Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.     

Glycoprofiling Bifidobacterial Consumption of Galacto-Oligosaccharides by Mass Spectrometry Reveals Strain-Specific,Preferential Consumption of Glycans

Galacto-oligosaccharides (GOS) are versatile food ingredients that possess prebiotic properties. However, at present there is a lack of precise analytical methods to demonstrate specific GOS consumption by bifidobacteria. To better understand the role of GOS as prebiotics, purified GOS (pGOS) without disaccharides and monosaccharides was prepared and used in bacterial fermentation experiments. Growth curves showed that all bifidobacteria assayed utilized and grew on pGOS preparations. We used a novel mass spectrometry approach involving matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) to determine the composition of oligosaccharides in GOS syrup preparations. MALDI-FTICR analysis of spent fermentation media demonstrated that there was preferential consumption of selected pGOS species by different bifidobacteria. The approach described here demonstrates that MALDI-FTICR is a rapid-throughput tool for comprehensive profiling of oligosaccharides in GOS mixtures. In addition, the selective consumption of certain GOS species by different bifidobacteria suggests a means for targeting prebiotics to enrich select bifidobacterial species.Galacto-oligosaccharides (GOS) are nondigestible carbohydrates and versatile food ingredients that possess prebiotic properties (1). In addition, other health benefits have been reported to result from consumption of these oligosaccharides, such as stimulation of intestinal mobility and mineral absorption, elimination of ammonium, and colon cancer prevention, as well as protection against certain pathogenic bacterial infections (6, 11, 19).The physicochemical characteristics of GOS have enabled them to be incorporated in food as prebiotic ingredients. GOS have been of interest in acidic beverages and fermented milk formulations since they exhibit increased thermal stability in acidic environments compared to fructo-oligosaccharides (16, 21). Thus, in the past decade, the applications of GOS in human food products have included dairy products, sugar replacements, diet supplements, and infant formula (11).Commercial GOS preparations are produced by enzymatic treatment of lactose with β-galactosidases from different sources, such as fungi, yeast, or bacteria, which results in a mixture of oligomers with various chain lengths (1). Thus, the basic structure of GOS includes a lactose core at the reducing end, which is typically elongated with up to six galactose residues. Structural diversity in GOS preparations is dependent on the enzyme used in the transgalactosylation reaction and the experimental conditions used, such as pH and temperature (5).Considerable effort has been made to understand the effects of GOS in vivo, and most studies have described the impact of GOS on intestinal bacterial population shifts and production of short-chain fatty acids attributed to bacterial fermentation. While there have a been a variety of in vitro studies characterizing the growth of different gut microbes on GOS, the majority of these studies used commercially available preparations of GOS. These commercial preparations contain high concentrations of monosaccharides (i.e., galactose and glucose) and the disaccharide lactose, both of which remain in the product after the transgalactosylation reaction. However, monosaccharides are the preferred substrates for most microorganisms when they are available in a mixed-carbon source (2). Thus, to evaluate growth on GOS, removal of monosaccharides and lactose is helpful (15).An analytical method currently used to measure GOS in food and feed products is high-pH anion-exchange chromatography (HPAEC) coupled to analysis with a pulse amperometric detector (PAD) (4). Van Laere and coworkers have used this method to monitor GOS fermentation in Bifidobacterium adolescentis cultures (20). However, HPAEC-PAD analysis is time-consuming and thus a low-throughput method. More importantly, due to the detector, in HPAEC-PAD analysis there is a differential response to oligosaccharides with higher degrees of polymerization (DP). Thus, new analytical approaches are needed to specifically characterize the consumption of GOS and other prebiotics by probiotic bacteria.We have previously developed analytical methods employing high-mass-accuracy and high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to characterize bacterial consumption of human milk oligosaccharides and fructo-oligosaccharides (9, 10, 14, 17). The matrix-assisted laser desorption ionization (MALDI)-FTICR method is a sensitive and robust analytical method with high-performance capability, and it allows rapid and unambiguous assignment of oligosaccharide signals.The aims of the present study were to investigate the oligosaccharide composition of GOS syrup preparations using MALDI-FTICR MS, to test lactose-free purified GOS (pGOS) as a sole carbon source in bifidobacterial fermentation experiments, and to determine the pGOS consumption profile by MALDI-FTICR MS. Four major bifidobacterial phylotypes, B. adolescentis, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, and Bifidobacterium longum subsp. longum, were used, and our results demonstrate that there is differential consumption of individual GOS species by various bifidobacteria, which provides a conceptual basis for targeted enrichment of specific bifidobacterial strains using specific GOS fractions.     

SAR and biological evaluation of SEN12333/WAY-317538: Novel alpha 7 nicotinic acetylcholine receptor agonist

Alpha 7 nicotinic acetylcholine receptor (α₇ nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment associated with a variety of disorders including Alzheimer’s disease and schizophrenia. Alpha 7 nAChRs are expressed in brain regions associated with cognitive function, regulate cholinergic neurotransmission and have been shown to be down regulated in both schizophrenia and Alzheimer’s disease. Herein we report a novel, potent small molecule agonist of the alpha 7 nAChR, SEN12333/WAY-317538. This compound is a selective agonist of the α₇ nAChR with excellent in vitro and in vivo profiles, excellent brain penetration and oral bioavailability, and demonstrates in vivo efficacy in multiple behavioural cognition models. The SAR and biological evaluation of this series of compounds are discussed.     

Effect of L-carnitine administration on the seminal characteristics of oligoasthenospermic stallions

The effect of orally administered l-carnitine on the quality of semen obtained from stallions with different semen qualities was investigated. Four stallions with proven fertility (high motility group, HM) and with normal seminal characteristics (>50% progressive motility and > 80 x 10(6) spermatozoa/ml), and four questionable breeders (low motility group, LM) with <50% of sperm progressive motility and < 80 x 10(6) spermatozoa/ml, received p.o. 20 g of l-carnitine for 60 days. Blood and semen samples were collected before treatment (T0) and after 30 (T1) and 60 days (T2). Semen evaluation were performed on five consecutive daily ejaculates (n = 120 ejaculates) and conventional semen analysis was carried out on each ejaculate, both at collection and after refrigeration for 24, 48, and 72 h. Furthermore l-carnitine, acetylcarnitine, pyruvate, and lactate concentrations, and carnitine acetyltransferase activity (CAT) were determined both in raw semen and seminal plasma. There were an increase in progressive motile spermatozoa only in the LM group (26.8 +/- 12.9, 39.1 +/- 15.5, and 48.8 +/- 8.6 for T0, T1, and T2, respectively). Free seminal plasma carnitine concentration was higher in the LM group compared to the HM one. Both pyruvate and lactate were higher in the LM group. Raw semen and seminal plasma carnitine and acetylcarnitine levels correlate positively with both sperm concentration and progressive motility; moreover, acetylcarnitine content was positively correlated with total motile morphologically normal spermatozoa. In conclusion, oral administration of l-carnitine to stallions with questionable seminal characteristics may improve spermatozoa kinetics and morphological characteristics; whereas, it seem to be ineffective in normospermic animals.     

Modeling Staphylococcus epidermidis-Induced Non-Unions: Subclinical and Clinical Evidence in Rats

S. epidermidis is one of the leading causes of orthopaedic infections associated with biofilm formation on implant devices. Open fractures are at risk of S. epidermidis transcutaneous contamination leading to higher non-union development compared to closed fractures. Although the role of infection in delaying fracture healing is well recognized, no in vivo models investigated the impact of subclinical low-grade infections on bone repair and non-union. We hypothesized that the non-union rate is directly related to the load of this commonly retrieved pathogen and that a low-grade contamination delays the fracture healing without clinically detectable infection. Rat femurs were osteotomized and stabilized with plates. Fractures were infected with a characterized clinical-derived methicillin-resistant S. epidermidis (10³, 10⁵, 10⁸ colony forming units) and compared to uninfected controls. After 56 days, bone healing and osteomyelitis were clinically assessed and further evaluated by micro-CT, microbiological and histological analyses. The biofilm formation was visualized by scanning electron microscopy. The control group showed no signs of infection and a complete bone healing. The 10³ group displayed variable response to infection with a 67% of altered bone healing and positive bacterial cultures, despite no clinical signs of infection present. The 10⁵ and 10⁸ groups showed severe signs of osteomyelitis and a non-union rate of 83–100%, respectively. The cortical bone reaction related to the periosteal elevation in the control group and the metal scattering detected by micro-CT represented limitations of this study. Our model showed that an intra-operative low-grade S. epidermidis contamination might prevent the bone healing, even in the absence of infectious signs. Our findings also pointed out a dose-dependent effect between the S. epidermidis inoculum and non-union rate. This pilot study identifies a relevant preclinical model to assess the role of subclinical infections in orthopaedic and trauma surgery and to test specifically designed diagnostic, prevention and therapeutic strategies.     

Site-Specific Integration in Mammalian Cells Mediated by a New Hybrid Baculovirus–Adeno-Associated Virus Vector

Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable cell lines. To prolong transgene expression, we have developed new hybrid vectors which associate key elements from adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectors contain a transgene cassette composed of the β-galactosidase (β-Gal) reporter gene and the hygromycin resistance (Hyg^r) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replication and integration in the host genome. Constructs were derived both with and without the AAV rep gene under the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrin promoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR–Hyg^r–β-Gal sequence was obtained with insect cells only when the rep gene was placed in an antisense orientation to the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10- to 50-fold increase in the number of Hyg^r stable cell clones. Additionally, rep expression determined the localization of the transgene cassette in the aavs1 site in approximately 41% of cases as detected by both Southern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITR-flanked DNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts. These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNA constructs for ex vivo treatment of primary human cells.     

Parasympathetic Stimuli on Bronchial and Cardiovascular Systems in Humans

Background

It is not known whether parasympathetic outflow simultaneously acts on bronchial tone and cardiovascular system waxing and waning both systems in parallel, or, alternatively, whether the regulation is more dependent on local factors and therefore independent on each system. The aim of this study was to evaluate the simultaneous effect of different kinds of stimulations, all associated with parasympathetic activation, on bronchomotor tone and cardiovascular autonomic regulation.

Methods

Respiratory system resistance (Rrs, forced oscillation technique) and cardio-vascular activity (heart rate, oxygen saturation, tissue oxygenation index, blood pressure) were assessed in 13 volunteers at baseline and during a series of parasympathetic stimuli: O₂ inhalation, stimulation of the carotid sinus baroreceptors by neck suction, slow breathing, and inhalation of methacholine.

Results

Pure cholinergic stimuli, like O₂ inhalation and baroreceptors stimulation, caused an increase in Rrs and a reduction in heart rate and blood pressure. Slow breathing led to bradycardia and hypotension, without significant changes in Rrs. However slow breathing was associated with deep inhalations, and Rrs evaluated at the baseline lung volumes was significantly increased, suggesting that the large tidal volumes reversed the airways narrowing effect of parasympathetic activation. Finally inhaled methacholine caused marked airway narrowing, while the cardiovascular variables were unaffected, presumably because of the sympathetic activity triggered in response to hypoxemia.

Conclusions

All parasympathetic stimuli affected bronchial tone and moderately affected also the cardiovascular system. However the response differed depending on the nature of the stimulus. Slow breathing was associated with large tidal volumes that reversed the airways narrowing effect of parasympathetic activation.