bluuebunny/crossref_metadata_2025 huggingface dataset:
Uterine artery embolization
Authors: Reidy JF
Published: February 2003

Percutaneous trans-catheter embolization has been practised by radiologists for well over 20 years. In many different clinical situations a great variety of embolization materials or agents has been used in all parts of the body, but generally these procedures have been performed rarely. An important indication is severe bleeding not responding to conservative measures, where the alternative treatment would involve major surgery. Embolization has also been used in tumours, particularly where they are hypervascular, when the role has often been to debulk and devascularize immediately prior to surgery. The third main indication is in arteriovenous malformations and fistulae. Thus, it is somewhat surprising that it was not until 1995 that uterine artery embolization (UAE) was first advocated as a treatment for uterine fibroid disease.
Human embryonic stem cells: prototypical pluripotential progenitors
Authors: Mollard R , Conley BJ , Trounson AO
Published: February 2003

Embryonic stem (ES) cells are a primitive cell type derived from the inner cell mass (ICM) of the developing embryo. When cultured for extended periods, ES cells maintain a high telomerase activity, normal karyotype and the pluripotential developmental capacity of their ICM derivatives. Such capacity is best demonstrated by mouse ES cells which can contribute to all tissues of the developing embryo following either their injection into host blastocysts or tetraploid embryo complimentation (for a review see Robertson). For both practical and ethical reasons it is not possible to inject human ES cells into blastocysts for the development of a term fetus. However, when injected beneath the testicular capsule of severe combined immunodeficient (SCID) mice, human ES cells form teratomas comprising tissue representatives of all three embryonic germ layers (ectoderm, mesoderm and endoderm) thus attesting to their pluripotency. Based upon morphological criteria, neuronal, cardiac, bone, squamous epithelium, skeletal muscle, gut and respiratory epithelia are readily identifiable within the human ES-cell-derived teratomas. With the demonstrated capability to isolate and maintain pluripotent human ES cells in vitro, their ability to give rise to tissue representatives of all three embryonic germ layers and the technical advances made possible by research on mouse ES cells, a rapid increase in human ES cell research aimed at drug discovery and human cell and gene therapies has occurred. Indeed in the mouse, dissociated embryoid bodies (EBs) have already been demonstrated capable of repopulating the haematopoietic system of recipient animals (for a review see Keller) and mouse ES cells are currently being used in attempts to repair mouse neural degenerative lesions.
Introduction to the special issue on evaluating word sense disambiguation systems
Authors: EDMONDS PHILIP , KILGARRIFF ADAM
Published: February 2003

Has system performance on Word Sense Disambiguation (WSD) reached a limit? Automatic systems don't perform nearly as well as humans on the task, and from the results of the SENSEVAL exercises, recent improvements in system performance appear negligible or even negative. Still, systems do perform much better than the baselines, so something is being done right. System evaluation is crucial to explain these results and to show the way forward. Indeed, the success of any project in WSD is tied to the evaluation methodology used, and especially to the formalization of the task that the systems perform. The evaluation of WSD has turned out to be as difficult as designing the systems in the first place.
Combining Classifiers for word sense disambiguation
Authors: FLORIAN RADU , CUCERZAN SILVIU , SCHAFER CHARLES , YAROWSKY DAVID
Published: February 2003

Classifier combination is an effective and broadly useful method of improving system performance. This article investigates in depth a large number of both well-established and novel classifier combination approaches for the word sense disambiguation task, studied over a diverse classifier pool which includes feature-enhanced Naïve Bayes, Cosine, Decision List, Transformation-based Learning and MMVC classifiers. Each classifier has access to the same rich feature space, comprised of distance weighted bag-of-lemmas, local ngram context and specific syntactic relations, such as Verb-Object and Noun-Modifier. This study examines several key issues in system combination for the word sense disambiguation task, ranging from algorithmic structure to parameter estimation. Experiments using the standard SENSEVAL2 lexical-sample data sets in four languages (English, Spanish, Swedish and Basque) demonstrate that the combination system obtains a significantly lower error rate when compared with other systems participating in the SENSEVAL2 exercise, yielding state-of-the-art performance on these data sets.
Word sense disambiguation with pattern learning and automatic feature selection
Authors: MIHALCEA RADA F.
Published: February 2003

This paper presents a novel approach for word sense disambiguation. The underlying algorithm has two main components: (1) pattern learning from available sense-tagged corpora (SemCor), from dictionary definitions (WordNet) and from a generated corpus (GenCor); and (2) instance based learning with automatic feature selection, when training data is available for a particular word. The ideas described in this paper were implemented in a system that achieves excellent performance on the data provided during the SENSEVAL-2 evaluation exercise, for both English all words and English lexical sample tasks.
Parameter optimization for machine-learning of word sense disambiguation
Authors: HOSTE V. , HENDRICKX I. , DAELEMANS W. , VAN DEN BOSCH A.
Published: February 2003

Various Machine Learning (ML) approaches have been demonstrated to produce relatively successful Word Sense Disambiguation (WSD) systems. There are still unexplained differences among the performance measurements of different algorithms, hence it is warranted to deepen the investigation into which algorithm has the right ‘bias’ for this task. In this paper, we show that this is not easy to accomplish, due to intricate interactions between information sources, parameter settings, and properties of the training data. We investigate the impact of parameter optimization on generalization accuracy in a memory-based learning approach to English and Dutch WSD. A ‘word-expert’ architecture was adopted, yielding a set of classifiers, each specialized in one single wordform. The experts consist of multiple memory-based learning classifiers, each taking different information sources as input, combined in a voting scheme. We optimized the architectural and parametric settings for each individual word-expert by performing cross-validation experiments on the learning material. The results of these experiments show that the variation of both the algorithmic parameters and the information sources available to the classifiers leads to large fluctuations in accuracy. We demonstrate that optimization per word-expert leads to an overall significant improvement in the generalization accuracies of the produced WSD systems.
Evaluating lexical resources using SENSEVAL
Authors: CALZOLARI NICOLETTA , SORIA CLAUDIA , BERTAGNA FRANCESCA , BARSOTTI FRANCESCO
Published: February 2003

The aim of our paper is twofold: to introduce some general reflections on the task of lexical semantic annotation and the adequacy of existing lexical-semantic reference resources, while giving an overall description of the Italian lexical sample task for the SENSEVAL-2 experiment. We suggest how the SENSEVAL exercise (and comparison between the two editions of the experiment) can be employed to evaluate the lexical reference resources used for annotation. We conclude with a few general remarks on the gap between the lexicon, a partially decontextualised object, and the corpus, where context plays a significant role.
The role of domain information in Word Sense Disambiguation
Authors: MAGNINI BERNARDO , STRAPPARAVA CARLO , PEZZULO GIOVANNI , GLIOZZO ALFIO
Published: February 2003

This paper explores the role of domain information in word sense disambiguation. The underlying hypothesis is that domain labels, such as MEDICINE, ARCHITECTURE and SPORT, provide a useful way to establish semantic relations among word senses, which can be profitably used during the disambiguation process. Results obtained at the SENSEVAL-2 initiative confirm that for a significant subset of words domain information can be used to disambiguate with a very high level of precision.
Dedication to William A. Gale
Authors: CHURCH KENNETH
Published: February 2003

Gale, as he liked to be called by his friends and family, had extremely broad interests, both professionally and otherwise. His professional career at Bell Labs included radio astronomy, economics, statistics and computational linguistics.
Editorial
Authors: Salkovskis Paul
Published: February 2003

Happy New Year! As the first issue of volume 31 thuds onto your mat, we are happy to declare the increase in page numbers a success in every respect. As you know, we have increased our published volume by 25% in order to keep up with the growing number of good quality manuscripts. The flow has continued, and in fact continues to increase. At the same time, after a bit of a hiccup, we have a working website that carries the additional material that usually accompanies brief clinical reports. For those of you thinking of submitting brief clinical reports, this is important because it means that you will no longer have to post out fuller reports or supporting documents. Also, documents posted in this way will be citable publications. (However, it is also important to note that the rules restricting “double publication” apply to such material).
WHY ARE CERTAIN INTRUSIVE THOUGHTS MORE UPSETTING THAN OTHERS?
Authors: Rowa Karen , Purdon Christine
Published: February 2003

Recent cognitive behavioural models of obsessive compulsive disorder (OCD) suggest that the misinterpretation of the meaning of intrusive thoughts plays a pivotal role in the escalation of these thoughts to clinical obsessions, but less attention has been paid to why only certain intrusive thoughts become the focus of these misappraisals. Theoretical speculation suggests that thoughts that have relevance for an individual's value system or sense of self may be particularly salient and upsetting for people. The role of thought appraisal and contradiction of valued aspects of self were examined in a nonclinical population. It was hypothesized that participants reporting on upsetting intrusive thoughts would appraise these thoughts negatively and would report that these thoughts contradict important aspects of self to a greater degree than participants reporting on less upsetting intrusive thoughts. Participants (N = 64) were randomly assigned to report on either the most or least upsetting intrusive thought they had experienced. They completed questionnaires on appraisals of these thoughts, valued aspects of self, and contradiction of self. Consistent with predictions, participants reporting on more upsetting thoughts appraised these thoughts in a more negative manner and reported that these thoughts contradicted valued aspects of self to a greater degree than participants in the least upsetting thought group. These results support Salkovskis' (1985) and Rachman's (1997, 1998) cognitive behavioural models of OCD, and suggest that the degree of contradiction of self may help us understand why some obsessional thoughts are much more upsetting than others.
COGNITION AND THE BODY: SOMATIC ATTRIBUTIONS IN IRRITABLE BOWEL SYNDROME
Authors: Martin Maryanne , Crane Catherine
Published: February 2003

How do somatic causal attributions for symptoms relate to treatment seeking behaviour in Irritable Bowel Syndrome (IBS)? How might a tendency to make somatic attributions influence an individual's cognitive representation of their illness once a diagnosis of IBS is established? In Study 1 attributions about symptoms were investigated in treatment-seekers and non treatment-seekers with IBS. Treatment-seekers had an increased tendency to make somatic attributions for both gastrointestinal symptoms and physiological symptoms characteristic of anxiety and depression, although they did not differ from non treatment-seekers in the severity of these symptoms or in their reports of psychological distress. Treatment-seekers also perceived themselves to be significantly less resistant to illness and to be significantly more likely to have poor health in the future than non treatment-seekers. In Study 2, 20 treatment seekers with chronic symptoms of IBS completed measures of mood and of the degree to which they viewed a range of symptoms as a part of their IBS. Physiological symptoms of anxiety and depression were seen as a part of IBS by a considerable proportion of the sample. Higher levels of depression were associated with an increased tendency to see physiological symptoms of anxiety and depression and even symptoms of colds as “a part of” IBS. It is concluded that a somatic attributional style may contribute both to initial treatment seeking for symptoms of IBS and the subsequent maintenance and exacerbation of the disorder once a diagnosis is established.
INFLUENCE OF THE SUPPRESSION OF SELF-DISCREPANT THOUGHTS ON THE VIVIDNESS OF PERCEP
TION OF AUDITORY ILLUSIONS
Authors: García-Montes José M. , Pérez-Álvarez Marino , Fidalgo Ángel M.
Published: February 2003

Based on the relationship between cognitive intrusions and auditory hallucinations established by Morrison and Baker (2000) and Morrison, Haddock and Tarrier (1995) the present study examines the possible effect of the repeated suppression of self-discrepant thoughts on the vividness of auditory illusions in a sample from a non-clinical population. Sixty-one participants were randomly assigned to a suppression of thoughts group (n = 31) or a focalization of thoughts group (n = 30) with different levels of self-discrepancy. After carrying out the task over a period of 48 hours, participants were presented with non-vocal auditory stimulation and asked to state whether they heard any verbalizations, and if so, how clearly. Results show how the repeated suppression of self-discrepant thoughts has a considerable effect on the vividness of illusions (F(1, 50) = 16.09; p < 0.001). The implications of these results for psychological therapy are analysed, with special emphasis on the importance of a research line based on acceptance.
COGNITIVE THERAPY FOR PREVENTING TRANSITION TO PSYCHOSIS IN HIGH RISK INDIVIDUALS: A CASE SERIES
Authors: French Paul , Morrison Anthony P. , Walford Lara , Knight Alice , Bentall Richard P.
Published: February 2003

Early interventions for psychosis have recently received a great deal of interest. This is because research findings have indicated the importance of duration of untreated psychosis (DUP) in influencing future prognosis. Most interventions have so far centered around the early identification of first episode cases in an attempt to minimize the DUP. A further development of the early intervention strategy aims to identify high-risk cases prior to the onset of psychosis. Treatment of this at risk group could potentially prevent the onset of psychosis. In this paper we describe three cases from a randomized controlled trial designed to test the feasibility of a cognitive therapy intervention, to prevent the onset of psychosis. These are presented with formulations based on a recent cognitive model conceptualizing the onset of psychosis. Implications of this approach for future research and clinical practice are also discussed.
GROUP COGNITIVE BEHAVIOUR THERAPY FOR ANGER: A PILOT STUDY
Authors: Siddle Ronald , Jones Freda , Awenat Fairuz
Published: February 2003

Patients referred with anger problems often do not attend for treatment. The aim of this study was to determine if group Cognitive Behaviour Therapy (CBT) was feasible. Patients referred for help with their anger were assessed, given 6 sessions of group CBT and re-assessed. Of 119 patients referred, 49 (41%) did not attend the initial appointment. Patients who attended for interview were invited to participate in the group CBT. Only 11 patients (9%) of those referred for therapy attended for the full course of CBT. Thirty-four patients (29%) were exposed to at least one session of CBT, while 66 patients (56%) did not attend for any therapy. Patients who attended for some or all of the CBT treatment reported reductions in the frequency and intensity of their anger outbursts. There was also a significant reduction in measures of their anger traits. It could be concluded that group CBT is an appropriate way to deliver this therapy to patients with anger problems, but it is clear that many of those referred are ambivalent about therapy and will not attend. Figures are given that will allow the planning of a randomized controlled trial to evaluate the difference between individual and group based CBT for patients with anger problems.
AN ILLUSTRATION OF DELIVERING EVIDENCE-BASED PRACTICE THROUGH STAFF TRAINING: MULTI-DIMENSIONAL PROCESS, OUTCOME AND ORGANIZATIONAL EVALUATION
Authors: Milne Derek , Woodward Katy , Hanner Seirian , Iceton John , Fitzsimmons Ann , Rochester Julie
Published: February 2003

Developing staff competence in evidence-based interventions for people with a severe mental illnesses is now a government priority. However, evidence concerning the nature and effectiveness of the training programmes that are designed to develop competence is decidedly limited. The purpose of the present study was, therefore, to evaluate systematically a manualized and evidence-based staff training programme. This programme was delivered over a 17-day period, in a modular and experiential workshop format. Staff (mostly mental health nurses) were allocated to either an experimental group (N = 18) who received the training or to a waiting-list, control group condition (N = 7). Multi-dimensional process and outcome evaluations indicated that the staff training was delivered competently, followed an experiential learning approach, and led to significant improvement in the participants' targeted knowledge and skills. The staff were also satisfied with the acceptability and delivery of the training. Generalization to the participants' clients was reported to be very good, which was congruent with evidence of a supportive work environment, the organizational evaluation aspect of this study. While these learning outcomes are typical of such training, the degree of generalization is exceptional. Possible reasons for this favourable outcome are discussed, and conclusions are drawn for developing staff competence in ways that maximize the delivery of evidence-based practice.
COGNITIVELY ENHANCED PARENT TRAINING
Authors: White Caroline , McNally Deborah , Cartwright-Hatton Sam
Published: February 2003

Parent training is one of the most effective interventions for behaviour problems in young children. Most models of parent training are largely behavioural in content and have been developed from social learning theory. As with developments in treatments for adult disorders, it is proposed that parent training implementation and parental engagement may be improved by the introduction of a cognitive component. In particular, the use of the “thoughts, feelings, behaviour cycle” throughout parent training is proposed as a tool for challenging parental beliefs and attributions regarding children's behaviour and increasing parental uptake of the behavioural strategies that are taught. Preliminary findings are discussed with reference to clinical implications and future research.
ASSESSING PREFERENCES FOR STAFF: SOME PILOT DATA
Authors: Sturmey Peter , Lee Ronald , Reyer Howie , Robek Adrienne
Published: February 2003

Stimulus preference assessment (SPA) methods have been used to identify stimuli that might function as reinforcers. These methods typically assess social stimuli, such as hugs and verbal praise, tangible stimuli, such as toys and edibles, and high-frequency activities. Individual preferences for staff personnel, however, have not been assessed. In this study, the paired-stimulus assessment method was adapted to assess consumer preferences for different staff. Four adults with intellectual disabilities, two children with autistic-spectrum disorders, and one child with emotional difficulties participated in the present study. The results showed that preferences for different staff were identified for five of the seven consumers. These results provide support for the use of this procedure for identifying preferences for different staff. Directions for future research are discussed in terms of systematizing the assessment procedure, validation of results, and practical considerations regarding the assessment of preferences for staff personnel.
A PSYCHO-PHYSIOLOGICAL COMPARISON OF POST-TRAUMATIC AND PROLONGED DURESS STRESS DISORDERS
Authors: Marsh Charlie
Published: February 2003

It has been proposed that post-traumatic stress disorder (PTSD) and so-called “prolonged duress stress disorder” (PDSD) have similar symptom profiles and differ only with regard to the presence or absence of a “traumatic event”. This single case experiment investigated whether PTSD can be distinguished from PDSD at the level of patho-physiology. The results indicate that both PTSD and PDSD imagery elicit physiological responses, but these are more readily and more strongly evoked by the former than the latter. These findings suggest that physiological response differences between PTSD and PDSD may be only a matter of degree. Implications are drawn for the psycho-physiological assessment of PDSD and recommendations for further research are made.
Editorial
Authors: Myatt Tony
Published: February 2003

This issue of Organised Sound focuses on mapping in computer music. Unlike many issues of the journal, all articles in this edition are related to the theme. The call for contributions resulted in a significant collection of work which covered a broad range of activity in the field. This number of articles would normally fill more than one issue of the journal but we decided to keep the collection together as a representative body of work.

We were delighted to welcome Marcelo Wanderley as Guest Editor for this issue. I would like to thank him for his hard and diligent work throughout the preparation of this issue.
Interpolated mappings for musical instruments
Authors: Goudeseune Camille
Published: February 2003

Software-based musical instruments have controls for input, a sound synthesizer for output,
and mappings connecting the two. An effective layout of controls considers how many degrees of
freedom each has, as well as the overhead of selecting each one while performing. An isolated
mapping from one control to one synthesis parameter needs an appropriate choice of proportional,
integral or derivative control (the control's value, or that value's rate of change, drives the
synthesis parameter's value, or that value's rate of change). Beyond this, a compound mapping
cross-coupling several controls and synthesis parameters can surprisingly increase the performer's
intuitive understanding of the instrument.
Mapping performer parameters to synthesis engines
Authors: Hunt Andy , Wanderley Marcelo M.
Published: February 2003

This paper considers the issues involved in the design of electronic and computer interfaces, specifically mapping - the designed link between an instrument's playing interface and its sound source. It defines the problem area, reviews the literature, and gives examples of specific system mappings. A general model is presented, with the aim of providing a framework for future discussions on what makes an effective mapping. Several guidelines for mapping strategies are given, based on existing work.
Mapping transparency through metaphor: towards more expressive musical instruments
Authors: Fels Sidney , Gadd Ashley , Mulder Axel
Published: February 2003

We define a two-axis transparency framework that can be used as a predictor of the expressivity of a musical device. One axis is the player's transparency scale, while the other is the audience's transparency scale. Through consideration of both traditional instruments and new technology-driven interfaces, we explore the role that metaphor plays in developing expressive devices. Metaphor depends on a literature, which forms the basis for making transparent device mappings. We examine four examples of systems that use metaphor: Iamascope, Sound Sculpting, MetaMuse and Glove-TalkII; and discuss implications on transparency and expressivity. We believe this theory provides a framework for design and evaluation of new human–machine and human–human interactions, including musical instruments.
Strategies of mapping between gesture data and synthesis model parameters using perceptual spaces
Authors: Arfib D. , Couturier J. M. , Kessous L. , Verfaille V.
Published: February 2003

This paper is about mapping strategies between gesture data and synthesis model parameters by means of perceptual spaces. We define three layers in the mapping chain: from gesture data to gesture perceptual space, from sound perceptual space to synthesis model parameters, and between the two perceptual spaces. This approach makes the implementation highly modular. Both perceptual spaces are developed and depicted with their features. To get a simple mapping between the gesture perceptual subspace and the sound perceptual subspace, we need to focus our attention on the two other mappings. We explain the mapping types: explicit/implicit, static/dynamic. We also present the technical and aesthetical limits introduced by mapping. Some practical examples are given of the use of perceptual spaces in experiments done at LMA in a musical context. Finally, we discuss several implications of the mapping strategies: the influence of chosen mapping limits onto performers' virtuosity, and the incidence of mapping on the learning process with virtual instruments and on improvisation possibilities.
Composers' views on mapping in algorithmic composition
Authors: Doornbusch Paul
Published: February 2003

Mapping concerns the connection between gestures, or structures and audible results in a musical performance or composition. While this is of intense interest to performers of new instruments and instrument designers, it has also been an area of interest for some composers. Algorithmic composition is sometimes the process of imagining a gesture or structure - perhaps physical or visual - and then applying a mapping process to turn that ‘gesture’ of the conceptual domain into sound which may display the original conception in some way. This article looks at mapping from the point of view of algorithmic composition, particularly where persistence is an issue, such that the gesture (conceptual domain) is embodied and perceptible in the musical result.
Strategies for interaction in construction 3
Authors: Myatt Tony
Published: February 2003

This paper describes the interactive strategies adopted during the composition and realisation of construction 3 for soprano saxophone and multiple media, by Tony Myatt and Peter Fluck. This work is an interactive multiple media composition for computer, computer-enhanced saxophone and computer graphics projected onto irregularly shaped screens. Derivation of the performer sensing system and mapping strategies to control signal processing, computer generated materials and communication gestures between the performer and computer are described. These processes include generative mapping techniques using neural networks for the recognition of gestural information and the development and application of wireless, wearable sensing technology. This work is described as a holistic approach to the derivation of performer sensing, data mapping and the application of reactive and interactive processes in the context of the creation of a new musical and multiple media composition.
Sensing and mapping for interactive performance
Authors: Ng Kia
Published: February 2003

This paper describes a trans-domain mapping (TDM) framework for translating meaningful activities from one creative domain onto another. The multi-disciplinary framework is designed to facilitate an intuitive and non-intrusive interactive multimedia performance interface that offers the users or performers real-time control of multimedia events using their physical movements. It is intended to be a highly dynamic real-time performance tool, sensing and tracking activities and changes, in order to provide interactive multimedia performances.

From a straightforward definition of the TDM framework, this paper reports several implementations and multi-disciplinary collaborative projects using the proposed framework, including a motion and colour-sensitive system, a sensor-based system for triggering musical events, and a distributed multimedia server for audio mapping of a real-time face tracker, and discusses different aspects of mapping strategies in their context.

Plausible future directions, developments and exploration with the proposed framework, including stage augmenta
tion, virtual and augmented reality, which involve sensing and mapping of physical and non-physical changes onto multimedia control events, are discussed.
The Metasaxophone: concept, implementation, and mapping strategies for a new computer music instrument
Authors: Burtner Matthew
Published: February 2003

The Metasaxophone is an acoustic tenor saxophone retrofitted with an onboard computer microprocessor and an array of sensors that convert performance data into MIDI control messages. The instrument has additionally been outfitted with a unique microphone system that allows for detailed control of the amplified sound. While maintaining full acoustic functionality it is also a versatile MIDI controller and an electric instrument. A primary motivation behind the Metasaxophone is to put signal processing under direct expressive control of the performer. Through the combination of gestural and audio performance control, employing both discrete and continuous multilayered mapping strategies, the Metasaxophone can be adapted for a wide range of musical purposes. This paper explores the artistic and technical development of the instrument, as well as new conceptions of musical mappings arising from the enhanced interface.
Improvement of Monoclonal Antibody Production in Hybridoma Cells by Dimethyl Sulfoxide
Authors: Ling Wai Lam W. , Deng Liang , Lepore Joseph , Cutler Collette , Cannon‐Carlson Susan , Wang Yan , Voloch Marcio
Published: February 2003

Hybridoma cultures are routinely used as a source for monoclonal antibody (mAb) production necessary for preclinical evaluation. However, these cultures typically have low volumetric and specific productivities. In this article, we examined the use and the timing of addition of dimethyl sulfoxide (DMSO) as a medium additive to improve mAb production in our hybridoma clone 19 (c19) cultures. From shake flask studies, we defined the optimal DMSO concentration and time of addition for improved productivity. This timing coordinated with high cell viability and density. Hybridoma cultures treated with DMSO up to 0.3% (v/v) possessed cell densities and viabilities comparable to untreated control. We demonstrated that 0.2% (v/v) DMSO added to shake flask cultures at their maximal viable cell densities resulted in a 2‐fold increase in specific mAb production. This procedure was scaleable up to 20 L Cellbags (Wave Bioreactors) with similar titer improvement. Moreover, DMSO treatment did not affect the bioactivity or glycosylation profiles of the mAb.
Development of a Chinese Hamster Ovary Cell Line for Recombinant Adenovirus‐Mediated Gene Expression
Authors: Condon Russell G. G. , Schaefer Eugene J. , Santoro Marc , Longley Robert , Tsao Yung‐Shyeng , Zurawski Sandra M. , Liu Zhong
Published: February 2003

Recombinant human adenovirus (rhAd) has been used extensively for functional protein expression in mammalian cells including those of human and nonhuman origin. High‐level protein production by rhAd vectors is expected in their permissive host cells, such as the human embryonic kidney 293 (HEK293) cell line. This is attributed primarily to the permissiveness of HEK293 to rhAd infection and their ability to support viral DNA replication by providing the missing El proteins. However, the HEK293 cells tend to suffer from cytopathic effect (CPE) as a result of virus replication. Under these circumstances, the host cell function is compromised and the culture viability will be reduced. Consequently, newly synthesized polypeptides may not be processed properly at posttranslational levels. Therefore, the usefulness of HEK293 cells for the expression of complex targets such as secreted proteins could be limited. In the search for a more robust cell line as a production host for rhAd expression vectors, a series of screening experiments was performed to isolate clones from Chinese hamster ovary‐K1 (CHO‐K1) cells. First, multiple rounds of infection of CHO‐K1 cells were performed utilizing an rhAd expressing GFP. After each cycle of infection, a small population of CHO cells with high GFP levels was enriched by FACS. Second, individual clones more permissive to human adenovirus infection were isolated from the highly enriched subpopulation by serial dilution. A single clone, designated CHO‐K1‐C5, was found to be particularly permissive to rhAd infection than the parental pool and has served as a production host in the successful expression of several secreted proteins.
Enhancement of Monoclonal Antibody Production by Lysine‐Containing Peptides
Authors: Franěk František , Eckschlager Tomáš , Katinger Hermann
Published: February 2003

In the search for peptides that could effectively enhance the monoclonal antibody production of a model hybridoma, the performance of five lysine‐containing peptides was compared. The capacity of the peptides to enhance the monoclonal antibody yield correlated with their growth‐suppressing activity. No correlation of the production‐enhancing activity with the character of the distribution of cell‐cycle phases could be found. All of the tested peptides, including the negative control peptide Gly‐Phe‐Gly, altered the cell‐cycle phases distribution in favor of the proportion of the S phase.The peptides added to the hybridoma culture were found to be gradually decomposed into dipeptides and free amino acids. Among the set of tested lysine‐containing di‐ to pentapeptides, the best results were obtained with the tripeptide Gly‐Lys‐Gly. The growth‐suppressing and production‐enhancing capacity of this peptide supplement was obviously associated with the temporary presence of the intact peptide molecule in the culture media, because the addition of a mixture of free amino acids constituting this peptide, i.e., glycine and lysine, displayed a different effect‐a slight promotion of cell growth.
Epstein‐Barr Virus Vectors Provide Prolonged Robust Factor IX Expression in Mice
Authors: Sclimenti Christopher R. , Neviaser Andrew S. , Baba Edward J. , Meuse Leonard , Kay Mark A. , Calos Michele P.
Published: February 2003

We demonstrate that vectors incorporating components from Epstein‐Barr virus (EBV) for retention and from human genomic DNA for replication greatly enhance the level and duration of marker gene expression in dividing cultured cells. The same types of vectors were tested in vivo by high‐pressure tail vein injection of naked DNA in mice, resulting in liver delivery and expression. The therapeutic gene was a human factor IX (hFIX) minigene comprising genomically derived 5′, 3′, and intronic sequences that provided relatively good gene expression in vivo. We demonstrated that addition of the EBV EBNA1 gene and its family of repeats binding sites provided a 10‐ to 100‐fold increase in prolonged hFIX expression in mouse liver. A single 25‐μg dose of vector DNA generated normal (>5 μg/mL) levels of hFIX throughout the 8 month duration of the experiment. Vector DNA with or without the EBV sequences was retained in liver cells, and vector replication was not a factor in these nondividing liver cells. Instead, it appears that enhancement of stable hFIX expression by the EBV components was responsible for the increased level and duration of therapeutic gene expression. The EBV sequences also significantly enhanced stable expression of a vector carrying the full genomic hFIX gene delivered to mouse liver. These results underline the crucial importance of appropriate gene expression signals on gene therapy vectors and the utility of EBV sequences in particular for increasing stable gene expression.
Sustained High‐Yield Production of Recombinant Proteins in Transiently Transfected COS‐7 Cells Grown on Trimethylamine‐Coated (Hillex) Microcarrier Beads
Authors: Knibbs Randall N. , Dame Michael , Allen Melissa R. , Ding Yunhong , Hillegas William J. , Varani James , Stoolman Lloyd M.
Published: February 2003

The present study shows that COS‐7 cells transiently transfected and maintained on positively charged (trimethylamine‐coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high‐level synthesis was observed with secreted chimeric proteins (murine E‐selectin– and P‐selectin‐human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte‐macrophage colony‐stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine‐coated microcarriers than to polystyrene flasks. After 10–14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5‐fold increase in protein production from a single transfection over two weeks. Thus, microcarrier‐based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.
Comparison of Batch and Perfusion Culture in Combination with Pilot‐Scale Expanded Bed Purification for the Production of Soluble Recombinant β‐Secretase
Authors: Lüllau E. , Kanttinen A. , Hassel J. , Berg M. , Haag‐Alvarsson A. , Cederbrant K. , Greenberg B. , Fenge C. , Schweikart F.
Published: February 2003

β‐Secretase is one of the prime targets for therapeutic intervention of Alzheimerapos;s disease. For the development of a secretase inhibitor a steady supply of large quantities of a homogeneous and active recombinant β‐secretase is a prerequisite. Therefore various culture modes were investigated using HEK‐293 cells stably transfected with soluble recombinant β‐secretase. The coupling of the Fc part of human IgG1 to the ectodomain of β‐secretase (residues 1–460) allowed a fast purification of the protein with rProtA expanded bed chromatography. Batch cultures of 5 to 50 L working volume run for 7 days showed reproducible cell growth and product yields of 3 mg/L purified protein. A 20 L perfusion culture was operated for 21 days, reaching a cell density of 30 × 106 cells/mL at a dilution rate of 2/d. The total product yield of the perfusion culture was 1.4 g of purified protein. The effect of different perfusion rates on cell growth, protein yield, and quality was investigated and compared to the results obtained in batch cultures. Protein quality was consistent as analyzed on 1D SDS‐PAGE, and the final product contained both the mature and the pro form of β‐secretase. Although the cell specific protein expression was slightly reduced in perfusion culture, a substantial increase in specific activity of over 75% was achieved. Some of the increase in activity can be explained by an increase in the percentage of the mature form of the recombinant protein.
Engineering mRNA Translation Initiation to Enhance Transient Gene Expression in Chinese Hamster Ovary Cells
Authors: Underhill Michèle F. , Coley Clare , Birch John R. , Findlay Alison , Kallmeier Robert , Proud Christopher G. , James David C.
Published: February 2003

To increase transient expression of recombinant proteins in Chinese hamster ovary cells, we have engineered their protein synthetic capacity by directed manipulation of mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser51Ala site‐directed mutant of eIF2α, a subunit of the trimeric eIF2 complex that is implicated in regulation of the global rate of mRNA translation initiation in eukaryotic cells. Phosphorylation of eIF2α by protein kinases inhibits eIF2 activity and is known to increase as cells perceive a range of stress conditions. Using single‐and dual‐gene plasmids introduced into CHO cells by electroporation, we found that transient expression of the eIF2α Ser51Ala mutant with firefly luciferase resulted in a 3‐fold increase in reporter activity, relative to cells transfected with reporter only. This effect was maintained in transfected cells for at least 48 h after transfection. Expression of the wild‐type eIF2α protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2α phosphorylation in cells transfected with the mutant eIF2α construct. Transfection of CHO cells with the luciferase‐only construct resulted in a marked decrease in the global rate of protein synthesis in the whole cell population 6 h post‐transfection. However, expression of the mutant Ser51Ala or wild‐type eIF2α proteins restored the rate of protein synthesis in transfected cells to a level equivalent to or exceeding that of control cells. Associated with this, entry of plasmid DNA into cells during electroporation was visualized by confocal microscopy using a rhodamine‐labeled plasmid construct expressing green fluorescent protein. Six hours after transfection, plasmid DNA was present in all cells, albeit to a variable extent. These data suggest that entry of naked DNA into the cell itself functions to inhibit protein synthesis by signaling mechanisms affecting control of mRNA translation by eIF2. This work therefore forms the basis of a rational strategy to generically up‐regulate transient expression of recombinant proteins by simultaneous host cell engineering.
Quantitative Monitoring for Secreted Production of Human Interleukin‐2 in Stable Insect Drosophila S2 Cells Using a Green Fluorescent Protein Fusion Partner
Authors: Shin Hwa Sung , Lim Hye Jung , Cha Hyung Joon
Published: February 2003

Human interleukin‐2 (hIL‐2) production in Escherichia coliand insect cell/baculovirus expression systems can be inefficient. Here we investigated secreted production of hIL‐2 fused with green fluorescent protein (GFP) as a versatile fusion partner in optimized stably transfected insect Drosophila melanogasterS2 cells. This nonlytic S2 insect cell expression system employs a plasmid vector and allows for secretion of functional human proteins. We report that, following stable transfection and induction, S2 cells secreted hIL‐2 as a fusion protein (∼2.3 μg/mL yield), with a secretion efficiency of approximately 90%. Regression analysis indicated a single linear relationship existed between GFP fluorescence and hIL‐2 mass in both whole cell and secreted medium samples, indicating that in vivo monitoring and quantification of target foreign protein expression and even secretion is possible using this system. The simple comparative measurement of GFP fluorescence also allowed monitoring of secretion efficiency during periods of high GFP/hIL‐2 expression.
Stability of Recombinant Protein Production in the GS‐NS0 Expression System Is Unaffected by Cryopreservation
Authors: Barnes Louise M. , Bentley Catherine M. , Dickson Alan J.
Published: February 2003

Mammalian cells form a very important part of the repertoire of production systems available to scientists involved in the production of recombinant proteins. During the production of therapeutic proteins it is vital for regulatory approval of products that no phenotypic or genetic changes are observed in the cell line or product. As part of the generation and development of therapeutic protein production, cell lines have to be frozen at various stages to create cell banks. If cryopreservation and revival of frozen stocks were to give rise to any phenotypic changes in the cells, this would again be detrimental to the further development of that particular cell line. This study uses one of the most industrially important expression systems, the GS‐NS0 expression system, to examine the effect of cryopreservation on the growth and productivity profile of cell lines that exhibit differential degrees of stability during prolonged (production) culture periods. Results show that cryopreservation and revival procedures do not alter the stability characteristics of cell lines. This type of information is of great value in definition of protocols for cell line development.
Influence of Carboxymethyl Dextran and Ferric Citrate on the Adhesion of CHO Cells on Microcarriers
Authors: Landauer Karlheinz , Wiederkum Susanne , Dürrschmid Markus , Klug Helga , Simic Gordana , Blüml Gerald , Doblhoff‐Dier Otto
Published: February 2003

Due to the inherent risks of animal‐derived raw materials, the biopharmaceutical industry has an increasing demand for serum‐free and protein‐free media for industrial cell culture bioprocesses. The absence of serum often changes the characteristics of mammalian cells, especially growth, productivity, and adherence properties. This study is mainly focused on the influence of media additives on cell adherence characteristics. An array of different carboxymethyl dextrans and different ferric citrate concentrations was tested with a number of CHO clones, using standard cell culture Roux‐flasks and Cytoline 1 macroporous microcarriers. A prototype mixing system with controlled shear force input was developed as a screening system for adherence characteristics. The results of this evaluation revealed a negatively correlated dose‐dependent influence on adhesion for ferric citrate. It was also found that certain carboxymethyl dextrans are capable of increasing the adherence on Roux‐flasks and microcarriers.
Protective Effect of Viral Homologues of bcl‐2 on Hybridoma Cells under Apoptosis‐Inducing Conditions
Authors: Vives Joaquim , Juanola Sandra , Cairó Jordi J. , Prats Eva , Cornudella Lluís , Gòdia Francesc
Published: February 2003

Targets for metabolic engineering have been identified in a hybridoma cell line to make it more robust in culture toward potential limitations inducing apoptosis. The cells were genetically modified with plasmids harboring endogenous bcl‐2 gene and also with viral Bcl‐2 homologues, particularly ksbcl‐2 and bhrf‐1 genes. When cells were exposed to apoptosis‐inducing conditions (i.e., glutamine‐free medium), the control cells exhibited a decrease in viable cell number within the first 12 h, whereas, for the bcl‐2 and ksbcl‐2 transfected cell cultures, the viable cell number did not exhibit any clear decrease until after 60 h. Furthermore, hybridoma cells expressing the viral homologue bhrf‐1 were even more resistant to cell death, showing a decrease in viability of only 50% at 72 h of culture in glutamine‐deprived medium, substantially lower than the 90% viability decrease observed for the control culture. In addition, and most relevant for further bioprocess applications, the cells genetically modified could be brought back to growth conditions even after being exposed to glutamine‐deprived conditions during a significant time window, up to 72 h.