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.     

Lack of sexual experience does not reduce the responses of LH, estrus or fertility in anestrous goats exposed to sexually active males

We investigated whether LH secretion, estrous behavior and fertility would differ between sexually inexperienced and experienced anestrous goats exposed to the males. Male goats were rendered sexually active during the reproductive rest season by exposure to 2.5 months of artificial long days. Two groups of anovulatory sexually inexperienced and sexually experienced does were exposed to males during 15 days (n = 20 per group). LH pulsatility was determined every 15 min from 4 h before to 8 h after introducing males (Day 0). Estrous behavior was recorded twice daily. Pregnancy rates were determined on Day 50. Fertility was determined at parturition. Male sexual behavior was registered on days 1 and 2 during 1 h. Before introducing the males, the number of LH pulses did not differ between groups. After introduction of the males, all females increased their LH pulsatility, but the number of pulses did not differ between sexually inexperienced and experienced goats. The proportion of females displaying estrous behavior with a high pregnancy rate and fertility did not differ between inexperienced and experienced goats. The sexual behavior of the males did not differ significantly between those interacting with sexually inexperienced or experienced goats. We conclude that goats can show substantial endocrine and reproductive responses to males, even in the absence of previous sexual experience, when sexually active bucks are used.     

Altered cortical synaptic morphology and impaired memory consolidation in forebrain- specific dominant-negative PAK transgenic mice

Molecular and cellular mechanisms for memory consolidation in the cortex are poorly known. To study the relationships between synaptic structure and function in the cortex and consolidation of long-term memory, we have generated transgenic mice in which catalytic activity of PAK, a critical regulator of actin remodeling, is inhibited in the postnatal forebrain. Cortical neurons in these mice displayed fewer dendritic spines and an increased proportion of larger synapses compared to wild-type controls. These alterations in basal synaptic morphology correlated with enhanced mean synaptic strength and impaired bidirectional synaptic modifiability (enhanced LTP and reduced LTD) in the cortex. By contrast, spine morphology and synaptic plasticity were normal in the hippocampus of these mice. Importantly, these mice exhibited specific deficits in the consolidation phase of hippocampus-dependent memory. Thus, our results provide evidence for critical relationships between synaptic morphology and bidirectional modifiability of synaptic strength in the cortex and consolidation of long-term memory.     

SERS Properties of Different Sized and Shaped Gold Nanoparticles Biosynthesized under Different Environmental Conditions by Neurospora crassa Extract

Surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. It is known that metal nanoparticles, especially gold and silver nanoparticles, exhibit great SERS properties, which make them very attractive for the development of biosensors and biocatalysts. On the other hand, the development of ecofriendly methods for the synthesis of metallic nanostructures has become the focus of research in several countries, and many microorganisms and plants have already been used to biosynthesize metallic nanostructures. However, the majority of these are pathogenic to plants or humans. Here, we report gold nanoparticles with good SERS properties, biosynthesized by Neurospora crassa extract under different environmental conditions, increasing Raman signals up to 40 times using methylene blue as a target molecule. Incubation of tetrachloroauric acid solution with the fungal extract at 60°C and a pH value of a) 3, b) 5.5, and c) 10 resulted in the formation of gold nanoparticles of a) different shapes like triangles, hexagons, pentagons etc. in a broad size range of about 10-200 nm, b) mostly quasi-spheres with some different shapes in a main size range of 6-23 nm, and c) only quasi-spheres of 3-12 nm. Analyses included TEM, HRTEM, and EDS in order to corroborate the shape and the elemental character of the gold nanoparticles, respectively. The results presented here show that these ‘green’ synthesized gold nanoparticles might have potential applicability in the field of biological sensing.     

Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2 +5G>C mutation in ABCA1 gene

Two point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i) G>C in intron 2 (IVS2 +5G>C) and ii) c.844 C>T in exon 9 (R282X). The IVS2 +5G>C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 +5G>C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2-/mRNA), exon 4 (Ex4-/mRNA), or both these exons (Ex2-/Ex4-/mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 +5G>C mutation, we constructed two ABCA1 minigenes encompassing Ex1-Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2-/mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly. These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations.     

Cytotoxic and Genotoxic Effects of <Emphasis Type="Italic">cis</Emphasis>-Tetraammine(oxalato)Ruthenium(III) Dithionate on the Root Meristem Cells of <Emphasis Type="Italic">Allium cepa</Emphasis>

Ruthenium complexes have attracted much attention as possible building blocks for new transition-metal-based antitumor agents. The present study examines the mitotoxic and clastogenic effects induced in the root tips of Allium cepa by cis-tetraammine(oxalato)ruthenium(III) dithionate {cis-[Ru(C₂O₂)(NH₃)₄]₂(S₂O₆)} at different exposure durations and concentrations. Correlation tests were performed to determine the effects of the time of exposure and concentration of ruthenium complex on mitotic index (MI) and mitotic aberration index. A comparison of MI results of cis-[Ru(C₂O₂)(NH₃)₄]₂(S₂O₆) to those of lead nitrate reveals that the ruthenium complex demonstrates an average mitotic inhibition eightfold higher than lead, with the frequency of cellular abnormalities almost fourfold lower and mitotic aberration threefold lower. A. cepa root cells exposed to a range of ruthenium complex concentrations did not display significant clastogenic effects. Cis-tetraammine(oxalato)ruthenium(III) dithionate therefore exhibits a remarkable capacity to inhibit mitosis, perhaps by inhibiting DNA synthesis or blocking the cell cycle in the G2 phase. Further investigation of the mechanisms of action of this ruthenium complex will be important to define its clinical potential and to contribute to a novel and rational approach to developing a new metal-based drug with antitumor properties complementary to those exhibited by the drugs already in clinical use.     

Lysophosphatidic acid induces endothelial cell death by modulating the redox environment

Oxidant stress plays a significant role in hypoxic-ischemic injury to the susceptible microvascular endothelial cells. During oxidant stress, lysophosphatidic acid (LPA) concentrations increase. We explored whether LPA caused cytotoxicity to neuromicrovascular cells and the potential mechanisms thereof. LPA caused a dose-dependent death of porcine cerebral microvascular as well as human umbilical vein endothelial cells; cell death appeared oncotic rather than apoptotic. LPA-induced cell death was mediated via LPA(1) receptor, because the specific LPA(1) receptor antagonist THG1603 fully abrogated LPA's effects. LPA decreased intracellular GSH levels and induced a p38 MAPK/JNK-dependent inducible nitric oxide synthase (NOS) expression. Pretreatment with the antioxidant GSH precursor N-acetyl-cysteine (NAC), as well as with inhibitors of NOS [N(omega)-nitro-l-arginine (l-NNA); 1400W], significantly prevented LPA-induced endothelial cell death (in vitro) to comparable extents; as expected, p38 MAPK (SB203580) and JNK (SP-600125) inhibitors also diminished cell death. LPA did not increase indexes of oxidation (isoprostanes, hydroperoxides, and protein nitration) but did augment protein nitrosylation. Endothelial cytotoxicity by LPA in vitro was reproduced ex vivo in brain and in vivo in retina; THG1603, NAC, l-NNA, and combined SB-203580 and SP600125 prevented the microvascular rarefaction. Data implicate novel properties for LPA as a modulator of the cell redox environment, which partakes in endothelial cell death and ensued neuromicrovascular rarefaction.