Challenges in the implementation of EAACI guidelines on allergen immunotherapy: A global perspective on the regulation of allergen products.

Regulatory approaches for allergen immunotherapy (AIT) products and the availability of high-quality AIT products are inherently linked to each other. While allergen products are available in many countries across the globe, their regulation is very heterogeneous. First, we describe the regulatory systems applicable for AIT products in the European Union (EU) and in the United States (US). For Europe, a depiction of the different types of relevant procedures, as well as the committees involved, is provided and the fundamental role of national agencies of the EU member states in this complex and unique network is highlighted. Furthermore, the regulatory agencies from Australia, Canada, Japan, Russia, and Switzerland provided information on the system implemented in their countries for the regulation of allergen products. While AIT products are commonly classified as biological medicinal products, they are made available by varying types of procedures, most commonly either by obtaining a marketing authorization or by being distributed as named patient products. Exemptions from marketing authorizations in exceptional cases, as well as import of allergen products from other countries, are additional tools applied by countries to ensure availability of needed AIT products. Several challenges for AIT products are apparent from this analysis and will require further consideration.

Allergen manufacturing and quality aspects for allergen immunotherapy in Europe and the United States: An analysis from the EAACI AIT Guidelines Project.

Adequate quality is essential for any medicinal product to be eligible for marketing. Quality includes verification of the identity, content and purity of a medicinal product in combination with a specified production process and its control. Allergen products derived from natural sources require particular considerations to ensure adequate quality. Here, we describe key aspects of the documentation on manufacturing and quality aspects for allergen immunotherapy products in the European Union and the United States. In some key parts, requirements in these areas are harmonized while other fields are regulated separately between both regions. Essential differences are found in the use of Reference Preparations, or the requirement to apply standardized assays for potency determination. As the types of products available are different in specific regions, regulatory guidance for such products may also be available in one specific region only, such as for allergoids in the European Union. Region-specific issues and priorities are a result of this. As allergen products derived from natural sources are inherently variable in their qualitative and quantitative composition, these products present special challenges to balance the variability and ensuring batch-to-batch consistency. Advancements in scientific knowledge on specific allergens and their role in allergic disease will consequentially find representation in future regulatory guidelines.

Time dependent responses of glycolytic intermediates in a detailed glycolytic model of Lactococcus lactis during glucose run-out experiments.

Glucose addition and subsequent run-out experiments were compared to simulations with a detailed glycolytic model of Lactococcus lactis. The model was constructed largely on bases of enzyme kinetic data taken from literature and not adjusted for the specific simulations shown here. Upon glucose depletion a rapid increase in PEP, inorganic phosphate and a gradual decrease in fructose 1,6-bisphosphate (FBP) were measured and predicted by simulation. The dynamic changes in these and other intermediate concentrations as measured in the experiments were well predicted by the kinetic model.

Spontaneous formation of a mannitol-producing variant of Leuconostoc pseudomesenteroides grown in the presence of fructose.

We report the spontaneous formation of a stable mannitol-producing variant of Leuconostoc pseudomesenteroides. The mannitol-producing variant showed mannitol dehydrogenase activity which was absent in the parental strain. It was also able to use fructose and glucose simultaneously, whereas the parental strain showed diauxic growth with these sugars. A possible explanation of these observations is discussed.

A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation.

The alternative oxidase is a quinol oxidase of the respiratory chain of plants and some fungi and protists. Its activity is regulated by redox-sensitive disulphide bond formation between neighbouring subunits and direct interaction with certain alpha-ketoacids. To investigate these regulatory mechanisms, we undertook site-directed mutagenesis of soybean and Arabidopsis alternative oxidase cDNAs, and expressed them in tobacco plants and Escherichia coli, respectively. The homologous C99 and C127 residues of GmAOX3 and AtAOX1a, respectively, were changed to serine. In the plant system, this substitution prevented oxidative inactivation of alternative oxidase and rendered the protein insensitive to pyruvate activation, in agreement with the recent results from other laboratories [Rhoads et al. (1998) J. Biol. Chem. 273, 30750-30756; Vanlerberghe et al. (1998) Plant Cell 10, 1551-1560]. However, the mutated protein is instead activated specifically by succinate. Measurements of AtAOX1a activity in bacterial membranes lacking succinate dehydrogenase confirmed that the stimulation of the mutant protein's activity by succinate did not involve its metabolism. Examples of alternative oxidase proteins with the C to S substitution occur in nature and these oxidases are expected to be activated under most conditions in vivo, with implications for the efficiency of respiration in the tissues which express them.

Activation of the plant alternative oxidase by high reduction levels of the Q-pool and pyruvate.

This report describes the activation of the alternative oxidase (AOX) of higher plant mitochondria by a high reduction level of the ubiquinone pool in the presence of pyruvate. In mitochondria from both thermogenic (Arum italicum spadices) and nonthermogenic (Glycine max cotyledons) tissues AOXis activated when the Q-pool becomes highly reduced in the presence of pyruvate. Pyruvate is essential for this activation. The enzyme is not activated when pyruvate is added after a transient high reduction level of the Q-pool, but is when pyruvate is added before the transient reduction. Pyruvate also protects the enzyme against inhibition during catalytic turnover. Although this activation is not accompanied by a reduction of the covalent disulfide bond, the same activation can be achieved with dithiothreitol (DTT). It is suggested that a part of the activation by DTT is not the result of reducing the covalent disulfide bond, and the relation between these types of activation is discussed. The importance of this activation for the in vivo regulation and its relation to previously reported activators is discussed. A mechanism is proposed in which it is suggested that AOX is inactivated by its product (oxidized ubiquinone) during catalysis and that this inhibition is prevented in the presence of pyruvate. The inhibition can be reversed by a reductive process, achieved by high levels of reduction of the Q-pool or by DTT, but not by pyruvate. This restoration of activity is not related to the redox process involved in reducing the covalent disulfide bond.

New inhibitors of the ubiquinol oxidase of higher plant mitochondria.

A screen has been performed of possible inhibitors of the ubiquinol oxidase of higher plant mitochondria by assaying their effects on cyanide-insensitive NADH oxidase of mitochondria of Arum maculatum. A number of compounds which have powerful inhibitory effects have been identified. Potent inhibition was found with compounds related to the previously described n-propyl gallate, but with the n-propyl sidechain replaced with alkyl chains of greater hydrophobicity. Titration of a range of partial reactions showed that the inhibitors act specifically on the ubiquinol oxidase. The concentrations of inhibitor required are dependent on the respiratory substrate and on the amount of mitochondria used in the assay. Octyl gallate also proved to be a potent inhibitor of the ubiquinol oxidase in tobacco cell suspensions. A second class of compounds which strongly inhibit cyanide-insensitive NADH oxidation is aurachin C and its analogues. Compounds related to aurachin D are much less effective. Titrations of a range of partial reactions indicate that inhibition is caused by a direct action on the ubiquinol oxidase. However, both types of aurachins also act strongly at the Qi site of the cytochrome bc1 complex, as already known to be the case in other systems, and so they are of more limited value for studies of the ubiquinol oxidase. Titration of the oxidation of NADH via the ubiquinol oxidase in a purified mitochondrial fraction from the spadices of Arum maculatum with octyl gallate gave a half-maximal effect at a concentration of around 6 nM when the protein concentration was 14 micrograms ml-1. A similar titre was obtained with a decyl derivative of aurachin C. This allowed us to estimate an upper limit for the concentration of ubiquinol oxidase in these mitochondria of 0.72 +/- 0.15 nmol mg-1 protein, or a ratio of ubiquinol oxidase/cytochrome oxidase of about 15 +/- 7:1. The measurements also provide a minimal turnover number for the ubiquinol oxidase of 186 +/- 42 electrons.s-1. Titration of the ubiquinol oxidase in soybean cotyledon mitochondria with these compounds gave the concentration of inhibitor required to elicit 50% of the maximum observed effect (I50) values about one order of magnitude higher than those found with Arum mitochondria, and again the values depended on the respiratory substrate. An explanation for the variation in I50 values may be found in terms of differences in oxidase concentrations in the different mitochondrial membranes and in the differences in rate-controlling steps with substrates of different activities.

Cytochrome and alternative respiratory pathways compete for electrons in the presence of pyruvate in soybean mitochondria.

The partitioning of electrons between the alternative oxidase and the cytochrome pathway of soybean mitochondria has been reassessed in the presence of the alternative oxidase activator pyruvate. In the presence of pyruvate and with succinate as substrate, the alternative oxidase became active at a much lower level of ubiquinone reduction than in the absence of pyruvate. Under state 4 (no ADP present) conditions, activation of the alternative oxidase with pyruvate resulted in an oxidation of b cytochromes, demonstrating switching of electrons away from the cytochrome chain. In the presence of ferricyanide and the cytochrome oxidase inhibitor KCN, cytochrome chain activity could be followed spectrophotometrically and that of the alternative pathway with an oxygen electrode. Under these conditions, the addition of pyruvate diverted electron flow from the cytochrome chain to the alternative pathway; subsequent inhibition of the alternative oxidase increased electron flow via the cytochrome chain. This indicates that electrons can be switched from one pathway to the other when the cytochrome chain is not saturated and this was confirmed by n-propylgallate titrations (p plots) of mitochondria oxidizing succinate. Decreases in ADP/O ratios and phosphorylation rate upon addition of pyruvate indicated that the alternative pathway could also contribute to respiration under state 3 conditions. The results indicate that when the alternative oxidase is activated by pyruvate, it can compete for electrons with the cytochrome chain and does not act as an overflow pathway. The significance of these observations for in vivo respiration is discussed.

Hexose uptake by Catharanthus roseus cell suspensions is inhibited by a high medium salt content.

The uptake of glucose and fructose from the medium by Catharanthus roseus cell suspensions was strongly inhibited by high medium salt concentration, such as found in LS (Linsmaier and Skoog 1965) medium. After inoculation into standard LS nutrient medium with less than 5 mM hexose no uptake occurred, while in low salt medium hexose was completely depleted. At a hexose concentration of 50 mM the uptake rate was higher in low salt medium than in standard medium. The lower rate of uptake at high salt concentration was not the result of a pH or osmotic effect of the salts. Probably the affinity of the hexose carrier is affected by the ion concentration of the medium. The decrease in medium salt concentration during normal batch culture probably will have a considerable effect on hexose uptake.