Myosin heavy chain fibre type composition in foals: analyses at the mRNA and protein level.

REASONS FOR PERFORMING STUDY: An optimal developed musculoskeletal system is vital for the performance of the horse. Previously, we showed that in the m. gluteus medius from adult untrained horses, identical mRNA and protein expression patterns were found in the majority of fibres. However, co-expression of IIa and IId/x myosin heavy chain (MyHC) was substantially more common at the protein than at the mRNA level, suggesting a transcriptionally controlled fine-tuning of these 2 genes. OBJECTIVE: To analyse the MyHC transcripts and proteins (including the cardiac alpha isoform) in the same muscle during post natal development when the muscle is adapting to movement and load. METHODS: Biopsies were taken from the m. gluteus medius of 2 Dutch Warmblood foals at 0, 2, 4, 22 and 48 weeks of age. mRNA was compared to protein expression on a fibre-to-fibre basis using in situ hybridisation and immunofluorescence. The MyHC slow (I), alpha, IIa and IId/x isoforms were analysed. RESULTS: At all ages the expression of the mRNA and protein MyHC isoforms was almost identical. Surprisingly, coexpression of the IIad isoform was also detected at the mRNA level especially early in life. The transcript of the alpha isoform was only detectable at young age, indicating silencing of the gene around birth. CONCLUSION: During the first year of life, MyHCs are continuously adapting at the mRNA and protein level. Additionally, the regulation of hybrid fibres is different from that in adult fibres. POTENTIAL RELEVANCE: We postulate that interfering in this process by e.g. early training will be levelled out by the maturation of the muscle.

Ethical issues of cost effectiveness analysis and guideline setting in mental health care.

This article discusses ethical issues which are raised as a result of the introduction of economic evidence in mental health care in order to rationalise clinical practice. Cost effectiveness studies and guidelines based on such studies are often seen as impartial, neutral instruments which try to reduce the influence of non-scientific factors. However, such rationalising instruments often hide normative assumptions about the goals of treatment, the selection of treatments, the role of the patient, and the just distribution of scarce resources. These issues are dealt with in the context of increased control over clinical practice by third parties. In particular, health insurers have a great interest in economic evidence in clinical care settings in order to control access to and quality of (mental) health care. The authors conclude that guideline setting and cost effectiveness analysis may be seen as important instruments for making choices in health care, including mental health care, but that such an approach should always go hand in hand with a social and political debate about the goals of medicine and (mental) health care. This article is partly based on the results of a research project on the normative aspects of guideline setting in psychiatry and cardiology which was conducted under the guidance of the Royal Dutch Medical Association.

Guidelines for appropriate care: the importance of empirical normative analysis.

The Royal Dutch Medical Association recently completed a research project aimed at investigating how guidelines for 'appropriate medical care' should be construed. The project took as a starting point that explicit attention should be given to ethical and political considerations in addition to data about costs and effectiveness. In the project, two research groups set out to design guidelines and cost-effectiveness analyses (CEAs) for two circumscribed medical areas (angina pectoris and major depression). Our third group was responsible for the normative analysis. We undertook an explorative, qualitative pilot study of the normative considerations that played a role in constructing the guidelines and CEAs, and simultaneously interviewed specialists about the normative considerations that guided their diagnostic and treatment decisions. Explicating normative considerations, we argue, is important democratically: the issues at stake should not be left to decision analysts and guideline developers to decide. Moreover, it is a necessary condition for a successful implementation of such tools: those who draw upon these tools will only accept them when they can recognize themselves in the considerations implied. Empirical normative analysis, we argue, is a crucial tool in developing guidelines for appropriate medical care.

Five days of oral topotecan (Hycamtin), a phase I and pharmacological study in adult patients with solid tumours.

Topotecan is a specific inhibitor to topoisomerase I. An oral formulation of topotecan is available with a bioavailability of 32-44% in humans. A phase I and pharmacological study of the oral formulation of topotecan administered daily for 5 days every 21 days was performed in adult patients with solid tumours to determine the maximum tolerated dose (MTD). Adult patients with a WHO performance status < or = 2 adequate haematological, hepatic and renal functions, with malignant solid tumours refractory to standard forms were entered into the study. Pharmacokinetics were performed on days 1 and 4 of the first course using a validated high performance liquid chromatographic assay. 29 patients entered the study, all patients were evaluable for toxicity and response. The doses studied in the 29 patients were 1.2, 1.8, 2.3, 2.7 mg/m2/day and a fixed dose of 4 mg/day without surface area adjustment. A total of 109 courses were given. Dose limiting toxicity (DLT) was reached at a dose of 2.7 mg/m2/day and consisted of CTC (NCI-Common Toxicity Criteria) grade IV granulocytopenia. The regimen was well tolerated. Non-haematological toxicities were mild, including fatigue, anorexia, nausea, vomiting and diarrhoea. A significant correlation was observed between the percentage decrease in white blood cells versus the area under the curve (AUC(t)) of topotecan lactone (R = 0.76 P < 0.01) which was modelled by a sigmoidal Emax function. The correlation coefficient between the absolute topotecan dose administered and the AUC(t) was R = 0.52 (P = 0.04). Pharmacokinetics of the fixed dose of 4 mg/day were comparable to the 2.3 mg/m2/day dose. DLT in this phase I study of five daily doses of oral topotecan every 21 days was granulocytopenia. The recommended dose for phase II studies is 2.3 mg/m2/day or alternatively, a fixed dose of 4 mg/day.

Development of a preparation and staining method for fetal erythroblasts in maternal blood: simultaneous immunocytochemical staining and FISH analysis.

In order to detect fetal nucleated red blood cells (NRBCs) in maternal blood, a protocol was developed which aimed at producing a reliable staining method for combined immunocytochemical and FISH analysis. The technique had to be suitable for eventual automated screening of slides. Chorionic villi washings, cord blood, and maternal blood samples were used for this study. After a density gradient separation and centrifugal cytology, slides were stained either with 3,3-diaminobenzidin (DAB), a marker for heme, or with antibodies against the gamma-chain of fetal hemoglobin (HbF). FISH analysis for both X- and Y-chromosomes was performed on the same slides. Cytocentrifugation provided a controlled cell density on the slides with good cell morphology. Both the DAB and HbF staining were suitable for manual screening of large numbers of slides. The HbF staining, although supposed to be more specific for fetal NRBCs, appeared to be more sensitive to minor changes in preparation. We were eventually able to combine HbF staining with FISH analysis, and produced a detection efficiency of >85% for both X- and Y-chromosome signals. This preparation protocol simplifies the detection of NRBCs in maternal blood. Immunocytochemical staining and FISH analysis can be performed on the same cell with good image contrast, thus facilitating both manual and automated image analysis. This will facilitate the use of this approach for prenatal diagnosis.