Environmental risk assessment for medicinal products containing genetically modified organisms.

Many gene therapy medicinal products and also some vaccines consist of, or contain, genetically modified organisms (GMOs), which require specific consideration in the environmental risk assessment (ERA) before marketing authorisation or clinical trial applications. The ERA is performed in order to identify the potential risks for public health and the environment, which may arise due to the clinical use of these medicinal products. If such environmental risks are identified and considered as not acceptable, the ERA should go on to propose appropriate risk management strategies capable to reduce these risks. This article will provide an overview of the legal basis and requirements for the ERA of GMO-containing medicinal products in the context of marketing authorisation in the EU and clinical trials in Germany. Furthermore, the scientific principles and methodology that generally need to be followed when preparing an ERA for GMOs are discussed.

Regulatory requirements for clinical trial and marketing authorisation application for gene therapy medicinal products.

Over the last two decades, clinical trials using gene therapy medicinal products (GTMPs) have been carried out for a large number of rare, inherited monogeneic disorders as well as common multigeneic diseases such as cancer, cardiovascular and infectious diseases including AIDS. Despite some early difficulties and setbacks, the gene therapy field has slowly progressed and, nowadays, offers the promise of novel treatments for a growing number of diseases. On the other hand, gene therapy approaches are often associated with additional risks due to limited clinical experience with a given gene transfer system, long-lasting effects of the therapeutic gene, and/or a complex mode of action. As a result, specific regulations and guidelines have been introduced within the EU to help address these uncertainties. This article summarises the legislative framework and will provide an overview on the regulatory requirements for clinical trials and marketing authorisation applications.

Recombinant adeno-associated virus-mediated expression of O6-alkylguanine-DNA-alkyltransferase protects human epithelial and hematopoietic cells against chloroethylating agent toxicity.

Recombinant adeno-associated virus (rAAV) encoding the human O6-alkylguanine-DNA-alkyltransferase (hAT) protein and a selectable marker (Neo(r)) was used to transduce human cervical carcinoma (HeLa) cells and erythroleukemic (K562) cells and clones were selected using G418 (0.4 mg/ml). Thirteen HeLa clones were isolated, 9 of which survived for 2-3 months before cell death ensued, presumably owing to the loss of G418 resistance. Northern blot analysis of the remaining four clones, using a neo probe, showed high levels of RNA equivalent in size to the bicistronic RNA expected to be produced from this construct. Analysis of hAT activity showed that 2000-5000 fmol/mg protein was expressed relative to untransduced cells (800-900 fmol/mg protein). Cell survival analysis following exposure to the chloroethylating agent mitozolomide revealed that expression of hAT at levels two- to fourfold higher than background conferred significant resistance (p < 0.001) to the toxic effects of this drug. Two days following infection of K562 cells with the rAAV vector, immunoblot analysis showed that hAT protein was being produced. Three K562 clones, isolated using G418 selection, were studied in detail and were shown to express hAT activities of 1500, 1010, and 890 fmol/mg protein, respectively, at 40 days posttransduction (mock-transduced K562 cells contain <2 fmol of hAT/mg protein). As with HeLa cells, Northern blot analysis showed the production of an appropriately sized transcript and immunoblot analysis indicated that hAT protein was being produced. These clones were assayed for cell survival following exposure to mitozolomide. Expression of hAT at levels 800- to 1500-fold higher than background conferred significant resistance (p < 0.001) to the toxic effects of mitozolomide. We have therefore successfully conferred a protective advantage against mitozolomide toxicity to cells by rAAV-mediated hAT expression.

Chemoprotective gene transfer II: multilineage in vivo protection of haemopoiesis against the effects of an antitumour agent by expression of a mutant human O6-alkylguanine-DNA alkyltransferase.

Murine bone marrow cells were transduced ex vivo with a retrovirus encoding an O6-benzylguanine (O6-beG) insensitive, double mutant form of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA). In animals reconstituted with the transduced bone marrow, about 50% of cells in the multipotent spleen colony-forming cells (CFU-S) and lineage restricted granulocyte-macrophage (GM-CFC) haemopoietic progenitor populations were found to be carrying the transgene and this correlated with the frequency of bone marrow cells and spleen colonies which stained positive for hATPA/GA by immunocyto-chemistry. Expression of hATPA/GA was associated with significant in vivo protection of both CFU-S (P = 0.001) and GM-CFC (P < 0.024) against the toxicity of the antitumour methylating agent, temozolomide, given in combination with O6-beG. Expression of hATPA/GA also led to a reduction in the frequency of combined O6-beG/temozolomide-induced micronuclei seen in polychromatic erythrocytes (P < 0.003). This study is the first to demonstrate in vivo protection of multipotent haemopoietic progenitors against the toxic and clastogenic effects of an O6-alkylating agent in the presence of O6-beG. It also represents the first report of reduced clastogenesis as a consequence of expression of an O6-beG-resistant ATase. In the accompanying article we report hATPA/GA-mediated resistance of human CD34+ haemopoietic progenitors to combined O6-beG/O6-alkylating agent toxicity. Together these two reports suggest that a gene therapy strategy whereby protection of normal haemopoietic tissue may be combined with O6-beG-mediated tumour sensitisation may be efficacious in achieving an increase in therapeutic index.

Working out of hours: the experiences and training needs of general practitioner registrars.

Out-of-hours work has been identified as a major concern for registrars, and as contributing to the steady decline both in the number of applicants to vocational training schemes and in those practising as principals on completion of their training. Until now, little has been known about registrars' views about their experience of working out of hours and how this might be improved. The present study describes general practitioner (GP) registrars' current patterns of out-of-hours working and their perceptions about training needs.

Eubacterial approach to the diagnosis of bacterial infection.

Computed tomography revealed a frontal empyema in an 8 month old boy who presented with a prolonged convulsion. Before admission he had received oral antibiotics and all specimens were negative on culture. The presence of bacteria in cerebrospinal and empyema fluid was demonstrated using the polymerase chain reaction (PCR) with 16S rRNA gene primers. A presumptive identification of Streptococcus pneumoniae was made by comparison of PCR products with those derived from known bacteria using denaturing gradient gel electrophoresis.

Using out-of-hours services: general practice or A&E?

BACKGROUND: General medical and accident and emergency (A&E) services are the two major providers of open access out-of-hours care, and there are widespread concerns about rising and non-urgent demand presented to both. METHODS: This paper examines the differential use of these services out of hours, in an audit and research study two A&E departments and 21 practices in South London. It focuses on aspects of demand, including time of contact, age-related usage and nature of presenting complaints. Through interviews with a subsample of 82 patients who attended A&E, it also provides a more qualitative focus on differential decision making. RESULTS: Findings show that there are differences in the way A&E and general medical services are used in terms of age-related demand and aspects of presenting complaints. Significantly more families with children aged under 10 contacted a GP, and whilst more digestive, respiratory and viral/non-specific complaints were presented to GPs, musculoskeletal problems constituted the largest category of complaints presented at the A&E departments. However, some usage relating to perceived and actual availability of services appeared to be interchangeable in terms of site-of-help seeking. CONCLUSION: There is a need for a collaborative multi-method approach to respond to and influence demand.

Identification of mutations contributing to the reduced virulence of a modified strain of respiratory syncytial virus.

The nucleotide sequences of the genome of the RSS-2 wild type strain of respiratory syncytial (RS) virus, which is known to induce upper respiratory tract infection in adults, and that of the attenuated ts1C candidate vaccine derived from it by three cycles of mutagenesis and selection of temperature-sensitive (ts) mutants, have been determined. Comparison of the sequences has located the genetic changes which contribute to the reduced pathogenicity in adults of the candidate vaccine. Thirty-seven nucleotide changes distinguish the wild type and ts1C, 13 of which confer amino acid substitutions; no mutations are present in extragenic regions. Partial nucleotide sequencing of the genomes of the first stage ts mutant (ts1A) and the second stage ts mutant (ts1B), which were intermediates in the derivation of the third stage mutant ts1C, established that five mutations resulting in amino acid substitutions had been induced in the first cycle of mutagenesis, one in the second cycle, and seven in the third cycle. The unique mutation differentiating ts1B from ts1A substitutes an alanine for a threonine at residue 736 in the polymerase (L) protein. The occurrence of a mutation in ts1C inducing substitution of a phenylalanine for a serine residue at an adjacent site (731) suggests that mutations in this region of the polymerase can have significant attenuating effects. The data suggest also that a mutation in the F gene may contribute to the attenuated phenotype.

Beta-2 microglobulin: a prognostic factor in diffuse aggressive non-Hodgkin's lymphomas.

beta-2 microglobulin levels were measured in stored serum taken at presentation from 262 patients treated with combination chemotherapy for Kiel classification high-grade lymphoma at a single centre over a 15 year period. A significant association was found between elevated levels and advanced (Ann Arbor stage III or IV) disease or hepatic infiltration, but not with other sites of extranodal involvement or bulky disease. Patients with normal levels at presentation had a 70% remission rate with treatment compared to 37% of those with elevated levels (P < 0.001). With median follow up of 6 years duration of remission was significantly greater in patients with normal beta-2 microglobulin at presentation (plateau at 70%, compared to median remission of 19 months in those with raised levels, P < 0.001). Survival overall was also better in the group with normal levels (actuarial median 9 years compared to 1 year, P < 0.001). Multivariate analyses including treatment type, age, sex, B symptoms, stage, bulk, albumin, sodium, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase and beta-2 microglobulin, placed beta-2 microglobulin among the three most influential independent variables for prediction of response rate, duration of remission and overall survival.

Measuring cytokine levels in blood. Importance of anticoagulants, processing, and storage conditions.

The stability and recovery of six human recombinant cytokines (tumour necrosis factor (TNF), interferon-alpha (IFN-alpha), IFN-gamma, interleukin-1 alpha (IL-1 alpha), IL-1 beta, and IL-6) from whole blood was investigated with a view to optimizing blood collection and storage procedures prior to performing immunoassays. Blood from healthy volunteers was subjected to various processing and storage procedures. Blood samples were treated with either: ethylenediamine tetraacetic acid (EDTA) (1.5 mg/ml blood) (E); EDTA/Trasylol (1.5 mg and 1000 KIU/ml blood) (ET); heparin (30 IU/ml) (H) or allowed to clot (serum). The bloods were spiked with individual cytokines, split into aliquots and kept at 4 degrees C or RT. In the first instance spiked bloods from healthy volunteers (n = 5 per cytokine) were processed using sterile and non-pyrogenic materials and procedures. At regular time intervals, samples were cold spun, separated, flash frozen and assayed for the appropriate cytokine using RIA/IRMA methods. In a further study, timed separation was repeated with spiked blood from healthy volunteers (n = 5 per cytokine) using normal commercially available blood collection materials and procedures. In a third study, spiked blood from healthy volunteers (n = 3 per cytokine) was processed under sterile and non-pyrogenic conditions, and the blood samples separated, aliquoted and flash frozen within half hour of collection. These were then subjected to repeated cycles of freeze thawing at 4 degrees C or RT before assaying. In general, the stability of cytokines in whole blood was improved by storage at 4 degrees C and/or rapid separation. There was no significant difference between samples handled under sterile, non-pyrogenic conditions and those collected using normal blood collection procedures. The blood collection procedures described in this paper did not induce any of the six cytokines in the unspiked blood. Overall, EDTA-treated samples performed most consistently. The addition of trasylol did not significantly affect the results. Most of the cytokines appeared unaffected by up to three freeze thaw cycles. The stability and recovery of the spiked cytokines varied from least stable to most stable spiked cytokine as follows; TNF-alpha less than IL-6 less than IFN-gamma less than IL-1 alpha less than IFN-alpha less than IL-1 beta. The recovery of spiked IFN-gamma from heparinized plasma samples was considerably higher than any other plasma or serum samples. The recovery of spiked TNF-alpha and IL-6 from serum samples was consistently lower than amounts recovered from plasma samples (anticoagulant treated).(ABSTRACT TRUNCATED AT 400 WORDS)