Anne-Marie Nagy

Effects of non-steroidal anti-inflammatory drugs on the luminol and lucigenin amplified chemiluminescence of human neutrophils

A panel of non-steroidal anti-inflammatory drugs commonly used for therapeutic purposes was assessed for their effects on the respiratory burst of isolated human polymorphonuclear neutrophils. Cells were stimulated with opsonised yeast and the production of reactive oxygen species was measured by amplified chemiluminescence with luminol and lucigenin which are two luminogenic agents measuring different cellular events. A special attention was devoted to the establishment of dose-effect curves and calculation of ED50. Some of the drugs tested (acemetacine, diclofenac, flufenamic acid and niflumic acid) were able to decrease both luminol and lucigenin chemiluminescence in a dose-dependent manner reflecting an inhibitory effect on the respiratory burst. The most potent derivative was flufenamic acid (ED50 8 and 78 microM, respectively, with luminol and lucigenin), followed by diclofenac (21 and 98 microM), niflumic acid (97 and 227 microM) and acemetacine (585 and 427 microM). In contrast, several other drugs (flurbiprofen, ibuprofen, ketoprofen, piroxicam) stimulated both luminol and lucigenin chemiluminescence, suggesting a pro-oxidant activity. Acetylsalicylic acid (up to 1250 microM) was a modest inhibitor (maximum 25% inhibition) showing no dose-dependent effect and tolmetin (up to 125 microM) had no significant effect in both systems. The results were in agreement using both luminogenic agents, except for indomethacin, naproxen and tenoxicam which showed different kinds of effects. The unspecific and complex nature of the measurement systems used did not allow to give a complete mechanistic interpretation of the results, but the comparison with literature data gave some pertinent explanations for both anti- and pro-oxidant effects.

The potential allergenicity of novel foods

Recombinant DNA technology provides a powerful tool to create new products in many different fields. Agriculture and the agro-food industry are two of them. Genetic engineering aims at improving the yield, the nutritional quality or the technological value of food crops. The composition of foods may also be modified in order to decrease the content of toxic substances sometimes present in certain food crops. The expression of recombinant proteins in foods must be carefully assessed as proteins may induce allergic reactions in humans. Currently, the potential allergenicity of a protein can be reasonably assessed only when the protein is known to trigger an immune response in sensitive subjects. By contrast, the potential allergenicity of a protein of unknown allergenicity cannot be easily predicted as no immunoserum of allergic subjects is available. That is why an allergenicity assessment model for any genetically engineered food should be designed. Even though the gene product is completely characterised in the transgenic product, this does not necessarily provide information on its potential allergenicity. Animal models, in vitro tests and protein structure should be taken into account. Besides, allergenicity is a biosafety issue, for it deals with human health. The issue of food allergies in food safety should not be neglected, as an allergic reaction can be life threatening.

Scientific and ethical issues of preimplantation diagnosis

Preimplantation diagnosis (PID) offers couples at high risk of having offspring affected with a genetic disorder the possibility of an early prenatal diagnosis. For many couples this approach will give the opportunity to avoid a selective termination of affected pregnancies. Substantial advances were made in PID since the report, in 1990, of the first birth obtained after PID. Yet, many technical hazards have to be solved for PID to become a standard clinical tool. The very close correlation existing between the forthcoming developments in the fields of PID and human genome mapping will improve the reliability and efficiency of genetic diagnosis. In the near future, the procedure may also become easier and safer. As a consequence, the indications for PID could be extended to other genetic defects, such as multifactorial diseases. They could also be extended to cases with no medical background, such as social gender selection or behavioural traits. In this perspective, it is now time for both the medical and scientific communities to identify the ethical issues related to these potential new indications.

Epitope mapping on intact, heated and reduced molecular variants of human chorionic gonadotrophin

Monoclonal antibodies (MAbs) raised against purified human chorionic gonadotrophin (hCG) (n = 30) and synthetic peptides derived from hCG (n = 3) were able to recognize by Western blotting several hCG dimers (57-47 and 42 kDa), free beta-subunits (35-32, 26 and 16 kDa) and free alpha-subunit (21 kDa) which coexist in commercially available hCG preparations. According to differences in the immunoreactivity of hCG-related molecular forms observed under native or denaturing conditions such as boiling or reducing hCG samples before or after gel electrophoresis, nine classes of MAbs able to recognize different immunoreactive domains were determined. Three domains corresponded to continuous epitopes recognized by MAbs raised against hCG-related peptides. The six remaining domains, recognized by the other MAbs, contained discontinuous epitopes from which three were surface-oriented and three disguised in the holo-hormone. This solid-phase approach, combining native and denaturing conditions, represents a simple and powerful tool to screen the specificity of MAbs from varying sources and to investigate molecular variants of proteic hormone.

Origin and significance of the heterogeneity of protein hormones

The heterogeneity of circulating protein hormone is the result of multiple steps including gene expression, mRNA maturation, post-translational processing and peripheral catabolism. As a consequence of these cumulative events, it seems difficult to evaluate the endocrine function by using specific radioimmunoassays for each circulating variant of a protein hormone. Moreover, the discovery of new molecular variants of protein hormones with unknown biological significance complicates the standardization and the clinical interpretation of immunoassays.

Human chorionic gonadotrophin early pregnancy levels are more closely related to changes in β-subunit trophoblast production than to variations in α-subunit production

Summary Human chorionic gonadotrophin (hCG) is composed of two non-covalently bound α and β subunits synthesized from separate mRNAs. The hCG heterodimer and uncombined subunits are secreted during early gestation by trophoblast into the maternal bloodstream and into the exocoelomic cavity on the opposite side of the trophoblastic layer. The aim was to compare the relative amount of hCG and its free subunits in these compartments. Levels of hCG were similar in coelomic fluid and in maternal serum collected from the same women. By contrast, levels of free subunits were higher in coelomic fluid than in maternal serum: 186-fold for free αhCG subunit and 34-fold for free βhCG subunit. These enormous gradients are likely to be related to differences in the clearance rates of hCG and its subunits between maternal and exocoelomic compartments. Considering coelomic fluid as a metabolic cul-de-sac into which hCG and its subunits accumulate and are slowly metabolized, their levels in this fluid may be reasonably considered as a direct reflection of their trophoblastic production. This hypothesis suggests that the amount of free α subunit is in formidable excess when compared to intact hCG and free βhCG subunit and that only a small fraction ( 90% of βhCG.