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The role of spatial mixing in the spread of foot-and-mouth disease.

A model of epidemic dispersal (based on the assumption that susceptible cattle were homogeneously mixed over space, or non-spatial model) was compared to a partially spatially explicit and discrete model (the spatial model), which was composed of differential equations and used geo-coded data (Euclidean distances between county centroids). While the spatial model accounted for intra- and inter-county epidemic spread, the non-spatial model did not assess regional differences. A geo-coded dataset that resembled conditions favouring homogeneous mixing assumptions (based on the 2001 Uruguayan foot-and-mouth disease epidemic), was used for testing. Significant differences between models were observed in the average transmission rate between farms, both before and after a control policy (animal movement ban) was imposed. They also differed in terms of daily number of infected farms: the non-spatial model revealed a single epidemic peak (at, approximately, 25 epidemic days); while the spatial model revealed two epidemic peaks (at, approximately, 12 and 28 days, respectively). While the spatial model fitted well with the observed cumulative number of infected farms, the non-spatial model did not (P<0.01). In addition, the spatial model: (a) indicated an early intra-county reproductive number R of approximately 87 (falling to <1 within 25 days), and an inter-county R<1; (b) predicted that, if animal movement restrictions had begun 3 days before/after the estimated initiation of such policy, cases would have decreased/increased by 23 or 26%, respectively. Spatial factors (such as inter-farm distance and coverage of vaccination campaigns, absent in non-spatial models) may explain why partially explicit spatial models describe epidemic spread more accurately than non-spatial models even at early epidemic phases. Integration of geo-coded data into mathematical models is recommended.

Animals↗

Gene expression omnibus: microarray data storage, submission, retrieval, and analysis.

The Gene Expression Omnibus (GEO) repository at the National Center for Biotechnology Information archives and freely distributes high-throughput molecular abundance data, predominantly gene expression data generated by DNA microarray technology. The database has a flexible design that can handle diverse styles of both unprocessed and processed data in a Minimum Information About a Microarray Experiment-supportive infrastructure that promotes fully annotated submissions. GEO currently stores about a billion individual gene expression measurements, derived from over 100 organisms, submitted by over 1500 laboratories, addressing a wide range of biological phenomena. To maximize the utility of these data, several user-friendly web-based interfaces and applications have been implemented that enable effective exploration, query, and visualization of these data at the level of individual genes or entire studies. This chapter describes how data are stored, submission procedures, and mechanisms for data retrieval and query. GEO is publicly accessible at http://www.ncbi.nlm.nih.gov/projects/geo/.

Animals↗

Metropolitan and non-metropolitan trends in coronary heart disease mortality within Appalachia, 1980-1997.

OBJECTIVES: In this article, we report on metropolitan and non-metropolitan trends in coronary heart disease (CHD) mortality within the Appalachian Region for the period 1980 to 1997. We hypothesized that trends in CHD mortality would be less favorable in non-metropolitan populations with diminished access to social, economic, and medical care resources at the community level. METHODS: Our study population consisted of adults aged 35 years and older who resided within the 399 counties of the Appalachian Region between 1980 and 1997. We examined mortality trends for sixteen geo-demographic groups, defined by gender, age, race, and metropolitan status of county of residence. For each geo-demographic group, we calculated annual age-adjusted CHD mortality rates. Line graphs of these temporal trends were created, and log-linear regression models provided estimates of the average annual percent change in CHD mortality from 1980 to 1997. Data on social, economic, and medical care resources for metropolitan vs. non-metropolitan counties were also analyzed. RESULTS: Rates of CHD mortality were consistently higher in non-metropolitan areas compared with metropolitan areas for blacks of all ages and for younger whites. CHD mortality declined among almost all geo-demographic groups, but rates of decline were slower among non-metropolitan vs. metropolitan residents, blacks vs. whites, women vs. men, and older vs. younger adults. Non-metropolitan areas had fewer socioeconomic and medical care resources than metropolitan areas in 1990. CONCLUSIONS: Appalachia, particularly non-metropolitan Appalachia, needs policies and programs that will enhance both primary and secondary prevention of CHD, and help diminish racial inequalities in CHD mortality trends.

Adult↗

How H-bonding affects aromaticity of the ring in variously substituted phenol complexes with bases. 4. Molecular geometry as a source of chemical information.

Aromaticity of the ring of variously substituted phenols in their H-bonded complexes with various bases was a subject of analysis based on 664 geometries retrieved from CSD and by use of the aromaticity index HOMA. GEO and EN, the components of the HOMA index, describing a decrease of aromaticity due to an increase of bond alternation (GEO term) and bond elongation (EN term), were also studied. There is an approximate monotonic dependence of HOMA and GEO on the H-bond strength estimated by the C-O bond length of the hydroxyl group in phenols.

Journal Article↗

Elevated urokinase-type plasminogen activator receptor expression in a colon cancer cell line is due to a constitutively activated extracellular signal-regulated kinase-1-dependent signaling cascade.

The urokinase-type plasminogen activator receptor (u-PAR) facilitates extracellular matrix degradation in part by accelerating plasmin formation at the cell surface. We previously reported that u-PAR expression is elevated in colon cancer cell lines characterized by their in vitro invasive capacity. Since, u-PAR expression is increased by a variety of growth factors, which signal through the extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2), we determined if these mitogen-activated protein kinases (MAPKs) regulate u-PAR expression in two cultured colon cancer cell lines. An in-gel kinase assay showed that ERK1 activity was considerably higher in RKO cells, which display > or = 10(5) receptors/cell, than the GEO cells which have approximately 10(4) urokinase receptors per cell. The expression of either an ERK-inactivating phosphatase (CL100), or a kinase-defective ERK1, decreased the activity of a u-PAR promoter-driven CAT reporter in RKO cells. Immune complex kinase assays indicated that the constitutive ERK1 activity in RKO cells was largely a result of an activated MEK1. Further, treatment of RKO cells with a specific inhibitor (PD 098059) of MEK1 activation, which diminished ERK1 activity, reduced the amount of urokinase specifically bound to the cell surface and this was associated with reduced laminin degradation. The expression of a dominant negative c-Raf-1 also reduced u-PAR promoter activity suggesting that MEK1 activation involved an activator at, or upstream, of this serine-threonine kinase. Transfection of the u-PAR-deficient GEO cells with a constitutively activated MEK1 expression construct up-regulated u-PAR promoter activity. Similarly treatment of GEO cells with a phosphatase inhibitor (sodium vanadate) caused a dose-dependent increase in ERK1 activity which paralleled increased cell surface binding of urokinase. Taken together, these data suggest that elevated u-PAR expression, in at least a sub-population of colon cancer, is partly a consequence of a constitutively activated ERK-1-dependent signaling cascade.

Calcium-Calmodulin-Dependent Protein Kinases↗

Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1.

A construct, MRE-beta Geo, with five metal response elements fused to a selectable reporter gene was transfected into BHK cells and a stable clone that could be induced up to 100-fold by zinc, cadmium, bismuth, silver, cobalt, copper, mercury, or nickle was isolated. Some, and perhaps all, of these metals induce MRE-beta Geo by displacing zinc. Transfection of these cells with a construct encoding the transcriptional activator MTF-1 resulted in constitutive expression of MRE-beta Geo, whereas expression of an antisense MTF-1 construct in these cells prevented induction by all of the metals. A variant cell line with high constitutive expression in the absence of added metals was isolated; normal regulation was restored by cell fusion. These results suggest that regulation of metallothionein genes by metals is mediated by MTF-1 interacting with metal response elements and that zinc functions to release MTF-1 from an inhibitor.

Animals↗

Sp3 is a transcriptional repressor of transforming growth factor-beta receptors.

MCF-7E breast cancer cells express transforming growth factor-beta (TGF-beta) receptors RI and RII in comparison to MCF-7L cells. We present data showing that Sp3 acts as a transcriptional repressor of RI and RII in MCF-7L cells and GEO colon cancer cells. MCF-7L and GEO cells express high levels of Sp3 protein. Gel shift analysis indicated enhanced binding of Sp3 from MCF-7L cells to a consensus Sp1 oligonucleotide. Southwestern data indicated increased binding of Sp3 to RI and RII promoters in MCF-7L cells, suggesting a correlation between Sp3 binding and reduced expression of TGF-beta receptors in MCF-7L cells. Cotransfection of CMV-Sp3 cDNA with RI and RII promoter-luciferase reporter constructs decreased RI and RII promoter activities by 70% in MCF-7E and GEO cells. Southwestern analysis detected the binding of transiently expressed Sp3 to RI and RII promoters in MCF-7E cells. Significantly, ectopic Sp3 expression led to repression of RI and RII transcripts in MCF-7E cells. This report demonstrates that inappropriate overexpression of Sp3 is a mechanism that contributes to repression of TGF-beta receptors.

DNA↗

cDNA molecular cloning of Geotrichum candidum lipase.

The cDNA clone of Geotrichum candidum (Geo.) lipase was isolated from the Geo. cDNA library by colony hybridization using 32P-labeled oligonucleotides corresponding to a partial amino acid sequence of this enzyme. The nucleotide sequence of the cDNA determined by the dideoxy chain terminating method included some partial amino acid sequences determined by Edman degradation, and the overall amino acid composition deduced from the cDNA coincided with that from amino acid analysis of this protein. The cloned cDNA coded a protein of 554 amino acids and a hydrophobic signal sequence of 19 amino acids. Geo. lipase contained the -Gly-X-Ser-X-Gly- sequence which is believed to form part of the interfacial lipid recognition site.

Amino Acid Sequence↗

Sinoatrial node dysfunction and early unexpected death of mice with a defect of klotho gene expression.

BACKGROUND: Homozygous mutant mice with a defect of klotho gene expression (kl/kl) show multiple age-related disorders and premature death from unknown causes. METHODS AND RESULTS: The kl/kl mice subjected to 20-hour restraint stress showed a high rate (20/30) of sudden death, which was associated with sinoatrial node dysfunction (conduction block or arrest). Heart rate and plasma norepinephrine of kl/kl mice, unlike those of wild-type (WT) mice, failed to increase during the stress. Intrinsic heart rate after pharmacological blockade of autonomic nerves in kl/kl mice was significantly lower than that in WT mice (380+/-33 versus 470+/-44 bpm; n=7). The sinus node recovery time after an overdrive pacing (600 bpm, 30 seconds) in kl/kl mice was significantly longer than in WT mice (392+/-37 versus 233+/-24 ms; n=6). In isolated sinoatrial node preparations, the positive chronotropic effect of isoproterenol was significantly less, whereas the negative chronotropic effect of acetylcholine was significantly greater in kl/kl than in WT mice. There was no degenerative structural change in the sinoatrial node of kl/kl mice. The precise localization of klotho was analyzed in newly prepared klotho-null mice with a reporter gene system (kl(-geo)). Homozygous kl(-geo) mice showed characteristic age-associated phenotypes that were almost identical to those of kl/kl mice. In the kl(-geo) mice, klotho expression was recognized exclusively in the sinoatrial node region in the heart in addition to parathyroid, kidney, and choroid plexus. CONCLUSIONS: In the heart, klotho is expressed solely at the sinoatrial node. klotho gene expression is essential for the sinoatrial node to function as a dependable pacemaker under conditions of stress.

Aging, Premature↗

Shock and vibration data acquisition system for off-road vehicle operator health and safety assessment.

A data acquisition software and hardware system was developed for acquiring geo-referenced shock and vibration data using National Instruments' LabView graphical programming language. This was used in conjunction with a modular data acquisition and signal conditioning system and a Differential Global Positioning System (DGPS) receiver. A prototype vehicle obstacle course, which introduced spatially varying shock events to vehicles as they traversed the course, was constructed. Obstacles consisted of both repetitious and single discrete events. A series of investigations was conducted on the obstacle course to evaluate the performance and characteristics of the DAQ system and the tractor when exposed to shock and vibration events. Spectral and time domain plots obtained from the geo-referenced data acquisition system (GDAQ) system under static, highway, and off-road obstacle course conditions were evaluated to demonstrate that the system performed as expected. The migration of experiments from laboratory to field gave confidence that this system could be used to collect shock and vibration data over a wide range of frequencies. The use of geo-referenced data records proved beneficial in isolating and extracting data segments of interest from a continuous data record.

Agriculture↗

Elucidating the Mechanism of Xiaoqinglong Decoction in Chronic Urticaria Treatment: An Integrated Approach of Network Pharmacology, Bioinformatics Analysis, Molecular Docking, and Molecular Dynamics Simulations.

INTRODUCTION: Xiaoqinglong Decoction (XQLD) is a traditional Chinese medicinal formula commonly used to treat chronic urticaria (CU). However, its underlying therapeutic mechanisms remain incompletely characterized. This study employed an integrated approach combining network pharmacology, bioinformatics, molecular docking, and molecular dynamics simulations to identify the active components, potential targets, and related signaling pathways involved in XQLD's therapeutic action against CU, thereby providing a mechanistic foundation for its clinical application. METHODS: The active components of XQLD and their corresponding targets were identified using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. CU-related targets were retrieved from the OMIM and GeneCards databases. Subsequently, core components and targets were determined via protein-protein interaction (PPI) network analysis and component-target-pathway network construction. Topological analyses were performed using Cytoscape software to prioritize core nodes within these networks. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted via the DAVID database to identify enriched biological processes and signaling pathways. Molecular docking was performed to evaluate binding interactions between key components and core targets, while molecular dynamics (MD) simulations were employed to assess the stability of the component-target complexes with the lowest binding energy. Finally, CU-related targets of XQLD were validated using datasets from the Gene Expression Omnibus (GEO) database. RESULTS: A total of 135 active components and 249 potential targets of XQLD were identified, alongside 1,711 CU-related targets. Core components, such as quercetin, kaempferol, beta-sitosterol, naringenin, stigmasterol, and luteolin, exhibited high degree values in the constructed networks. The core targets identified included AKT1, TNF, IL6, TP53, PTGS2, CASP3, BCL2, ESR1, PPARG, and MAPK3. GO and KEGG pathway enrichment analyses revealed the PI3K-Akt signaling pathway as a central regulatory mechanism. Molecular docking studies demonstrated strong binding affinities between active components and core targets, with the stigmasterol-AKT1 complex exhibiting the lowest binding energy (-11.4 kcal/mol) and high stability in MD simulations. Validation using GEO datasets identified 12 core genes shared between CU-related targets and XQLD-associated targets, including PTGS2 and IL6, which were also prioritized as core targets in the network pharmacology analyses. DISCUSSION: This study comprehensively integrates multidisciplinary approaches to clarify the potential molecular mechanisms of XQLD in treating CU, highlighting its multitarget and multipathway synergistic effects. Molecular docking and dynamics simulations confirm the stable interaction between stigmasterol and the core target AKT1. Additionally, GEO dataset analysis verifies the pathogenic relevance of targets such as PTGS2 and IL6, significantly enhancing the credibility of our findings. These results provide a modern scientific basis for the traditional therapeutic effects of XQLD on CU and have important implications for developing multitarget treatments for this condition. However, this study mainly relies on database mining and computational simulations. Further in vitro and in vivo experimental validations are needed to confirm the predicted component-target-pathway interactions. CONCLUSION: This study identifies the active components, potential targets, and pathways through which XQLD exerts therapeutic effects on CU. These findings provide a theoretical foundation for further mechanistic studies and support their clinical application in the treatment of CU.

Molecular Docking Simulation↗

Antitumor activity of sequential treatment with topotecan and anti-epidermal growth factor receptor monoclonal antibody C225.

Epidermal growth factor (EGF)-related proteins such as transforming growth factor alpha (TGF-alpha) control cancer cell growth through autocrine and paracrine pathways. Overexpression of TGF-alpha and/or its receptor (EGFR) has been associated with a more aggressive disease and a poor prognosis. The blockade of EGFR activation has been proposed as a target for anticancer therapy. Monoclonal antibody (MAb) C225 is an anti-EGFR humanized chimeric mouse MAb that is presently in Phase II clinical trials in cancer patients. Previous studies have suggested the potentiation of the antitumor activity of certain cytotoxic drugs, such as cisplatin and doxorubicin, in human cancer cell lines by treatment with anti-EGFR antibodies. We have evaluated in human ovarian, breast, and colon cancer cell lines, which express functional EGFR, the antiproliferative activity of MAb C225 in combination with topotecan, a cytotoxic drug that specifically inhibits topoisomerase I and that has shown antitumor activity in these malignancies. A dose-dependent supraadditive increase of growth inhibition in vitro was observed when cancer cells were treated with topotecan and MAb C225 in a sequential schedule. In this respect, the cooperativity quotient, defined as the ratio between the actual growth inhibition obtained by treatment with topotecan followed by MAb C225 and the sum of the growth inhibition achieved by each agent, ranged from 1.2 to 3, depending on drug concentration and cancer cell line. Treatment with MAb C225 also markedly enhanced apoptotic cell death induced by topotecan. For example, in GEO colon cancer cells, 5 nM topotecan, followed by 0.5 microg/ml MAb C225, induced apoptosis in 45% cells as compared with untreated cells (6%) or to 5 nM topotecan-treated cells (22%). Treatment of mice bearing established human GEO colon cancer xenografts with topotecan or with MAb C225 determined a transient inhibition of tumor growth because GEO tumors resumed the growth rate of untreated tumors at the end of the treatment period. In contrast, an almost complete tumor regression was observed in all mice treated with the two agents in combination. This determined a prolonged life span of the mice that was significantly different as compared with controls (P < 0.001), to MAb C225-treated group (P < 0.001), or to the topotecan-treated group (P < 0.001). All mice of the topotecan plus MAb C225 group were the only animals alive 14 weeks after tumor cell injection. Furthermore, 20% of mice in this group were still alive after 19 weeks. The combined treatment with MAb C225 and topotecan was well tolerated by mice with no signs of acute or delayed toxicity. These results provide a rationale for the evaluation of the anticancer activity of the combination of topoisomerase I inhibitors and anti-EGFR blocking MAbs in clinical trials.

Animals↗

CA 19-9 monosialoganglioside content of human colorectal tumor cells correlates with tumor cell-induced platelet aggregation.

BACKGROUND: Gangliosides are involved in tumor cell-induced platelet aggregation (TCIPA). CA 19-9 is a monosialoganglioside detected in colon carcinoma cells. Therefore, this study was designed to evaluate whether there was any correlation between the CA 19-9 monosialoganglioside content and TCIPA. MATERIALS AND METHODS: The CA 19-9 content was determined on gangliosidic extracts of human colorectal tumor cells (GEO, WiDr, DLD-1, MIP) in comparison with the degree of TCIPA. RESULTS: CA 19-9 was detected in all cell lines, except MIP. The mean CA 19-9 content was 10.2, 30.2 and 82.6 U/microgram of sialic acid for GEO, DLD-1 and WiDr, respectively. CA 19-9 content directly correlated with TCIPA (p < 0.0001). Furthermore, incorporation of exogenous gangliosides in GEO cells resulted in an increase of CA 19-9 content, paralleled by a concomitant increase of TCIPA. CONCLUSIONS: The CA 19-9 monosialoganglioside may be involved in platelet/tumor cell interactions, thus playing an important role in the haematogenous metastases of colorectal cancer.

CA-19-9 Antigen↗

The utility of anonymised postcoded registration data provided by the Dental Practice Board.

OBJECTIVE: To evaluate the completeness of the postcodes held in the data sets of the Dental Practice Board (DPB), and the use of the location of the dental practice at which a patient is registered, as a proxy for the location of their residential address. BASIC RESEARCH DESIGN: A cross-sectional evaluation of anonymised person-based registration data held by the DPB. The study population was all children aged under 6 years registered with a dentist in the North West Region of England. The percentage of records that could be geo-referenced from the postcodes contained within the DPB's data sets was calculated. For all records which could be geo-referenced, the proportion of children who lived in the same health authority as the dental practice they were registered with was also calculated. MAIN OUTCOME MEASURES: A total of 193,466 children in the North West Region were registered with a general dental practitioner (GDP). Of this total 117,607 (60.8 percent) could be geo-referenced from the contents of the postcode field. However, there was considerable variation in the match across the region. The majority of children lived in the same health authority as the dental practice they were registered with. It was noticeable that a greater proportion (28.5 percent) of residents registered with a dentist located within the boundaries of Manchester Health Authority were not Manchester residents. CONCLUSIONS: Due to a significant proportion of missing postcodes, data presently held by the DPB cannot be used to calculate accurate registration rates at a small area level. Using the dental practice location as a proxy for residential address is inaccurate at health authority level, especially for large conurbations. This inaccuracy will increase at small area level. Registration data held by DPB are a potentially valuable information source, but before this information can be of use to commissioners the problem of poor postcode coverage will need to be resolved.

Child, Preschool↗

Muscular dystrophy in mice caused by two different alterations of dystrophin gene.

The study addressed the question of the functional significance of various isoforms of dystrophin. Mice bearing two different alterations of the dystrophin gene, mdx and mdx-beta geo, were examined. Dystrophic mice with mdx mutation do not express full-length dystrophins, while they express short dystrophins; on the other hand, the dystrophic mice with mdx-beta geo mutation express neither full-length nor short dystrophins. We found pathological changes typical for muscular dystrophy in the diaphragm of both mutant strains. No pathological changes were found in brains of either mdx or mdx-beta geo mutants. We concluded tentatively that short isoforms of dystrophin do not contribute to the muscular dystrophy and that they do not play any role in the development and maintenance of histological structure of the brain.

Animals↗

Antitumor effects of ZD6474, a small molecule vascular endothelial growth factor receptor tyrosine kinase inhibitor, with additional activity against epidermal growth factor receptor tyrosine kinase.

PURPOSE: Vascular endothelial growth factor (VEGF) is a major mitogen for endothelial cells and enhances vascular permeability. Enhanced VEGF secretion is found in human cancers and correlates with increased tumor neovascularization. ZD6474 is a p.o. bioavailable, VEGF flk-1/KDR receptor (VEGFR-2) tyrosine kinase inhibitor with antitumor activity in many human cancer xenografts and is currently in Phase I clinical development. EXPERIMENTAL DESIGN: We tested the effects of ZD6474 on EGFR phosphorylation in cell expressing functional epidermal growth factor receptor (EGFR) and the antiproliferative and the proapoptotic activity of ZD6474 alone or in combination taxanes in human cancer cell lines with functional EGFR but lacking VEGFR-2. The antitumor activity of this drug was also tested in nude mice bearing established GEO colon cancer xenografts. RESULTS: ZD6474 causes a dose-dependent inhibition of EGFR phosphorylation in mouse NIH-EGFR fibroblasts and human MCF-10A ras breast cancer cells, two cell lines that overexpress the human EGFR. ZD6474 treatment resulted in a dose-dependent inhibition of soft agar growth in seven human cell lines (breast, colon, gastric, and ovarian) with functional EGFR but lacking VEGFR-2. A dose-dependent supra-additive effect in growth inhibition and in apoptosis in vitro was observed by the combined treatment with ZD6474 and paclitaxel or docetaxel. ZD6474 treatment of nude mice bearing palpable GEO colon cancer xenografts (which are sensitive to inhibition of EGFR signaling) induced dose-dependent tumor growth inhibition. Immunohistochemical analysis revealed a significant dose-dependent reduction of neoangiogenesis. The antitumor activity of ZD6474 in GEO tumor xenografts was also found to be enhanced when combined with paclitaxel. Tumor regression was observed in all mice after treatment with ZD6474 plus paclitaxel, and it was accompanied by a significant potentiation in inhibition of angiogenesis. Six of 20 mice had no histological evidence of tumors after treatment with ZD6474 plus paclitaxel. CONCLUSIONS: This study suggests that in addition to inhibiting endothelial cell proliferation by blocking VEGF-induced signaling, ZD6474 may also be able to inhibit cancer cell growth by blocking EGFR autocrine signaling. These results provide also a rationale for the clinical evaluation of ZD6474 combined with taxanes in cancer patients.

Agar↗

Regulation of the urokinase receptor by its plasminogen activator.

There is now ample evidence that the proteolytic action of urokinase (UK) is potentiated by a specific cell surface receptor. The present study was undertaken to assess the role of UK as a modulator of its receptor. GEO colonic cells, which secrete relatively low levels of UK (congruent to 0.1 nM/72 h per 10(6) cells) and display approximately 10(4) receptors per cell, 10% of which are "tagged" with the endogenous plasminogen activator (PA), was selected for the study. A 90% reduction in the specific binding of radioactive DFP-UK was observed for cells cultivated in the presence of two-chain (TC) UK (Mr 55,000). This only partly reflected occupation of the receptors with UK supplied in the culture medium, since the specific binding of the radioligand was still reduced by 60% after an acid pretreatment, which dissociates receptor-bound UK. The reduction in radioactive DFP-UK binding to cells treated with high molecular weight UK, either in the single or two-chain form, was both concentration and time dependent. Maximum reductions (70%) were achieved by treatment of the cells for 24 h with 1 nM of the plasminogen activator. In contrast, low molecular weight UK, which lacks part of the UK A chain, had no effect on ligand binding. Attenuation of radioactive DFP-UK binding to UK treated GEO cells was a consequence of a 60% reduction in the number of binding sites. Treatment of GEO cells with an antibody, which blocks the binding of endogenous UK to its receptor, augmented radioactive DFP-UK binding by two-fold. These data indicate that for one colonic cell line, at least, UK down-regulates its own binding site subsequent to it being bound to the receptor.

Amino Acid Sequence↗

Simultaneous production of IL-2, IL-4, and IFN-gamma by activated human CD4+ and CD8+ T cell clones.

In the present study, we have investigated the ability of human T cells to secrete IL-2, IL-4, and IFN-gamma. IL-4 and IFN-gamma were quantified with enzymatic immunoassays and IL-2 with a biologic assay by using the murine IL-2-dependent cell line CTLL-2. PBL, stimulated with Con A or with a combination of the phorbol ester 13-O-tetradecanoylphorbol-12-acetate and the Ca2+ ionophore A23187 secreted IL-2, IL-4, and IFN-gamma. The kinetics of the secretion of the three lymphokines was investigated with two CD4+ clones; one (GEO-2) that produced IL-2, IL-4, and IFN-gamma and another (HY640), that produced only IL-2 and IFN-gamma. Significant IL-2, IL-4, and IFN-gamma production was observed after only 8 h of activation. Maximal levels of IL-2 and IL-4 were found 20 h after the onset of the stimulation which subsequently decreased. In contrast, IFN-gamma levels continued to increase in a period up to 40 h and then leveled off. In spite of these differences in secretion, the kinetics of accumulation of mRNA did not differ. The IL-2, IL-4, and IFN-gamma mRNA were detectable 2 h after stimulation and continued to accumulate for a period up to 20 h. In a series of 22 CD4+ clones, 21 were able to secrete all three lymphokines upon stimulation. Almost all CD8+ clones were able to produce IL-2 and IFN-gamma, but only six of the 23 CD8+ T cell clones secreted IL-4. In addition, five CD4+ (allo)antigen-specific T cell clones were tested for IL-2, IL-4, and IFN-gamma secretion upon specific stimulation. Two alloantigen-specific and two tetanus toxoid-specific T cell clones secreted IL-2, IL-4, and IFN-gamma simultaneously, whereas one alloantigen-specific T cell clone secreted IL-2 and IFN-gamma, but not IL-4. A supernatant of the CD4+ T cell clone GEO-2, that contained high levels of IFN-gamma and IL-4, was unable to induce the low affinity receptor for IgE, CD23, on a Burkitt lymphoma cell line. However, after separation of IL-4 from IFN-gamma by using HPLC, the IL-4-containing fraction-induced CD23, which could be blocked by the fraction that contained IFN-gamma and by a polyclonal rabbit anti-IL-4 antiserum. Finally, the partly purified IL-4, that was devoid of IL-2, promoted the growth of the clone GEO-2.

Antigens, Differentiation, T-Lymphocyte↗