Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

Cortical potential imaging using L-curve and GCV method to choose the regularisation parameter

Background

The electroencephalography (EEG) is an attractive and a simple technique to measure the brain activity. It is attractive due its excellent temporal resolution and simple due to its non-invasiveness and sensor design. However, the spatial resolution of EEG is reduced due to the low conducting skull. In this paper, we compute the potential distribution over the closed surface covering the brain (cortex) from the EEG scalp potential. We compare two methods – L-curve and generalised cross validation (GCV) used to obtain the regularisation parameter and also investigate the feasibility in applying such techniques to N170 component of the visually evoked potential (VEP) data.

Methods

Using the image data set of the visible human man (VHM), a finite difference method (FDM) model of the head was constructed. The EEG dataset (256-channel) used was the N170 component of the VEP. A forward transfer matrix relating the cortical potential to the scalp potential was obtained. Using Tikhonov regularisation, the potential distribution over the cortex was obtained.

Results

The cortical potential distribution for three subjects was solved using both L-curve and GCV method. A total of 18 cortical potential distributions were obtained (3 subjects with three stimuli each – fearful face, neutral face, control objects).

Conclusions

The GCV method is a more robust method compared to L-curve to find the optimal regularisation parameter. Cortical potential imaging is a reliable method to obtain the potential distribution over cortex for VEP data.

     

Reduced natural selection associated with low recombination in Drosophila melanogaster

Synonymous codons are not used equally in many organisms, and the extent of codon bias varies among loci. Earlier studies have suggested that more highly expressed loci in Drosophila melanogaster are more biased, consistent with findings from several prokaryotes and unicellular eukaryotes that codon bias is partly due to natural selection for translational efficiency. We link this model of varying selection intensity to the population-genetics prediction that the effectiveness of natural selection is decreased under reduced recombination. In analyses of 385 D. melanogaster loci, we find that codon bias is reduced in regions of low recombination (i.e., near centromeres and telomeres and on the fourth chromosome). The effect does not appear to be a linear function of recombination rate; rather, it seems limited to regions with the very lowest levels of recombination. The large majority of the genome apparently experiences recombination at a sufficiently high rate for effective natural selection against suboptimal codons. These findings support models of the Hill-Robertson effect and genetic hitchhiking and are largely consistent with multiple reports of low levels of DNA sequence variation in regions of low recombination.      

Treatment of adult MPSI mouse brains with IDUA-expressing mesenchymal stem cells decreases GAG deposition and improves exploratory behavior

Background

Mucopolysaccharidosis type I (MPSI) is caused by a deficiency in alpha-L iduronidase (IDUA), which leads to lysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. While the currently available therapies have good systemic effects, they only minimally affect the neurodegenerative process. Based on the neuroprotective and tissue regenerative properties of mesenchymal stem cells (MSCs), we hypothesized that the administration of MSCs transduced with a murine leukemia virus (MLV) vector expressing IDUA to IDUA KO mouse brains could reduce GAG deposition in the brain and, as a result, improve neurofunctionality, as measured by exploratory activity.

Methods

MSCs infected with an MLV vector encoding IDUA were injected into the left ventricle of the brain of 12- or 25-month-old IDUA KO mice. The behavior of the treated mice in the elevated plus maze and open field tests was observed for 1 to 2 months. Following these observations, the brains were removed for biochemical and histological analyses.

Results

After 1 or 2 months of observation, the presence of the transgene in the brain tissue of almost all of the treated mice was confirmed using PCR, and a significant reduction in GAG deposition was observed. This reduction was directly reflected in an improvement in exploratory activity in the open field and the elevated plus maze tests. Despite these behavioral improvements and the reduction in GAG deposition, IDUA activity was undetectable in these samples. Overall, these results indicate that while the initial level of IDUA was not sustainable for a month, it was enough to reduce and maintain low GAG deposition and improve the exploratory activity for months.

Conclusions

These data show that gene therapy, via the direct injection of IDUA-expressing MSCs into the brain, is an effective way to treat neurodegeneration in MPSI mice.     

Kappa-chain constant-region gene sequences in genus Rattus: coding regions are diverging more rapidly than noncoding regions

We have determined the nucleotide sequence of a 1,200-base pair (bp) genomic fragment that includes the kappa-chain constant-region gene (C kappa) from two species of native Australian rodents, Rattus leucopus cooktownensis and Rattus colletti. Comparison of these sequences with each other and with other rodent C kappa genes shows three surprising features. First, the coding regions are diverging at a rate severalfold higher than that of the nearby noncoding regions. Second, replacement changes within the coding region are accumulating at a rate at least as great as that of silent changes. Third, most of the amino acid replacements are localized in one region of the C kappa domain--namely, the carboxy-terminal "bends" in the alpha-carbon backbone. These three features have previously been described from comparisons of the two allelic forms of C kappa genes in R. norvegicus. These data imply the existence of considerable evolutionary constraints on the noncoding regions (based on as yet undetermined functions) or powerful positive selection to diversify a portion of the constant-region domain (whose physiological significance is not known). These surprising features of C kappa evolution appear to be characteristic only of closely related C kappa genes, since comparison of rodent with human sequences shows the expected greater conservation of coding regions, as well as a predominance of silent nucleotide substitutions within the coding regions.      

Quantification of the virus-host interaction in human T lymphotropic virus I infection

Background

Cellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection.

Results

In this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 ± 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells.

Conclusion

These observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.     

Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.     

A Preliminary Report on a Combined Gram-Pappen-Heim Stain for Formalin Fixed Tissues

Although the color of indigo is strongly dependent on its environment, it is blue in most commonly encountered situations. Indigo's absorption at such long wavelengths for such a small molecule is unique, and I provide here an overview of the concepts advanced to account for this feature. A traditional valence–bond approach may be used to provide a reasonable qualitative explanation. A more rigorous, quantitative explanation is provided by molecular orbital methods of varying degrees of sophistication and several explanations have been proposed based on these models. Commonly, it is suggested that the important structural unit in determining color is based on the cross-conjugated “H-chromophore” concept. A second closely related explanation describes it as two symmetrically coupled merocyanine chains. Another proposal suggests that the basic chromophore may be interpreted as the aza analogue of two coupled anti aromatic-cyclopentadienyl ions. PiSYSTEM, a commercially available quantum mechanics program, has been used to provide a successful quantitative account of the colors of indigo and indirubin, a red isomer.     

A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata

Beta-globin gene families in eutherians (placental mammals) consist of a set of four or more developmentally regulated genes which are closely linked and, in general, arranged in the order 5'-embryonic/fetal genes- adult genes-3'. This cluster of genes is proposed to have arisen by tandem duplication of ancestral beta-globin genes, with the first duplication occurring 200 to 155 MYBP just prior to a period in mammalian evolution when eutherians and marsupials diverged from a common ancestor. In this paper we trace the evolutionary history of the beta-globin gene family back to the origins of these mammals by molecular characterization of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata. Using Southern and restriction analysis of total genomic DNA and bacteriophage clones of beta-like globin genes, we provide evidence that just two functional beta-like globin genes exist in this marsupial, including one embryonic- expressed gene (S.c-epsilon) and one adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3'. The entire DNA sequence of the adult beta-globin gene is reported and shown to be orthologous to the adult beta-globin genes of the North American marsupial Didelphis virginiana and eutherian mammals. These results, together with results from a phylogenetic analysis of mammalian beta-like globin genes, confirm the hypothesis that a two-gene cluster, containing an embryonic- and an adult-expressed beta-like globin gene, existed in the most recent common ancester of marsupials and eutherians. Northern analysis of total RNA isolated from embryos and neonatals indicates that a switch from embryonic to adult gene expression occurs at the time of birth, coinciding with the transfer of the marsupial from a uterus to a pouch environment.