that gene therapy with type I interferon (IFN) in an adenovirus vector is a powerful tool to suppress the growth of human tumors transplanted in immune-deficient mice. However, in these studies the host immune-mediated effects, which may be important in mediating the long-term control of tumor growth by these cytokines, was not studied. In this paper, we evaluate the antitumor efficacy of different adenoviral vectors containing mouse IFN-alpha genes (i.e., a first-generation replication-defective vector containing IFN-alpha1 and two different second-generation vectors containing IFN-alpha2) in immunocompetent DBA/2 mice transplanted with highly metastatic Friend leukemic cells resistant in vitro to type I IFN. We found that injection of all the different adenovirus vectors containing mouse IFN-alpha( genes resulted in a marked antitumor response in mice transplanted either subcutaneously or intravenously with IFN-resistant Friend leukemic cells compared to tumor-bearing animals inoculated with a control vector. Tumor growth inhibition after injection of IFN-adenovirus vectors was associated with a prolonged presence of high IFN levels in the sera of the injected mice. Suppression of metastatic tumor growth was also observed after a single injection of the IFN--adenovirus recombinant vectors, whereas a comparable antitumor response generally required several injections of high doses of IFN. Altogether, these results demonstrate that IFN--adenoviral vectors can efficiently inhibit metastatic tumor growth by host-mediated mechanisms and suggest that adenovirus-mediated IFN-alpha gene therapy may represent an attractive alternative to the conventional clinical use of this cytokine, which generally requires multiple injections of high IFN doses for a prolonged period of time.

Adenoviridae↗
PubMed2000

Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation.

Dendritic cells (DCs) play a key role in the activation and regulation of B and T lymphocytes. Production of indoleamine 2, 3-dioxygenase (IDO) by macrophages has recently been described to result in inhibition of T cell proliferation through tryptophan degradation. Since DCs can be derived from monocytes, we sought to determine whether DCs could produce IDO which could potentially regulate T cell proliferation. Northern blot analysis of RNA from cultured monocyte-derived human DC revealed that IDO mRNA was induced upon activation with CD40 ligand and IFN-gamma. IDO produced from activated DCs was functionally active and capable of metabolizing tryptophan to kynurenine. Activated T cells were also capable of inducing IDO production by DCs, which was inhibited by a neutralizing Ab against IFN-gamma. DC production of IDO resulted in inhibition of T cell proliferation, which could be prevented using the IDO inhibitor 1-methyl-dl -tryptophan. These results suggest that activation of DCs induces the production of functional IDO, which causes depletion of tryptophan and subsequent inhibition of T cell proliferation. This may represent a potential mechanism for DCs to regulate the immune response.

CD40 Ligand↗
PubMed2000

Analysis of transcription factors regulating induction of indoleamine 2,3-dioxygenase by IFN-gamma.

IFN-gamma treatment of the human carcinoma cell line ME180 causes cell death due to induction of indoleamine 2,3-dioxygenase (IDO) and resulting starvation for tryptophan. A mutant cell line 3B6A derived from ME180 was resistant to IFN-gamma because of loss of IDO activity. Cotransfecting an IDO promoter-chloramphenicol acetyl transferase (CAT) construct with IFN regulatory factor-1 (IRF-1) resulted in induction of CAT activity in both ME180 and 3B6A cells even in the absence of IFN-gamma. This induction was reduced by cotransfection with IRF-2. However, IRF-1 was not able to restore IDO activity, suggesting a possible repressor site outside the IDO promoter region. Stat1alpha (p91) restored both CAT and IDO activities in 3B6A cells following IFN-gamma treatment. 3B6A cells doubly treated with IFN-gamma and IFN-alpha or IFN-beta restored IDO activity, although neither cytokine on its own could induce IDO. Western blot analysis showed that both constitutive expression and induction of Stat1alpha by IFN-gamma were reduced in 3B6A cells, and double treatment of IFN-gamma with IFN-alpha or IFN-beta restored the expression level of Statla. Electrophoretic mobility shift assays indicated that Stat1 binds to the IFN-gamma-activated sequence (GAS) region in the IDO promoter in ME180 cells following IFN-gamma treatment. Our results indicated that the defect in 3B6A cells was reduced expression of Stat1alpha and that IRF-1, NF-kappaB, and PKR were all involved to some extent in the induction of IDO following IFN-gamma treatment.

Base Sequence↗
PubMed2000

An indoleamine 2,3-dioxygenase-negative mutant is defective in stat1 DNA binding: differential response to IFN-gamma and IFN-alpha.

We have previously reported the isolation of mutant cell lines from the human carcinoma line ME180 that are resistant to the antiproliferative effect of interferon-gamma (IFN-gamma). These cell lines were defective in the induction of indoleamine 2,3-dioxygenase (IDO), a key enzyme of tryptophan catabolism. One of these cell lines, 3B6A, was chosen for further study. This cell line was also defective in the ability of IFN-gamma to protect against vesicular stomatitis virus (VSV) infection. However it maintained a normal antiviral response to IFN-alpha. A promoter-chloramphenicol acetyltransferase (CAT) construct containing the promoter region of IDO, which includes IFN-gamma activation site (GAS), IFN-stimulated response element-1 (ISRE-1), and ISRE-2 regions, was not expressed in 3B6A in the presence of IFN-gamma, indicating that the defect was likely to be in either Stat1 or IFN regulatory factor-1 (IRF-1), transcription factors known to bind to these cis-acting sequences. The induction of other IFN-gamma-inducible genes, such as tryptophanyl-tRNA synthetase (hWRS), was also affected. Electrophoretic mobility shift assays (EMSA) comparing nuclear extracts from parental and mutant cells indicated that Stat1 from the mutant did not bind to GAS sequences. However, Western blot analysis indicated that Stat1 protein was present. This IDO-negative phenotype can be reversed by transfection with a Stat1 expression vector. DNA sequencing of the Stat1 cDNA from wild-type and 3B6A cells indicated that an amino acid change occurred in the Stat1 protein of the mutant at W573, a tryptophan conserved in all known Stat proteins. We hypothesize that a change in this region of the Stat protein affects the response to IFN-gamma but not to IFN-alpha.

Chloramphenicol O-Acetyltransferase↗
PubMed2000

The effects of interferon on the expression of human papillomavirus oncogenes.

The effects of interferon (IFN)-alpha, IFN-beta and IFN-gamma on human papillomavirus (HPV) oncogene expression were studied in various cervical carcinoma cell lines containing integrated copies of either HPV type 16 or HPV type 18. The levels of E6 and E7 transcripts were examined 6 h and 30 h after treatment with IFN. In HeLa cells, all three classes of IFNs effected a decrease in the level of HPV-18 E6 and E7 transcripts. On the other hand, none of the IFNs altered the level of these transcripts in C-4II cells. Only IFN-gamma decreased the level of HPV-16 E6 and E7 transcripts in CaSki and HPK1A cells, while IFN-gamma actually increased the level of these transcripts in SiHa cells. This differential IFN regulation of HPV expression in various cervical cancer cell lines may account for the contradictory clinical results observed after treatment of cervical cancer with IFN.

Female↗
PubMed1999

APT1, but not APT2, codes for a functional adenine phosphoribosyltransferase in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has two separate genes (APT1 and APT2) that encode two potentially different forms of adenine phosphoribosyltransferase (APRT). However, genetic analysis indicated that only APT1 could code for a complementing activity. Cloning and expression of both the APT1 and APT2 genes in Escherichia coli showed that although discrete proteins (APRT1 and APRT2) were made by these genes, only APRT1 had detectable APRT activity. Northern and Western blot analyses demonstrated that only APT1 was transcribed and translated under normal physiological conditions in yeast. Phylogenetic analysis revealed that APRT1 and APRT2 are evolutionary closely related and that they arise from a gene duplication event. We conclude that APT1 is the functional gene in S. cerevisiae and that APT2 is a pseudogene.

Adenine Phosphoribosyltransferase↗
PubMed1999

Isolation and characterization of the Saccharomyces cerevisiae XPT1 gene encoding xanthine phosphoribosyl transferase.

A new Saccharomyces cerevisiae gene, XPT1, was isolated as a multicopy suppressor of a hypoxanthine phosphoribosyl transferase (HPRT) defect. Disruption of XPT1 affects xanthine utilization in vivo and results in a severe reduction of xanthine phosphoribosyl transferase (XPRT) activity while HPRT is unaffected. We conclude that XPT1 encodes XPRT in yeast.

Genes, Fungal↗
PubMed1998

Induction of spermidine/spermine N1-acetyltransferase by interferon type I in cells of hematopoietic origin.

A number of novel genes activated by type I interferons (IFNs) were identified by differential display. Of five induced genes examined, four were of unknown function. However, one gene sequence was identical to the human spermidine/spermine N1-acetyltransferase, the rate-limiting enzyme in polyamine catabolism. This enzyme was induced by type I IFNs in a series of hematopoietic cell lines, including Daudi, HL-60, HPBMa, and Wil-2. No induction above constitutive levels occurred in a cell line of epithelial (ME180) or liver (HepG2) origin following treatment with type I IFN. Spermidine/spermine N1-acetyltransferase was not induced by IFN-gamma in Daudi or ME180 cells. That induction occurred not only at the level of transcription but also at the enzyme level was confirmed by direct enzyme assays. As the levels of polyamines are related to cell viability, we propose that induction of this enzyme by IFN may be directly related to the anti-proliferative response to type I IFNs.

Acetyltransferases↗
PubMed1998

Audit of populations in general practice: the creation of a national resource for the study of morbidity in Scottish general practice.

STUDY OBJECTIVE: To create a national data resource for studying morbidity in Scottish general practice, complementary to existing information systems and available for management and research purposes at national and local levels. DESIGN: The Department of General Practice, University of Aberdeen has worked since 1988 to collect and analyse computerised information at practice, regional, and national levels by distribution of a floppy disk-based software program, which extracts a predetermined dataset from each general practice computer system. SETTING: Almost 100% of patients in Scotland are registered with a general practitioner. Scotland has a national computer system, General Practice Administration System for Scotland (GPASS), used by over 75% of all Scottish practices. Escalating costs of health care and demographic changes in the national population emphasise the monetary value of the gatekeeper role of general medical practice. General practitioners' increasing involvement in the provision and purchasing of care has raised the importance of the management of populations as well as the care of individual patients. PATIENTS: Collection of major morbidity and prescribing data from up to 2.4 million patients, approximately half the population of Scotland, takes place biannually. A subset of practices (population 282,700 patients; 52 practices) are continuously collecting doctor/patient contact information (symptoms or diagnoses). MAIN RESULTS: The data collected provide information at the level of the individual patient. Morbidity, prescribing, screening, and administrative data can be linked by patient, date or postcode. The sample population studied is representative by age, sex, deprivation, and sparsity (using the postcode) of the national population. Large sub-populations of patients satisfying a selected criteria can be extracted for further study of needs assessment or of epidemiological research. CONCLUSIONS: The gatekeeping role of Scottish general practice and the predominance of GPASS favours standardisation of methods of data capture and the construction of large regional, national, and Continuous Morbidity databases. Analysis by geographical, demographic, and temporal distributions allows the changing patterns of illness and provision of health care to be studied in substantial detail to the benefit of patients, doctors, and the national health service.

Adolescent↗
PubMed1997

Purification and characterization of adenine phosphoribosyltransferase from Saccharomyces cerevisiae.

Adenine phosphoribosyltransferase (APRT) from Saccharomyces cerevisiae was purified approximately 1500-fold. The enzyme catalyzes the Mg-dependent condensation of adenine and 5-phosphoribosylpyrophosphate (PRPP) to yield AMP. The purification procedure included anion exchange chromatography, chromatofocusing and gel filtration. Elution of the enzyme from the chromatofocusing column indicated a pI value of 4.7. The molecular mass for the native enzyme was 50 kDa; however, upon electrophoresis under denaturing conditions two bands of apparent molecular mass of 29 and 20 kDa were observed. We have previously reported the presence of two separate coding sequences for APRT, APT1 and APT2 in S. cerevisiae. The appearance of two bands under denaturing conditions suggests that, unlike other APRTs, this enzyme could form heterodimers. This may be the basis for substrate specificity differences between this enzyme and other APRTs. Substrate kinetics and product inhibition patterns are consistent with a ping-pong mechanism. The Km for adenine and PRPP were 6 microM and 15 microM, respectively and the Vmax was 15 micromol/min. These kinetic constants are comparable to the constants of APRT from other organisms.

Adenine↗
PubMed1996

Importance of the two interferon-stimulated response element (ISRE) sequences in the regulation of the human indoleamine 2,3-dioxygenase gene.

Indoleamine 2,3-dioxygenase (INDO) is the rate-limiting enzyme in the catabolism of the essential amino acid L-tryptophan. It is induced strongly in many cell lines following interferon-gamma treatment. We report the cloning and characterization of the full-length human INDO promoter. This promoter is 1,245 base pairs long and includes two interferon-stimulated response elements (ISRE) separated by an approximately 1-kilobase sequence. The presence of these two ISREs is critical for maximum INDO promoter activity (50-fold induction). When the ISREs are present in two separate fragments cloned upstream of the chloramphenicol acetyltransferase (CAT) reporter vector, the INDO promoter activity drops significantly (7-fold induction). 5' end deletions of the wild type promoter sequence indicate that removal of the ISRE (ISRE1) at position -1126 reduces the induction level to approximately 25-fold. This activity does not change appreciably when the promoter is deleted down to position -241. Furthermore, site-directed mutagenesis of ISRE1 also decreases the promoter activity in a similar way. When ISRE1 is kept intact, deletion of the second ISRE (ISRE2) at position -111 leads to only 11-fold induction of the promoter. A similar result is obtained when substitution mutations are introduced in ISRE2. Deletion of a 748-base pair sequence between the two ISREs only shows a slight decrease in the INDO promoter activity. These data indicate that the two ISRE sequences are required for the full transcriptional induction of the interferon-gamma-inducible human INDO gene. INDO activity is not induced in the hepatic cell line HepG2. An analysis of INDO-CAT activity in this cell line indicated that the lack of INDO activity was at the transcriptional level and could reflect either the presence of a repressor or lack of a transcription factor. This lack of induction could be correlated with a truncated or unstable IRF-1. However, the levels of IRF-2, JAK2, and STAT 91 were similar in both ME180 and HepG2 cells.

Base Sequence↗
PubMed1996

Treatment of a human breast cancer xenograft with an adenovirus vector containing an interferon gene results in rapid regression due to viral oncolysis and gene therapy.

Treatment of a human breast cancer cell line (MDA-MB-435) in nude mice with a recombinant adenovirus containing the human interferon (IFN) consensus gene, IFN-con1 (ad5/IFN), resulted in tumor regression in 100% of the animals. Tumor regression occurred when virus was injected either within 24 hr of tumor cell implantation or with established tumors. However, regression of the tumor was also observed in controls in which either the wild-type virus or a recombinant virus containing the luciferase gene was used, although tumor growth was not completely suppressed. Tumor regression was accompanied by a decrease in p53 expression. Two other tumors, the human myelogenous leukemic cell line K562 and the hamster melanoma tumor RPMI 1846, also responded to treatment but only with ad5/IFN. In the case of K562 tumors, there was complete regression of the tumor, and tumors derived from RPMI 1846 showed partial regression. We propose that the complete regression of the breast cancer with the recombinant virus ad5/IFN was the result of two events: viral oncolysis in which tumor cells are being selectively lysed by the replication-competent virus and the enhanced effect of expression of the IFN-con1 gene. K562 and RPMI 1846 tumors regressed only as a result of IFN gene therapy. This was confirmed by in vitro analysis. Our results indicate that a combination of viral oncolysis with a virus of low pathogenicity, itself resistant to the effects of IFN and IFN gene therapy, might be a fruitful approach to the treatment of a variety of different tumors, in particular breast cancers.

Adenoviruses, Human↗
PubMed1996

Cell surface binding characteristics correlate with consensus type I interferon enhanced activity.

The binding characteristics of a genetically engineered consensus interferon with unusually high biologic activity were compared to the characteristics of recombinant interferon-alpha 2. Both interferon-alpha 2 and the consensus interferon produced typical biphasic Scatchard plots, indicating multiple independent binding sites. The consensus interferon, which exhibited a biologic potency more than 10-fold greater than all other type I interferons, also exhibited binding site affinities greater than those for IFN-alpha 2b. In addition, a larger number of high, and low-affinity cell surface sites were recognized by the consensus interferon, resulting in equivalent numbers of sites at reduced molar concentrations compared to IFN-a2b. Thus, at any given biologic activity, similar numbers of sites were bound by the consensus interferon and IFN-alpha 2, despite differences in their molar concentrations. No differences in internalization kinetics were identified between the two interferons, indicating that the differences in cell surface binding may be sufficient to produce the differences in biologic activity.

Cell Line↗
PubMed1996

The biologic activity and molecular characterization of a novel synthetic interferon-alpha species, consensus interferon.

Consensus interferon (Infergen) is a wholly synthetic type I interferon (IFN), developed by scanning several interferon-alpha nonallelic subtypes and assigning the most frequently observed amino acid in each position, resulting in a consensus sequence. The antiviral, antiproliferative, NK cell activation activity, cytokine induction, and interferon-stimulated gene-induction activity of consensus interferon has been compared with naturally occurring type I interferons. In all of these comparisons, consensus interferon had a higher activity when compared, on a mass basis, with IFN-alpha 2a and IFN-alpha 2b, although the activity was the same for all of these parameters on an antiviral unit basis. That a synthetic type I interferon could have higher activities than naturally occurring molecules is surprising and may be a result of the higher affinity for the array of type I interferon receptors demonstrated for consensus interferon when compared with IFN-alpha. In contrast, consensus interferon was shown to be an inferior inducer of IL-1 beta when compared with IFN-alpha. These results may reflect differential binding to multiple accessory proteins interacting with a type I interferon receptor. These unique biologic properties may lead to a favorable clinical benefit for consensus interferon when compared with the naturally occurring recombinant molecules. Ongoing clinical trials will ascertain whether consensus interferon can be used in a wide array of disease situations, such as chronic viral infections and certain malignancies.

Amino Acid Sequence↗
PubMed1996

Treatment of ME180 cells with interferon-gamma causes apoptosis as a result of tryptophan starvation.

Indoleamine 2'3 dioxygenase (INDO), the rate-limiting enzyme in the catabolism of the essential amino acid L-tryptophan, is induced in many cell lines following interferon gamma (IFN-gamma) treatment. The induction of this enzyme has been associated with the antiparasitic and cytotoxic activities of human IFN-gamma. DNA analysis coupled to morphologic studies indicated that ME180 cells underwent apoptosis within 48 h of treatment with IFN-gamma. We hypothesized that apoptosis results from L-tryptophan starvation following INDO induction. This was confirmed by the prevention of apoptosis on adding back tryptophan to IFN-gamma-treated cells and the induction of apoptosis by removing tryptophan from the medium in the absence of IFN-gamma.

Apoptosis↗