Production and characterization of a chimeric protein targeting synuclein epitopes for immunotherapy against synucleinopathies

The aggregation and spread of alpha-synuclein (αSyn) is associated with several pathogenic pathways that lead to neurodegeneration and, ultimately, to synucleinopathies development. Hence, the establishment of a safe and effective disease-modifying therapy that limits or prevents the spread of toxic αSyn aggregation could lead to positive clinical outcomes. A rational vaccine design can be focused on the selection of specific epitopes able to induce the immune response desired, for example, antibodies able to mediate the clearance of αSyn aggregates without the induction of inflammatory responses. To develop a rapid system for the evaluation of a vaccine candidate against synucleinopathies, rLTB-Syn (an antigen based on three B cell epitopes from αSyn and the B subunit of the heat-labile Escherichia coli enterotoxin [LTB] as adjuvant/carrier) was produced using recombinant E. coli (Rosetta DE3) as the expression host. The bacterial version of rLTB-Syn was produced as soluble protein at yields up to 1.72 mg/g biomass. A method for the purification of rLTB-Syn (~18 kDa) was developed based on ion exchange chromatography, reaching purity >93% with a final concentration of 82.6 μg/mL. Furthermore, the purified soluble rLTB-Syn retained GM1 binding activity, suggesting proper folding and pentameric structure. The results from this study establish a fast and effective method to obtain rLTB-Syn, making it useful in the design of novel vaccine formulations targeting synucleinopathies.     

The Detection of Inherent Homologous Recombination Between Repeat Sequences in H. pylori 26695 by the PCR-Based Method

Helicobacter pylori infects more than half of the world’s population, making it the most widespread infection of bacteria. It has high genetic diversity and has been considered as one of the most variable bacterial species. In the present study, a PCR-based method was used to detect the presence and the relative frequency of homologous recombination between repeat sequences (>500 bp) in H. pylori 26695. All the recombinant structures have been confirmed by sequencing. The inversion generated between inverted repeats showed distinct features from the recombination for duplication or deletion between direct repeats. Meanwhile, we gave the mathematic reasoning of a general formula for the calculation of relative recombination frequency and indicated the conditions for its application. This formula could be extensively applied to detect the frequency of homologous recombination, site-specific recombination, and other types of predictable recombination. Our results should be helpful for better understanding the genome evolution and adaptation of bacteria.     

Improving Mycobacterium bovis Bacillus Calmette-Guèrin as a Vaccine Delivery Vector for Viral Antigens by Incorporation of Glycolipid Activators of NKT Cells

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8⁺ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8⁺ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8⁺ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8⁺ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8⁺ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.     

Randomized controlled trial of recombinant human granulocyte-macrophage colony-stimulating factor for the treatment of chronic hepatitis c

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered subcutaneously to 45 chronic hepatitis C patients, randomly assigned to receive 0.5, 1 or 2 microg GM-CSF/kg b.w. daily/6 weeks (n=30), or no treatment (n=15). Alanine transaminase (ALT) values normalized in four out of 10 (40%) patients administered 2 microg GM-CSF [1 cleared hepatitis C virus (HCV) RNA] but in none given 0.5 or 1 microg or untreated controls (P=0.0079). Following 4 weeks of rest, patients received 5 million units of interferon (IFN)alpha2b every other day/6 months, alone (n=30), or combined with 2 microg GM-CSF/daily for 3 months (n=15). At treatment end, ALT levels in patients administered the combination normalized more frequently than in those given monotherapy (73% vs 47%, P=0.089). Viraemia decreased significantly in 11/15 (73%) patients administered GM-CSF/IFNalpha2b combination (mean log HCV RNA copies/ml+/-SEM: 4.13+/-0.40 vs 5.29+/-0.23;P=0.011), and in 20/30 (67%) receiving IFNalpha2b monotherapy (4.27+/-0.28 vs 5. 31+/-0.14;P=0.004); 27% and 20% of patients given the combination and monotherapy, respectively, cleared HCV RNA. One patient in each regime had a sustained response after 12 months. 2', 5'-Oligoadenylate synthetase activity (2-5AS) increased during GM-CSF therapy (P=0.033 with the 2 microg dose). 2-5AS increased more in the GM-CSF/IFN-alpha2b combination than with IFNalpha2b monotherapy (P<0.02). GM-CSF provoked a skin reaction at the injection site, accompanied by moderate and reversible rises in eosinophil and leucocyte counts. In summary, daily s.c. GM-CSF administration is safe and shows effects against HCV; the GM-CSF/IFNalpha2b combination has an additional-but transient-antiviral activity in chronic hepatitis C.     

Observability in strategic models of viability selection

Strategic models of frequency-dependent viability selection, in terms of mathematical systems theory, are considered as a dynamic observation system. Using a general sufficient condition for observability of nonlinear systems with invariant manifold, it is studied whether, observing certain phenotypic characteristics of the population, the development of its genetic state can be recovered, at least near equilibrium.     

The intrastriatal injection of thrombin in rat induced a retrograde apoptotic degeneration of nigral dopaminergic neurons through synaptic elimination

We have performed intrastriatal injection of thrombin and searched for distant effects in the cell body region. In striatum, thrombin produced a slight loss of striatal neurons as demonstrated by neural nuclei immunostaining – a non-specific neuronal marker – and the expression of glutamic acid decarboxylase 67 mRNA, a specific marker for striatal GABAergic interneurons, the most abundant phenotype in this brain area. Interestingly, striatal neuropil contained many boutons immunostained for synaptic vesicle protein 2 and synaptophysin which colocalize with tyrosine hydroxylase (TH), suggesting a degenerative process with pre-synaptic accumulation of synaptic vesicles. When we studied the effects on substantia nigra, we found the disappearance of dopaminergic neurons, shown by loss of TH immunoreactivity, loss of expression of TH and dopamine transporter mRNAs, and disappearance of FluoroGold-labelled nigral neurons. The degeneration of substantia nigra dopaminergic neurons was produced through up-regulation of cFos mRNA, apoptosis and accumulation of α-synuclein shown by colocalization experiments. Thrombin effects could be mediated by protease-activated receptor 4 activation, as protease-activated receptor 4-activating peptide mimicked thrombin effects. Our results point out the possible relationship between synapse elimination and retrograde degeneration in the nigral dopaminergic system.     

Density and population structure of the jaguar (<Emphasis Type="Italic">Panthera onca</Emphasis>) in a protected area of Los Llanos,Venezuela, from 1 year of camera trap monitoring

Density is crucial for understanding large carnivore ecology and conservation, but estimating it has proven methodologically difficult. We conducted 1 year of camera trapping to estimate jaguar (Panthera onca) density and population structure in the Los Llanos region of Venezuela on the Hato Piñero ranch, where hunting is prohibited and livestock are excluded from half of ranch lands. We identified 42 different jaguars and determined their sex, age class, and reproductive status. We estimated adult jaguar densities with spatial capture-recapture models, using sex/reproductive state and session as covariates. Models without temporal variation received more support than models that allowed variation between sessions. Males, reproductive females, and nonreproductive females differed in their density, baseline detectability, and movement. The best estimate of total adult jaguar population density was 4.44 individuals/100 km². Based on reproductive female density and mean number of offspring per female, we estimated cub density at 3.23 individuals/100 km² and an overall density of 7.67 jaguars/100 km². Estimated jaguar population structure was 21% males, 11% nonreproductive females, 26% reproductive females, and 42% cubs. We conclude that extending the sampling period to 1 year increases the detectability of females and cubs and makes density estimates more robust as compared to the more common short studies. Our results demonstrate that the Venezuelan Llanos represent important jaguar habitat, and further, they emphasize the importance of protected areas and hunting restrictions for carnivore conservation.     

Effects of different metabolites of vitamin D3 and of calcium concentration on the intestinal absorption of strontium]

The effect of calcium concentration and vitamin B3 25 OHD3 and 1.25 diOHD3 upon intestinal strontium transport was studied in vitamin D deficient rats with duodenal perfusion in situ. When the calcium concentration was increased, the strontium passive absorption was decreased. The vitamin D3 and its derivatives increased intestinal strontium transport significantly and this increment was not modified by calcium. In our experiment, calcium is competitive with strontium only in passive absorption.     

The effects of cytocentrifugation on differential cell counts in samples obtained by bronchoalveolar lavage

Quantification of the differential cell count and total number of cells recovered from the lower respiratory tract by bronchoalveolar lavage is a valuable technique for the diagnostic study of interstitial lung diseases. To examine the effect on the cell counts of different methods of processing the lavage fluid, two comparisons were performed. First, two methods of differential cell counting were compared using 28 fluids. One count was performed in a Malassez hemocytometer after incubation of the living cells with neutral red for five minutes at room temperature; large cells and some small cells that had incorporated neutral red were identified as macrophages. Another count was performed on cytocentrifuge preparations made using the Shandon Cytospin I and Cytospin II and stained by the May-Grünwald-Giemsa method. The percentage of cells identified as lymphocytes was significantly lower on the cytocentrifuge preparations than with the Malassez hemocytometer. In the second study, the differential cell counts on smears prepared by the two types of cytocentrifuge (Cytospin I and Cytospin II) were compared for 32 bronchoalveolar lavage fluids. The percentage of small cells (especially lymphocytes) was lower on preparations made with the Cytospin I than on those made with the Cytospin II, but the difference was not significant. The results indicate that (1) cytocentrifugation of bronchoalveolar lavage fluids does result in a significant loss of small cells, especially lymphocytes, and (2) this loss is not significantly lessened by the use of the Cytospin II.     

Aquatic birds as bioindicators of trophic changes and ecosystem deterioration in the Mar Menor lagoon (SE Spain)

The Mar Menor is the largest coastal lagoon in the Western Mediterranean and it is an important site for wintering and breeding waterfowl. During recent decades several hydrological and land-use changes in the watershed have increasingly threatened the conservation of the lagoon due to the development of urban areas, tourism and agriculture. A dynamic system model has been developed at the watershed scale to estimate the annual load of nutrients reaching the Mar Menor-associated wetlands. At present, mean annual loadings of approximately 2000 tonnes of nitrogen and 60 tonnes of phosphorus are delivered to the lagoon. The simulation results emphasize the role of heavy rainfall events and floods in the formation of the total nutrient load. The composition of aquatic bird communities has been used to assess the nutrient impact on the lagoon food-web. The Great Crested Grebe is apparently the species most closely dependant on local trophic conditions. The related Black-necked Grebe, that dominates the waterbird community of the lagoon, plays a similar role, but its more opportunistic response to changes in food resources, reduces its indicator value. The abundance of the two species of grebes seems to closely track the nitrogen load curve, especially during the first phase of enrichment, suggesting the existence of a direct trophic relationship. In the following phase, jellyfish blooms coincide with the bird decline. Jellyfishes seem to have a buffering effect towards nutrients, determining a bottom up limitation to other trophic compartments. In recent years, this buffering capacity has probably been overloaded, favouring the growth of new food resources available to the grebes. Unlike grebes, Mergus serrator, a typical piscivorous bird, does not seem to be affected positively by eutrophication since it shows a long-term stability in numbers or even a slight decline. Since this suspected decline would parallel a long-term reduction of fish catches, the species could be regarded as a potential indicator of habitat deterioration.