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[New polymers, surface treatments, bioactive materials: value of vascular access devices].

Complications associated with hemodialysis vascular access are mainly due to a relative lack of biocompatibility and hemocompatibility of the materials used for the making of these devices, and to infection phenomena which may be induced by these devices. New compositions which can be processed into polymers, and a functionalisation of the latter aimed at endowing them with a specific ability to interact with molecular and cellular actors involved in the already mentioned lack of fiability on the one hand, and with infectious pathogens on the other, open the way to some improvement of the situation. Let us hope that device manufacturers will make sufficient efforts in order to see the technology transfers undertaken and completed.

Bacterial Adhesion↗

Three Rs potential in the development and quality control of pharmaceuticals.

The intention of a pharmaceutical company is to develop new, efficient products quick and with a minimum of costs. Compared to in vitro methods, animal experiments in general consume much more time and resources (costs as well as time to the market) than in vitro methods. Therefore, the use of whole animal models depends primarily on the judgement of their efficacy in the screening process, but the willingness to incorporate in vitro methods in general is high and is furthered by new developments such as high-throughput screening. Nevertheless, in vitro tests might be politically promoted by increasing their costs (quality controls, requested housing conditions) and duration (time to start of an experiment, sequential performance). Which models are favoured by industry to include them in a screening process: They have to be based on our most recent understanding of the respective disease, well characterised to allow interpretation of results and require only limited development time. All these aspects argue in favour of collaboration between industry and academia, where our understanding of pathophysiology is generated and mechanism based models are developed and characterised. However, technology transfer towards industry represents a bottle-neck for industrial use of these new in vitro models. New platforms to promote this transfer should be developed in order to bring together developer and user of novel in vitro systems and promote demonstration projects. Financing of such collaborations is not the key problem (the development of a single drug makes up to 500 million $) but the dilemma of publication of results: The development advantage compared to competitors depends on the exclusive use of novel models. The protection of intellectual property rights and the public interest in spreading alternatives to animal experiments must be balanced, e.g. by delayed but indispensable publication or advantages for companies employing alternatives in the regulatory approval process for a new drug. Quality control of therapeutic drugs (except hormones and blood products) represents a minor field of animal consumption with the exception of pyrogenicity testing. Despite considerable progress due to the introduction of the Limulus assay which represents the most successful in vitro alternative in use so far. However, some limitations of this in vitro test might be overcome in the near future by the currently validated human whole blood assay. During the last few years considerable progress has been made in the replacement (and deletion) of animal tests required for the potency and safety testing of hormones. This has been made possible by biotechnical production methods, by better-defined products, and because physico-chemical methods can be used for the potency testing of these products. In general, the better defined a drug is, the easier chemical, physical or in vitro techniques can be used for batch control. Control authorities should therefore urge the use of highly standardised components.

Animal Testing Alternatives↗

Quantitative reverse transcription-polymerase chain reaction measurement of HASH1 (ASCL1), a marker for small cell lung carcinomas with neuroendocrine features.

PURPOSE: The Human Achaete-Scute homologue 1 (HASH1, ASCL1), a lineage-specific basic helix-loop-helix member of the achaete-scute family, is essential for the generation of pulmonary neuroendocrine (NE) cells during lung development. In small cell lung cancer (SCLC), the most lethal form of lung cancer, the gene is highly expressed and the expression of HASH1 correlates with NE features found in SCLCs. Here we describe a highly sensitive reverse transcription-PCR method for quantifying HASH1 mRNA in clinical samples, using real-time fluorescence resonance energy transfer technology (LightCycler). EXPERIMENTAL DESIGN: The HASH1-positive NE cell line NCI-H187 was compared with the non-NE cell line NCI-N417 by quantitative reverse transcription-PCR. Signals were normalized using the housekeeping gene PBGD, which is pseudogene free. Subsequently, HASH1 expression in RNA isolated from biopsies from SCLC patients (n = 4) was compared with biopsies from non-SCLC (NSCLC) patients (n = 2) or normal bronchus (n = 2). RESULTS: The HASH1-positive NE cell line NCI-H187 showed 50,000-fold higher normalized expression of HASH1 than did the non-NE cell line NCI-N417, indicating that the method is applicable over a wide dynamic range. Normalized average mRNA expression levels in SCLC clinical samples were 1,000-fold higher than in the NSCLC samples. Expression in normal bronchus was comparable to the expression levels in the NSCLC. CONCLUSIONS: These results show that marked and measurable differences exist between SCLCs and other lung tissues (either NSCLC or normal bronchus). We show that the method is applicable to small biopsy samples and can discriminate SCLC from NSCLC. This method could contribute to diagnosis based on molecular profiling of tumors.

Basic Helix-Loop-Helix Proteins↗

Regulation and function of ascorbate peroxidase isoenzymes.

Even under optimal conditions, many metabolic processes, including the chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems of higher plants, produce active oxygen species (AOS). Furthermore, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of AOS, resulting in oxidative damage at the cellular level. Therefore, antioxidants and antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation in each organelle. Ascorbate peroxidase (APX) exists as isoenzymes and plays an important role in the metabolism of H(2)O(2) in higher plants. APX is also found in eukaryotic algae. The characterization of APX isoenzymes and the sequence analysis of their clones have led to a number of investigations that have yielded interesting and novel information on these enzymes. Interestingly, APX isoenzymes of chloroplasts in higher plants are encoded by only one gene, and their mRNAs are generated by alternative splicing of the gene's two 3'-terminal exons. Manipulation of the expression of the enzymes involved in the AOS-scavenging systems by gene-transfer technology has provided a powerful tool for increasing the present understanding of the potential of the defence network against oxidative damage caused by environmental stresses. Transgenic plants expressing E. coli catalase to chloroplasts with increased tolerance to oxidative stress indicate that AOS-scavenging enzymes, especially chloroplastic APX isoenzymes are sensitive under oxidative stress conditions. It is clear that a high level of endogenous ascorbate is essential effectively to maintain the antioxidant system that protects plants from oxidative damage due to biotic and abiotic stresses.

Ascorbate Peroxidases↗

Do we need a "Chair of alternative methods", and where?

During the last two decades, the field of in vitro technology has been successfully developed and its use is continuously growing. Advanced tests avoiding animal experiments will be increasingly required for routine industrial applications e.g. for pharmacological high-throughput screening. Moreover and even more importantly, the availability of human cell based methods is essential for future quality assurance and risk assessment in the fields of health and consumer protection as well as environmental protection. Thereby, the potential of such advanced in vitro methods extends far beyond the mere replacement of regulated tests. In practice, the introduction and expansion of this technology has been achieved predominantly by offering funding and awards to the scientific community. After this initiation phase, the next consequent step to exploit this knowledge clearly consists in academic promotion of this new scientific culture in an institutionalised form. The tasks of such a chair focussed on advanced in vitro tests - most probably the first of its kind world-wide - would cover in addition to (a) research and (b) teaching, (c) the sharpening of social conscience for the topic. (a) While the validation of alternative methods was formally established by founding institutions like ZEBET in Berlin on the national and ECVAM in Ispra on the European level, the development of further new and more sophisticated in vitro methods to date emerge predominantly as a by-product of basic research. A considerable push might now be given by the structured search for new methods with a spill-over for research-based up-to-date teaching. (b) The field of alternative methods is more than a panel of advanced in vitro techniques: A culture of systematic evaluation and validation of in vitro tests has been developed, which has bearing far beyond the replacement of animal experiments. In vitro systems inherently prone to artefacts require the highest level of quality control and assurance. A successful initiative to establish a Good Cell Culture Practice (GCCP) in analogy to Good Laboratory Practice (GLP) has evolved out of the field of in vitro alternatives. The concept of validating the relevance of an in vitro test in comparison to the respective in vivo situation represents a consequent translation of evidence-based medicine into in vitro biomedicine. In other words: It does no longer suffice that an in vitro model is plausible - it has to prove its suitability and quality. (c) The broad implementation of advanced in vitro technology into curricula implies development of lectures, courses and other teaching materials including virtual education offers. Such a basis will allow efficient spreading of knowledge and ease transnational acceptance. Last but not least, taking over the leadership for erecting a chair for alternative methods represents a major political signal that demonstrates to the public the willingness to adapt academic education to modern social awareness. A location for such an initiative needs to be found that is in the centre of Europe, has the necessary infrastructure of surrounding biomedical research, international networks for the evaluation and validation of tests, technology transfer to industrial use and access to relevant publication organs. The unequivocal answer to the question in the heading is therefore: we need a chair for in vitro alternatives because (i) the patient is our primary concern but the animal is not just secondary (ii) man's responsibility for the integrity of all creatures including the own species makes it mandatory.

Animal Testing Alternatives↗

[Making census data accessible to local users in the public and private sectors].

The authors outline ways in which government statistical offices can make census data available to local public- and private-sector users. Several computer programs, including REDATAM-Plus and Geographical Information Systems, are discussed. "The successful implementation of the use of the census data at the local level will require that...national agencies facilitate the institutionalization of technology transfer by providing, among other things, technical support, training at a distance, data and cartography standards, and solutions to data protection issues." The geographical focus is on Latin America and the Caribbean. (SUMMARY IN ENG)

Americas↗

Hemophilia gene therapy: novel rAAV vectors and RNA repair strategy.

Hemophilia results from a deficiency of coagulation Factor VIII or IX and manifests clinically as spontaneous bleeding into the large joints and soft tissue. Current treatment relies on the intravenous infusion of recombinant or purified Factor proteins. Factor infusion is effective, but transient due to the short half-life of Factor proteins. Recent developments in gene transfer technology have led to new strategies using molecular therapeutics as permanent treatment for bleeding disorders. This review describes recent novel molecular strategies for the treatment of the hemophilias.

Dependovirus↗

Current status of gene therapy for hemophilia.

The hemophilias are an attractive model for gene therapy because their clinical manifestations are attributable to the lack of a single protein that circulates in minute amounts in the plasma. Sustained therapeutic expression of factors VIII and IX has been achieved in preclinical studies using a wide range of gene transfer technologies targeted at different tissues. This achievement has led to six different phase I/II clinical trials that resulted in limited efficacy but minimal toxicity. Recombinant adeno-associated viral vectors appear most promising for hemophilia gene therapy; however, this review summarizes all the major gene therapy approaches used and outlines the future challenges.

Factor IX↗

A fast track to IAIMS: the Vanderbilt University strategy.

In July 1991, Vanderbilt University Medical Center (VUMC) initiated a fast track approach to the implementation of an Integrated Academic Information Management System (IAIMS). The fast track approach has four elements: 1) an integrated organizational structure combining various operational information management units and the academic informatics program into a single entity to enhance efficiency; 2) technology transfer and network access to remote resources in preference to de novo development; 3) parallel IAIMS planning and infrastructure construction; 4) restriction of the scope of the initial IAIMS to permit a manageable implementation project. The fast track approach is intended to provide a truly functional IAIMS within a time period (7 years) associated with other major construction projects such as the building of a replacement hospital.

Integrated Advanced Information Management Systems↗

Development shows some backbone. HFSP Workshop on Genetic Control of Vertebrate Development cosponsored by the Human Frontier Science Program, European Science Foundation, and European Molecular Biology Organization, Les Diablerets, Switzerland, May 26-30, 1991.

This meeting aptly illustrated the power of a combined analysis of development in a range of vertebrate systems. Each system has its own inherent strengths: the mouse has gene transfer technology and targeted mutagenesis, the frog and chick have experimental embryology, and the zebrafish has genetics. It is the synergistic effect of considering all of these systems in combination that is without measure. In the past, the study of vertebrate development has been relegated to a largely descriptive phase. Initially, this was through analysis of morphological changes taking place during development. More recently, this has taken the form of cataloging the expression patterns of genes transcribed in development. It is clear that we are now entering an era when a functional analysis of development can get underway.

Animals↗

Progress toward skeletal gene therapy.

Skeletal gene therapy is an attractive new approach to the treatment of bone disorders. Impressive advances in our knowledge of the molecular genetic basis of skeletal disorders and fracture healing have led to the development of novel therapeutics based on ectopic expression of one or more genes in patient cells that can influence repair or regenerative processes in bone. Although still a relatively immature field, proof-of-principle for enhanced bone formation through skeletal gene therapy has already been established. The challenge now is to more precisely define optimal cellular targets and therapeutic genes, and to develop safe and efficient ways to deliver therapeutic genes to target cells. In this review, we will highlight some of the exciting advances that have been made in skeletal gene therapy in recent years, with a focus on treatment of localized skeletal lesions. Strengths and weaknesses of current approaches will be discussed, as will strategies for improved safety and therapeutic outcome in the future. Skeletal gene therapy can have an enormous impact on patient care. The next 5 years will present us with unparalleled opportunities to develop more effective therapeutic strategies and overcome obstacles presented by current gene transfer technologies.

Bone Diseases↗

The Trypanosoma cruzi genome initiative.

An initiative was launched in 1994 by the Special Programme for Research and Training in Tropical Diseases (TDR) of the WHO to analyse the genomes of the parasites Filaria, Schistosoma, Leishmania, Trypanosoma brucei and Trypanosoma cruzi. Five networks were established through wide publicity, holding meetings of key laboratories and developing proposals which were then reviewed by the Steering Committee of Strategic Research for financial support. The aim of the Programme was to use the platform of these networks to: (1) train scientists from tropical disease-endemic countries; (2) transfer technology and share material and expertise, thereby reducing costs and increasing efficiency; and (3) provide an information system that is accessible globally as soon as the results become available. The initial target was to produce a low-resolution genome map for each of the parasites, but it soon became evident that by using rapidly developing technologies, it might be feasible to complete DNA-sequence analysis for some of the parasites in the next decade, as discussed here by Alberto Carlos Frasch and colleagues, with particular focus on the T. cruzi genome initiative.

Journal Article↗

BioAsia Licensing and Deal-Making Summit-SRI Conference. Life science partnering and investment on the Pacific Rim 2-3 August, 2004, Coronado, CA, USA..

The Strategic Research Institute's inaugural BioAsia Licensing and Deal-Making Summit, co-organized by the BioMinerva Group, attracted industrial leaders in biotechnology and pharmaceuticals from both sides of the Pacific Ocean. Topics discussed at the 2-day conference spanned from trans-Pacific licensing and partnering trends led by Japan-US deals, the changing landscape of the Japanese pharmaceutical industry, and trans-Pacific partnering strategies to perspectives of Asia-Pacific markets and successful investment strategies. The emerging Chinese biotechnology and pharmaceutical industry was also covered prominently, including assessments of the Chinese market, discussions on intellectual property, regulatory and tax issues, as well as case studies of Sino-US collaborations and technology showcases from Chinese biotech companies.

Biotechnology↗

Alternatives to animal experimentation in basic research.

In contrast to animal testing required by law to guarantee minimum safety standards for the licensing of drugs and chemicals, there are no regulations in basic research forcing scientists to perform animal tests. By (usually) free choice, questions are posed and hypotheses are examined which, in many cases, can only be answered by means of animal tests. Just as easily, different questions could be asked or different hypotheses could be examined which do not require animal tests. The only criterion for the choice of a topic is its relevance which cannot necessarily be judged in the short-term. Thus, it is up to the individual scientist to judge what is worth studying and therefore worth animal consumption. The educated mind will consider ethical aspects of this choice. However, on the other hand, this decision is largely influenced by questions of efficacy or (in a negative sense) by the obstacles posed to an animal consuming approach. Here, peer review and general attitude will strongly influence the methodology chosen. Availability and awareness of adequate in vitro techniques represent the prerequisites for the use of alternative methods. The least one can do in basic research is to avoid tests which cause severe suffering to animals, as is required in Switzerland and other European countries by binding ethical principles and guidelines. The increasing standard of approval and control procedures has improved the situation over the years. There are many examples of successful alternative methods in basic research. But, the application of such methods is in most cases limited to the laboratories in which they were developed, calling for technology transfer. Exceptions are procedures that are used worldwide, like the production of monoclonal antibodies, which instead of using the ascites mouse can also be performed in vitro with some good will. In these cases, commercialisation of the techniques has aided their spread within the scientific community. Sadly, many methods, even if published in the scientific literature, are little standardised and reproducible. The suggestion is put forward that publicly accessible databases should make available more detailed descriptions of methodologies. Due to limitations in space, many scientific journals cannot publish detailed methodological descriptions. However, nowadays a supplementary central deposit of methods could easily be linked to the respective article. In numerous cases though, there is simply a lack of will to change procedures to methods without animal tests or to pose questions differently in order to avoid the use of animals or to reduce their number or, at least, to reduce stress. In other cases, researchers are simply not aware of the limitations of the animal experiment as such. A thorough review of the validity of critical animal experiments should be carried out and made available publicly. For example, many animal experiments are dramatically "under-powered", i.e. carried out with groups that are too small to allow conclusions to be drawn from the outcome. This stands in marked contrast to in vitro experiments where replicate experiments usually represent no major problem. Since in vitro models are generally more prone to artefacts due to the numerous variables, e.g. of cell culture, the key requirement for their application is their validation and quality control. Guided by the experience from validation studies for alternative methods in toxicology, concepts of a Good Cell Culture Practice (GCCP) are currently being developed which aim to define minimum quality standards for in vitro techniques. This initiative aiming to increase quality must be complemented by a concept to systematically assess the relevance of the tests in order to finally achieve an evidence-based biomedical research. A change in this direction is only possible if those public funds, which were previously assigned predominantly to alternatives to the animal tests required by law, are now channelled increasingly into developing those for basic research. A financial incentive is necessary to change procedures in basic research to animal free procedures. Ethical considerations alone will bring little movement or change. It is unacceptable that, while numbers of animal tests decrease in development and notification of drugs and chemicals, they are increasing in basic research. Due to the central role of publishing scientific results, the key options for control are the respective rules of journals for the acceptance of articles. By demanding certain standards in the instructions for authors, e.g. of quality (GCCP), relevance and in case of animal experiments proof that no alternative is available, pressure could be dramatically increased. It is suggested to hold a consensus conference of journals in the life sciences on this topic.

Animal Testing Alternatives↗

[Carotenoid biosynthesis in plants and application of its relative genes in gene engineering].

In recent years, the cloning and characterization of carotenogenic genes has provided new gene resources and molecular tools, which can be utilized to genetically alter carotenoid composition and enrich the carotenoid content in vivo by gene engineering technique for plants. Increasing evidences in the studies of biochemistry of carotenogenesis and its regulation in vivo make the genetically manipulating carotenoid biosynthesis pathway to be possible in plants. In this review, the carotenoid biosynthesis pathways and the some genes involved in this pathway in plants are summarized, and some progresses including author's work in the successful manipulation of carotenoid composition and content in plants using gene-transfer technology are also covered.

Carotenoids↗