Optimising Immunogenicity with Viral Vectors: Mixing MVA and HAdV-5 Expressing the Mycobacterial Antigen Ag85A in a Single Injection

The Bacillus Calmette - Guerin (BCG) vaccine provides a critical but limited defense against Mycobacterium tuberculosis (M.tb). More than 60 years after the widespread introduction of BCG, there is an urgent need for a better vaccine. A large body of pre-clinical research continues to support ongoing clinical trials to assess whether viral vectors expressing M.tb antigens that are shared by BCG and M.tb, can be used alongside BCG to enhance protection. A major focus involves using multiple unique viral vectors to limit anti-vector immunity and thereby enhance responses to the insert antigen delivered. The successful introduction of viral vector vaccines to target M.tb and other pathogens will be reliant on reducing the costs when using multiple vectors and inhibiting the development of unwanted anti-vector responses that interfere with the response to insert antigen. This study examines methods to reduce the logistical costs of vaccination by mixing different viral vectors that share the same insert antigen in one vaccine; and whether combining different viral vectors reduces anti-vector immunity to improve immunogenicity to the insert antigen. Here we show that a homologous prime-boost regimen with a mixture of MVA (Modified Vaccinia virus Ankara) and Ad5 (human adenovirus type 5) vectors both expressing Ag85A in a single vaccine preparation is able to reduce anti-vector immunity, compared with a homologous prime-boost regimen with either vector alone. However, the level of immunogenicity induced by the homologous mixture remained comparable to that induced with single viral vectors and was less immunogenic than a heterologous Ad5 prime-MVA-boost regimen. These findings advance the understanding of how anti-vector immunity maybe reduced in viral vector vaccination regimens. Furthermore, an insight is provided to the impact on vaccine immunogenicity from altering vaccination methods to reduce the logistical demands of using separate vaccine preparations in the field.     

Effect of antioxidants on the genetic stability of cryopreserved mint shoot tips by encapsulation–dehydration

The effect of antioxidants applied in one step of a cryopreservation protocol by encapsulation–dehydration on recovery and genetic stability of mint shoot tips has been studied. Glutathione (0.16 or 0.24 mM), ascorbic acid (0.28 or 0.43 mM) and α-tocopherol (vitamin E) were added to the preculture medium (0.3 M sucrose). DNA was extracted from three different types of samples: leaves from shoots, callus at the base of shoots and callus. RAPD and AFLP markers were used to assess the genetic stability. The use of antioxidants did not improve recovery after cryopreservation. One of the genotypes, ‘MEN 198’, showed higher percentage of stable samples than the other one, ‘MEN 186’ (56 vs. 37?%; considering all treatments and types of explant). The use of vitamin E improved the percentage of stable samples with respect to control treatment (no antioxidant) in ‘MEN 186’. No differences in the percentages of stable samples were observed among cryopreserved and non-cryopreserved (treated similarly without immersion in liquid nitrogen) plant material. Recovered shoots of both genotypes showed higher stability (76–80?% stable samples) than callus samples (14–22?%).     

Safety of ethanolic kava extract: Results of a study of chronic toxicity in rats

BACKGROUNDS: Recently, potential liver toxicity was discussed with the intake of kava extract preparations (Piper methysticum) as anxiolytic drugs. The aim of this study was to test chronic toxicity in rats by oral application of an ethanolic kava full extract. METHODS: Wistar rats of both sexes were fed 7.3 or 73 mg/kg body weight of ethanolic kava extract for 3 and 6 months. The animals were examined for changes in body weight, hematological and liver parameters, and macroscopical and microscopical histological changes in the major organs. RESULTS: No signs of toxicity could be found. CONCLUSIONS: The results are in accordance with the medical experience regarding the use of kava preparations and the long tradition of kava drinking in the South Pacific island states. Specifically, the results do not back the suspicion of potential liver toxicity.     

Plasmid transformation of Ruminococcus albus by means of high-voltage electroporation

To apply recombinant DNA techniques to the genetic manipulation of cellulolytic ruminal bacteria, a plasmid vector transformation system must be available. The objective of this work was to develop a system for plasmid transformation of Ruminococcus albus. Using high voltage electrotransformation, pSC22 and pCK17 plasmid vectors, derived from lactic acid bacteria plasmids and replicating via single-stranded DNA intermediate, were successfully introduced into three freshly isolated R. albus strains and into R. albus type strain ATCC 27210. The optimization of the electrotransformation condition raised the electroporation efficiency up to 3 x 10(5) transformants per microgram of pSC22 plasmid.     

Human urine: Epicatechin metabolites and antioxidant activity after cocoa beverage intake

Associations between cocoa consumption in humans, excreted metabolites and total antioxidant capacity (TAC) have been scarcely investigated. The aims of the study were to investigate the epicatechin (( ? )-Ec) metabolites excreted in urine samples after an intake of 40 g of cocoa powder along with the TAC of these urine samples and the relation between both the analyses. Each of the 21 volunteers received two interventions, one with a polyphenol-rich food (PRF) and one with a polyphenol-free food (PFF) in a randomized cross-over study. Urine samples were taken before and during 24 h at 0–6, 6–12 and 12–24 h periods after test intake. The excreted ( ? )-Ec metabolites and the TAC were determined in urine samples by LC-MS/MS and TEAC assay, respectively. The maximum excretion of ( ? )-Ec metabolites and the maximum TAC value were observed in urine samples excreted between 6 and 12 h after PRF consumption. Significance of TAC increase was found in urine samples excreted during 0–6 and 6–12 h (66.6 and 72.67%, respectively, with respect to the 0 h).     

Excitotoxic Insult Results in a Long-Lasting Activation of CaMKIIα and Mitochondrial Damage in Living Hippocampal Neurons

Over-activation of excitatory NMDA receptors and the resulting Ca²⁺ overload is the main cause of neuronal toxicity during stroke. CaMKII becomes misregulated during such events. Biochemical studies show either a dramatic loss of CaMKII activity or its persistent autonomous activation after stroke, with both of these processes being implicated in cell toxicity. To complement the biochemical data, we monitored CaMKII activation in living hippocampal neurons in slice cultures using high spatial/temporal resolution two-photon imaging of the CaMKIIα FRET sensor, Camui. CaMKII activation state was estimated by measuring Camui fluorescence lifetime. Short NMDA insult resulted in Camui activation followed by a redistribution of its protein localization: an increase in spines, a decrease in dendritic shafts, and concentration into numerous clusters in the cell soma. Camui activation was either persistent (> 1–3 hours) or transient (~20 min) and, in general, correlated with its protein redistribution. After longer NMDA insult, however, Camui redistribution persisted longer than its activation, suggesting distinct regulation/phases of these processes. Mutational and pharmacological analysis suggested that persistent Camui activation was due to prolonged Ca²⁺ elevation, with little impact of autonomous states produced by T286 autophosphorylation and/or by C280/M281 oxidation. Cell injury was monitored using expressible mitochondrial marker mito-dsRed. Shortly after Camui activation and clustering, NMDA treatment resulted in mitochondrial swelling, with persistence of the swelling temporarily linked to the persistence of Camui activation. The results suggest that in living neurons excitotoxic insult produces long-lasting Ca²⁺-dependent active state of CaMKII temporarily linked to cell injury. CaMKII function, however, is to be restricted due to strong clustering. The study provides the first characterization of CaMKII activation dynamics in living neurons during excitotoxic insults.     

Long-term treatment with fluoxetine induces desensitization of 5-HT4 receptor-dependent signalling and functionality in rat brain

The mode of action of antidepressant drugs may be related to mechanisms of monoamines receptor adaptation, including serotonin 5-HT₄ receptor subtypes. Here we investigated the effects of repeated treatment with the selective serotonin reuptake inhibitor fluoxetine for 21 days (5 and 10 mg/kg, p.o., once daily) on the sensitivity of 5-HT₄ receptors by using receptor autoradiography, adenylate cyclase assays and extracellular recording techniques in rat brain. Fluoxetine treatment decreased the density of 5-HT₄ receptor binding in the CA1 field of hippocampus as well as in several areas of the striatum over the doses of 5–10 mg/kg. In a similar way, we found a significant lower response to zacopride-stimulated adenylate cyclase activity in the fluoxetine 10 mg/kg/day treated group. Furthermore, post-synaptic 5-HT₄ receptor activity in hippocampus-measured as the excitatory action of zacopride in the pyramidal cells of CA1 evoked by Schaffer collateral stimulation was attenuated in rats treated with both doses of fluoxetine. Taken together, these results support the concept that a net decrease in the signalization pathway of 5-HT₄ receptors occurs after chronic selective serotonin reuptake inhibitor treatment: this effect may underlie the therapeutic efficacy of these drugs.