Liposome-induced hypersensitivity reactions: Risk reduction by design of safe infusion protocols in pigs.

Intravenous administration of liposomal drugs can entail infusion reactions, also known as hypersensitivity reactions (HSRs), that can be severe and sometimes life-threatening in a small portion of patients. One empirical approach to prevent these reactions consists of lowering the infusion speed and extending the infusion time of the drug. However, different liposomal drugs have different levels of reactogenicity, which means that the optimal protocol for each liposomal drug may differ and should be identified and evaluated to make the treatment as safe and convenient as possible. The goal of the present study was to explore the use of pigs for the above purpose, using PEGylated liposomal prednisolone (PLP) as a model drug. We compared the reactogenicities of bolus versus infusion protocols involving 2-, 3- and 4-step dose escalations for a clinically relevant total dose, also varying the duration of infusions. The strength of HSRs was measured via continuous recording of hemodynamic parameters and blood thromboxane B2 levels. We showed that bolus administration or rapid infusion of PLP caused transient changes in systemic and pulmonary blood pressure and heart rate, most notably pulmonary hypertension with paralleling rises in plasma thromboxane B2. These adverse responses could be significantly reduced or eliminated by slow infusion of PLP, with the 3-h 3-step dose escalation protocol being the least reactogenic. These data suggest that the pig model enables the development of safe infusion protocols for reactogenic nanomedicines.

Pseudo-anaphylaxis to Polyethylene Glycol (PEG)-Coated Liposomes: Roles of Anti-PEG IgM and Complement Activation in a Porcine Model of Human Infusion Reactions.

Polyethylene glycol (PEG)-coated nanopharmaceuticals can cause mild to severe hypersensitivity reactions (HSRs), which can occasionally be life threatening or even lethal. The phenomenon represents an unsolved immune barrier to the use of these drugs, yet its mechanism is poorly understood. This study showed that a single i.v. injection in pigs of a low dose of PEGylated liposomes (Doxebo) induced a massive rise of anti-PEG IgM in blood, peaking at days 7-9 and declining over 6 weeks. Bolus injections of PEG-liposomes during seroconversion resulted in anaphylactoid shock (pseudo-anaphylaxis) within 2-3 min, although similar treatments of naïve animals led to only mild hemodynamic disturbance. Parallel measurement of pulmonary arterial pressure (PAP) and sC5b-9 in blood, taken as measures of HSR and complement activation, respectively, showed a concordant rise of the two variables within 3 min and a decline within 15 min, suggesting a causal relationship between complement activation and pulmonary hypertension. We also observed a rapid decline of anti-PEG IgM in the blood within minutes, increased binding of PEGylated liposomes to IgM+ B cells in the spleen of immunized animals compared to control, and increased C3 conversion by PEGylated liposomes in the serum of immunized pigs. These observations taken together suggest rapid binding of anti-PEG IgM to PEGylated liposomes, leading to complement activation via the classical pathway, entailing anaphylactoid shock and accelerated blood clearance of liposome-IgM complexes. These data suggest that complement activation plays a causal role in severe HSRs to PEGylated nanomedicines and that pigs can be used as a hazard identification model to assess the risk of HSRs in preclinical safety studies.

Comparison of redox and gene expression changes during vegetative/generative transition in the crowns and leaves of chromosome 5A substitution lines of wheat under low-temperature condition.

The aim of our experiments was to investigate the effect of chromosome 5A on the thiol-dependent redox environment and on the transcription of cold- and vernalization-related genes during the vegetative/generative transition in crowns and leaves of wheat. Chinese Spring, a moderately freezing-tolerant variety, and its more and less tolerant substitution lines - [CS(Ch5A)] and [CS(Tsp5A)], respectively - with different combinations of vernalization alleles were compared. At low temperature, the amount of cystine and glutathione disulphide and the related redox potentials increased in the crowns but not in the leaves. In the crowns of the substitution lines, the concentration and redox state of thiols were different only at the vegetative and double ridge (start of the generative transition) stages. The expression of the vernalization-related VRN1 gene increased significantly during the transition both in the crowns and leaves. The transcription of the freezing tolerance-related CBF14, COR14b and COR39 genes markedly increased in both organs after 2 weeks at 4 °C when the seedlings were still in the vegetative stage. This increment was greater in CS(Ch5A) than in CS(Tsp5A). The Ch5A chromosome in CS genetic background enhanced the expression of CBF regulon even in the generative phase in crown that is the key organ for overwintering and freezing tolerance. At certain developmental stages, both the thiol and the transcript levels differed significantly in the two substitution lines.

The cold response of CBF genes in barley is regulated by distinct signaling mechanisms.

Cold acclimation ability is crucial in the winter survival of cereals. In this process CBF transcription factors play key role, therefore understanding the regulation of these genes might provide useful knowledge for molecular breeding. In the present study the signal transduction pathways leading to the cold induction of different CBF genes were investigated in barley cv. Nure using pharmacological approach. Our results showed that the cold induced expression of CBF9 and CBF14 transcription factors is regulated by phospholipase C, phospholipase D pathways and calcium. On the contrary, these pathways have negative effect on the cold induction of CBF12 that is regulated by a different, as yet unidentified pathway. The diversity in the regulation of these transcription factors corresponds to their sequence based phylogenetic relationships suggesting that their evolutionary separation happened on structural, functional and regulational levels as well. On the CBF effector gene level, the signaling regulation is more complex, resultant effect of multiple pathways.

Heavy metal accumulation and tolerance of energy grass (Elymus elongatus subsp. ponticus cv. Szarvasi-1) grown in hydroponic culture.

Phytoremediation is a plant based, cost effective technology to detoxify or stabilise contaminated soils. Fast growing, high biomass, perennial plants may be used not only in phytoremediation but also in energy production. Szarvasi-1 energy grass (Elymus elongatus subsp. ponticus cv. Szarvasi-1), a good candidate for this combined application, was grown in nutrient solution in order to assess its Cd, Cu, Ni, Pb and Zn accumulation and tolerance. Its shoot metal accumulation showed the order Pb < Ni < Cu ∼ Cd < Zn. In parallel with this, Pb and Ni had no or very little influence on the growth, dry matter content, chlorophyll concentration and transpiration of the plants. Cu and Cd treatment resulted in significant decreases in all these parameters that can be attributed to Fe plaque formation in the roots suggested by markedly increased Fe and Cu accumulation. This came together with decreased shoot and root Mn concentrations in both treatments while shoot Cu and Zn concentrations decreased under Cd and Cu exposure, respectively. Zn treatment had no effect or even slightly stimulated the plants. This may be due to a slight stimulation of Fe translocation and a very efficient detoxification mechanism. Based on the average 300 mg kg⁻¹ (dry mass) Zn concentration which is 0.03% of the shoot dry mass the variety is suggested to be classified as Zn accumulator.

Transgenic barley lines prove the involvement of TaCBF14 and TaCBF15 in the cold acclimation process and in frost tolerance.

The enhancement of winter hardiness is one of the most important tasks facing breeders of winter cereals. For this reason, the examination of those regulatory genes involved in the cold acclimation processes is of central importance. The aim of the present work was the functional analysis of two wheat CBF transcription factors, namely TaCBF14 and TaCBF15, shown by previous experiments to play a role in the development of frost tolerance. These genes were isolated from winter wheat and then transformed into spring barley, after which the effect of the transgenes on low temperature stress tolerance was examined. Two different types of frost tests were applied; plants were hardened at low temperature before freezing, or plants were subjected to frost without a hardening period. The analysis showed that TaCBF14 and TaCBF15 transgenes improve the frost tolerance to such an extent that the transgenic lines were able to survive freezing temperatures several degrees lower than that which proved lethal for the wild-type spring barley. After freezing, lower ion leakage was measured in transgenic leaves, showing that these plants were less damaged by the frost. Additionally, a higher Fv/Fm parameter was determined, indicating that photosystem II worked more efficiently in the transgenics. Gene expression studies showed that HvCOR14b, HvDHN5, and HvDHN8 genes were up-regulated by TaCBF14 and TaCBF15. Beyond that, transgenic lines exhibited moderate retarded development, slower growth, and minor late flowering compared with the wild type, with enhanced transcript level of the gibberellin catabolic HvGA2ox5 gene.

Cold response of dedifferentiated barley cells at the gene expression, hormone composition, and freezing tolerance levels: studies on callus cultures.

In this study, data is presented how dark-grown, embryogenic barley callus cells respond to cold without any light-dependent, chloroplast-related mechanism, independently of the systemic signals. The expression of HvCBF9, HvCBF14, and HvCOR14b genes, members of one of the most important cold-inducible regulatory system, was measured by real-time PCR. Characteristic of the cold response was similar in the crowns of seedlings and in dark-grown callus cultures, however, gene expression levels were lower in calli. Endogenous concentration of auxins, abscisic acid, and salicylic acid did not change, but phaseic acid and neophaseic acid showed robust accumulation after cold acclimation. Freezing tolerance of the cultures was also higher after 7 days of cold-hardening. The results suggest the presence of a basal, light-independent, cold-responsive activation of the CBF-COR14b pathway in barley cultures. The effects of Dicamba, the exogenous auxin analog used for maintaining tissue cultures were also studied. Dicamba seems to be a general enhancer of the gene expression and physiological responses to cold stress, but has no specific effect on the activation. Our data along with previous findings show that this system might be a suitable model for studying certain basic cellular mechanisms involved in the cold acclimation process in cereals.

Different accumulation of free amino acids during short- and long-term osmotic stress in wheat.

The effect of wheat chromosome 5A on free amino acid accumulation induced by osmotic stress was compared in chromosome 5A substitution lines with different freezing tolerance. Treatment with 15% polyethylene glycol (PEG) resulted in greater total free amino acid content even after 3 days compared to the controls. The ratio of amino acids belonging to various amino acid families differed after 3-week treatment in the control and PEG-treated plants only in the case of the freezing-sensitive substitution line. There was a transient increase with a maximum after 3 days in the amounts of several amino acids, after which their concentrations exhibited a more gradual increase. During the first days of osmotic stress the Glu, Gln, Asp, Asn, Thr, Ser, Leu, and His concentrations were greater in the tolerant substitution line than in the sensitive one, while the opposite relationship was observed at the end of the PEG treatment. The coordinated changes in the levels of individual amino acids indicated that they are involved in both the short- and long-term responses to osmotic stress. The alterations differed in the two chromosome 5A substitution lines, depending on the stress tolerance of the chromosome donor genotype.

Chelator-enhanced lead accumulation in Agropyron elongatum cv Szarvasi-1 in hydroponic culture.

Hydroponic culture was applied to compare the efficiency of K2EDTA and citrate in mobilizing Pb for accumulation in Agropyron elongatum cv. Szarvasi-1 and their effects on some physiological characteristics of the plants. The plants were grown in nutrient solutions containing 0, 10, and 100 microM Pb(NO3)2 combined with chelating agents added to the nutrient solutions after 21 days of growth, in 3 concentrations (0, 100, and 500 microM). The effects were measured after eight days. The energy grass proved to be greatly resistant to the treatments, as was reflected in the slight inhibition of growth, the resistance of the photosynthetic electron transport chain and the chlorophyll composition and the lack of change in the malone-dialdehyde content. Fundamental differences can be identified between the effects of EDTA and citrate. Citrate had only a little effect on the physiological parameters, which may be due to the strongly increasing lead accumulation with increasing concentration of Pb in the nutrient solution. Additionally, citrate ensured a higher biomass yield with higher shoot Pb accumulation compared to EDTA in almost all treatments. Concerning biomass reduction, 10 microM Pb applied together with K2EDTA has the most deleterious effects on energy grass. The effects correlated with the concentration of EDTA.

Regulation of freezing tolerance and flowering in temperate cereals: the VRN-1 connection.

In winter wheat (Triticum spp.) and barley (Hordeum vulgare) varieties, long exposures to nonfreezing cold temperatures accelerate flowering time (vernalization) and improve freezing tolerance (cold acclimation). However, when plants initiate their reproductive development, freezing tolerance decreases, suggesting a connection between the two processes. To better understand this connection, we used two diploid wheat (Triticum monococcum) mutants, maintained vegetative phase (mvp), that carry deletions encompassing VRN-1, the major vernalization gene in temperate cereals. Homozygous mvp/mvp plants never flower, whereas plants carrying at least one functional VRN-1 copy (Mvp/-) exhibit normal flowering and high transcript levels of VRN-1 under long days. The Mvp/- plants showed reduced freezing tolerance and reduced transcript levels of several cold-induced C-REPEAT BINDING FACTOR transcription factors and COLD REGULATED genes (COR) relative to the mvp/mvp plants. Diploid wheat accessions with mutations in the VRN-1 promoter, resulting in high transcript levels under both long and short days, showed a significant down-regulation of COR14b under long days but not under short days. Taken together, these studies suggest that VRN-1 is required for the initiation of the regulatory cascade that down-regulates the cold acclimation pathway but that additional genes regulated by long days are required for the down-regulation of the COR genes. In addition, our results show that allelic variation in VRN-1 is sufficient to determine differences in freezing tolerance, suggesting that quantitative trait loci for freezing tolerance previously mapped on this chromosome region are likely a pleiotropic effect of VRN-1 rather than the effect of a separate closely linked locus (FROST RESISTANCE-1), as proposed in early freezing tolerance studies.

Regulation of gene expression by chromosome 5A during cold hardening in wheat.

Cold hardening is necessary to achieve the genetically determined maximum freezing tolerance and to reduce yield losses in winter cereals. The aim of the present study was to determine a set of genes with an important role in this process, by comparing of chromosome 5A substitution lines with different levels of freezing tolerance, since chromosome 5A is a major regulator of this trait. During 21 days of treatment at 2 degrees C, 303 genes were up-regulated, while 222 were down-regulated at most sampling points, and 156 at around half of them (out of the 10,297 unigenes studied). The freezing-tolerant substitution line exhibited 1.5 times as many differentially expressed genes than the sensitive one. The transcription of 78 genes (39 up-regulated) proved to be chromosome 5A-dependent. These genes encoded proteins involved in transcriptional regulation, defence processes and carbohydrate metabolism. Three of the chromosome 5A-related genes, coding for a cold-responsive, a Ca-binding and an embryo and meristem-related protein, were genetically mapped and characterized in further detail. The present experimental system was appropriate for the selection of chromosome 5A-related genes involved in short- and long-term cold acclimation in wheat. By modifying the expression of these genes it may be possible to improve freezing tolerance.