Potency testing of veterinary vaccines: The way from in vivo to in vitro

Current quality control of inactivated animal vaccines still focuses on the potency of final products in a batch-wise manner. Animal welfare concerns as well as scientific considerations have led to the ‘3Rs-concept’ that comprises the refinement of animal procedures, the reduction of animal numbers, and the replacement of animal models. Although the 3Rs-concept has been widely accepted as a fundamental principle, the number of approved alternatives for in vivo tests is still limited. To promote further progress, the international scientific workshop ‘Potency Testing of Veterinary Vaccines: The Way from in vivo to in vitro’ was held at the Paul-Ehrlich-Institut in Langen, Germany, on 01-03 December 2010. More than 130 participants from industry, academia and regulatory authorities discussed the current state of the 3Rs-concept, examples of its successful implementation as well as still existing hurdles. Special emphasis was laid on the ‘consistency approach’ that aims to ensure relevant quality attributes of vaccine batches by in vitro analyses during production rather than by in vivo potency tests on the final product. This report provides an overview of the insights gained, including the recommendations produced at the end of the workshop.     

Obesity-Susceptibility Loci and Their Influence on Adiposity-Related Traits in Transition from Adolescence to Adulthood - The HUNT Study

Introduction

Obesity-susceptibility loci have been related to adiposity traits in adults and may affect body fat estimates in adolescence. There are indications that different sets of obesity-susceptibility loci influence level of and change in obesity-related traits from adolescence to adulthood.

Objectives

To investigate whether previously reported obesity-susceptible loci in adults influence adiposity traits in adolescence and change in BMI and waist circumference (WC) from adolescence into young adulthood. We also examined whether physical activity modifies the effects of these genetic loci on adiposity-related traits.

Methods

Nine obesity-susceptibility variants were genotyped in 1 643 adolescents (13–19 years old) from the HUNT study, Norway, who were followed-up into young adulthood. Lifestyle was assessed using questionnaires and anthropometric measurements were taken. The effects of genetic variants individually and combined in a genetic predisposition score (GPS) on obesity-related traits were studied cross-sectionally and longitudinally. A modifying effect of physical activity was tested.

Results

The GPS was significantly associated to BMI (B: 0.046 SD/allele [0.020, 0.073], p = 0.001) in adolescence and in young adulthood (B: 0.041 SD/allele [0.015, 0.067], p = 0.002) as it was to waist circumference (WC). The GPS was not associated to change in BMI (p = 0.762) or WC (p = 0.726). We found no significant interaction effect between the GPS and physical activity.

Conclusions

Our observations suggest that obesity-susceptibility loci established in adults affect BMI and WC already in adolescence. However, an association with change in adiposity-related traits from adolescence to adulthood could not be verified for these loci. Neither could an attenuating effect of physical activity on the association between the obesity-susceptibility genes and body fat estimates be revealed.     

Use of commercially available monoclonal antibodies for immunoenzyme double staining

Summary An immunoenzyme double-staining method for the simultaneous detection of two cellular epitopes, using commercially available mouse monoclonal antibodies, is described. The method employs a combination of the suppression of endogenous biotin and two successive indirect techniques with a blocking step in between. The first indirect method involves an unlabelled monoclonal antibody followed by an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin. After a blocking step with normal mouse serum, the second indirect method is applied using a biotinylated monoclonal antibody followed by the visualization of this antibody by avidin-biotinylated peroxidase complex (ABC) or rabbit anti-biotin and peroxidase-conjugated swine anti-rabbit immunoglobulin in successive steps. Using these methods in combination with the introduction of dioctyl sodium sulphosuccinate and tetramethylbenzidine as chromogens for peroxidase activity, two cellular epitopes could be distinguished clearly in tissue sections by the green-and violet-stained peroxidase and alkaline phosphatase activities, respectively. The expression of two epitopes on the same cellular constituent is outlined by the coappearance of both enzyme activities as a bluish-purple colour. This method allows for the simultaneous identification, localization and enumeration of two cellular epitopes. These can serve as parameters for a number of pathological processes.     

Amyloid P component--a special type of collagen?

The localization of amyloid P-components is demonstrated by immunofluorescence microscopy in normal human tissue (kidney, spleen, liver). The relation to collagen and to amyloidosis is discussed.     

The influence of zygosity and chorion type on fat distribution in young adult twins consequences for twin studies.

An adverse intra-uterine environment has been associated with abdominal fat distribution in singletons. Twins often have a low birth weight and a short gestation. Therefore, they may have an increased risk to develop abdominal obesity. Furthermore, monozygotic monochorionic twins (MZ MC) have a larger intra-pair birth weight difference compared to monozygotic dichorionic twins (MZ DC). If adult anthropometry is programmed in utero, this may affect the intra-pair correlations in adulthood and, consequently, also the results from the classic twin method to estimate genetic and environmental influences. In the present study, we compared the absolute values, the intra-pair differences, and the intra-pair correlations of body mass, height, BMI, and abdominal fat distribution of 424 MZ MC, MZ DC and dizygotic (DZ) twin pairs (aged 18-34 yrs). DZ, MZ DC and MZ MC twins did not differ for most anthropometric characteristics. Only MZ women tended (p = 0.03) to accumulate more abdominal fat compared to DZ twins. Overall, the contribution of zygosity and chorion type to adult anthropometry was rather low (< or = 1.7%). Although the intra-pair birth weight difference of MZ MC pairs (10.5% in men, 12.3% in women) was significantly larger compared to that of MZ DC pairs (6.9% and 9.2% resp.), the intra-pair differences in adult anthropometry were similar for both MZ twin types. Also the intra-pair correlations of MZ MC and MZ DC pairs were strikingly alike, suggesting no significant influence of the prenatal environment on adult concordance. In conclusion, the substantial difference in the prenatal environment of MZ MC and MZ DC twins did not result in a difference in intra-pair concordance of adult anthropometry and fat distribution. Therefore, we suggest that the chorion type of MZ twins does not bias the twin design and the estimation of the genetic contribution to adult anthropometry.     

Genetic and environmental determination of tracking in static strength during adolescence.

The purpose of this study was to determine whether the observed phenotypic stability in static strength during adolescence, as measured by interage correlations in arm pull, is mainly caused by genetic and/or environmental factors. Subjects were from the Leuven Longitudinal Twin Study (n = 105 pairs, equally divided over 5 zygosity groups). Arm-pull data were aligned on age at peak height velocity to attenuate the temporal fluctuations in interage correlations caused by differences in timing of the adolescent growth spurt. Developmental genetic models were fitted using structural equation modeling. After the data were aligned on age at peak height velocity, the annual interage correlations conformed to a quasi-simplex structure over a 4-yr interval. The best-fitting models included additive genetic and unique environmental sources of variation. Additive genetic factors that already explained a significant amount of variation at previous measurement occasions explained 44.3 and 22.5% of the total variation at the last measurement occasion in boys and girls, respectively. Corresponding values for unique environmental sources of variance are 31.2 and 44.5%, respectively. In conclusion, the observed stability of static strength during adolescence is caused by both stable genetic influences and stable unique environmental influences in boys and girls. Additive genetic factors seem to be the most important source of stability in boys, whereas unique environmental factors appear to be more predominant in girls.     

A confirmation of the proposed model for the hexose uptake system ofChlorella vulgaris. Anaerobic studies in the light and in the dark

Summary Energy for accumulation of sugar analogues inChlorella vulgaris can be supplied by respiration and by light. Under anaerobic conditions a quantum efficiency of close to one has been determined for light-dependent uptake of 6-deoxyglucose at 712 nm. Although the rate of uptake under anaerobic conditions in the dark is less than 15% of that in the light, the steady-state plateau of accumulation does not change when the light is turned off. This is explained by the observation that efflux of sugar is inhibited by lack of energy to the same extent as influx. This agrees with a model proposed earlier (Komor, Haass & Tanner,Biochim. Biophys. Acta 266:649, 1972). Under aerobic conditions during the steady-state plateau in the dark, the rate of influx of 6-deoxyglucose is twice that of initial influx (positive transmembrance effect). By anaerobiosis the initial influx is almost completely inhibited, whereas the steady-state influx is affected only to 50%. This points to an energyless homoexchange. However, this flux is completely inhibited by uncoupling agents. To explain these results, the existence of an energygenerating efflux has been assumed, which is affected by uncouplers. The efficiency of energy production by efflux and the re-use of this energy for influx is inversely related to the amount of energy supplied by other energy-generating processes in the cells; under anaerobic conditions in the dark, 90% of the efflux energy seems to be available for influx.