In vivo evidence for instability of episomal human immunodeficiency virus type 1 cDNA

Current regimens for the management of human immunodeficiency virus type 1 (HIV-1) infection suppress plasma viremia to below detectable levels for prolonged intervals. Nevertheless, there is a rapid resumption in plasma viremia if therapy is interrupted. Attempts to characterize the extent of viral replication under conditions of potent suppression and undetectable plasma viremia have been hampered by a lack of convenient assays that can distinguish latent from ongoing viral replication. Using episomal viral cDNA as a surrogate for ongoing replication, we previously presented evidence that viral replication persists in the majority of infected individuals with a sustained aviremic status. The labile nature of viral episomes and hence their validity as surrogate markers of ongoing replication in individuals with long-term-suppressed HIV-1 infection have been analyzed in short-term in vitro experiments with conflicting results. Since these in vitro experiments do not shed light on the long-term in vivo dynamics of episomal cDNA or recapitulate the natural targets of infection in vivo, we have analyzed the dynamics of episomal cDNA turnover in vivo by following the emergence of an M184V polymorphism in plasma viral RNA, in episomal cDNA, and in proviral DNA in patients on suboptimal therapies. We demonstrate that during acquisition of drug resistance, wild-type episomal cDNAs are replaced by M184V-harboring episomes. Importantly, a complete replacement of wild-type episomes with M184V-containing episomes occurred while proviruses remained wild type. This indicates that episomal cDNAs are turned over by degradation rather than through death or tissue redistribution of the infected cell itself. Therefore, evolution of episomal viral cDNAs is a valid surrogate of ongoing viral replication in HIV-1-infected individuals.     

Effects of Morris water maze testing on the neuroendocrine stress response and intrahypothalamic release of vasopressin and oxytocin in the rat

Adult male Wistar rats were trained in the Morris water maze (MWM) on 3 consecutive days to find a visible platform. Concomitantly, microdialysis samples from the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei were collected in order to monitor local release of the neuropeptides vasopressin (AVP) and oxytocin (OXT), respectively, during controllable swim stress. Additionally, a separate set of animals was equipped with chronic jugular venous catheters to collect blood samples for analyzing plasma concentrations of corticotropin (ACTH) and corticosterone during training in the MWM. As measured by microdialysis, swimming in the MWM caused a significantly increased release of AVP within the PVN and of OXT within the SON on each of the 3 test sessions. In contrast to OXT in the SON, basal AVP concentrations in the PVN tended to rise from day to day. Plasma ACTH and corticosterone were found to be similarly elevated in response to MWM exposure on each of the test sessions. Taken together, these data demonstrate that testing in the MWM is not only associated with a significant activation of the hypothalamo-pituitary-adrenal axis but also with an intrahypothalamic release of AVP and OXT. If compared with findings using repeated forced swimming as an uncontrollable stressor (Wotjak, C.T., Ganster, J., Kohl, G., Holsboer, F., Landgraf, R., Engelmann, M., 1998. Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons. Neuroscience 85, 1209-1222), the present results suggest that (1) similarities in the release profiles of AVP in the PVN and plasma hormone levels are fairly independent from the controllability of the stressor and seem, thus, to primarily relate to the physical demands of the task, whereas (2) the different intra-SON OXT release profiles might be linked to the controllability of the stressor.     

Unusual evolution of a catalytic core element in CCA-adding enzymes

CCA-adding enzymes are polymerases existing in two distinct enzyme classes that both synthesize the C-C-A triplet at tRNA 3′-ends. Class II enzymes (found in bacteria and eukaryotes) carry a flexible loop in their catalytic core required for switching the specificity of the nucleotide binding pocket from CTP- to ATP-recognition. Despite this important function, the loop sequence varies strongly between individual class II CCA-adding enzymes. To investigate whether this loop operates as a discrete functional entity or whether it depends on the sequence context of the enzyme, we introduced reciprocal loop replacements in several enzymes. Surprisingly, many of these replacements are incompatible with enzymatic activity and inhibit ATP-incorporation. A phylogenetic analysis revealed the existence of conserved loop families. Loop replacements within families did not interfere with enzymatic activity, indicating that the loop function depends on a sequence context specific for individual enzyme families. Accordingly, modeling experiments suggest specific interactions of loop positions with important elements of the protein, forming a lever-like structure. Hence, although being part of the enzyme’s catalytic core, the loop region follows an extraordinary evolutionary path, independent of other highly conserved catalytic core elements, but depending on specific sequence features in the context of the individual enzymes.     

Vector Choice Determines Immunogenicity and Potency of Genetic Vaccines against Angola Marburg Virus in Nonhuman Primates

The immunogenicity and durability of genetic vaccines are influenced by the composition of gene inserts and choice of delivery vector. DNA vectors are a promising vaccine approach showing efficacy when combined in prime-boost regimens with recombinant protein or viral vectors, but they have shown limited comparative efficacy as a stand-alone platform in primates, due possibly to suboptimal gene expression or cell targeting. Here, regimens using DNA plasmids modified for optimal antigen expression and recombinant adenovirus (rAd) vectors, all encoding the glycoprotein (GP) gene from Angola Marburg virus (MARV), were compared for their ability to provide immune protection against lethal MARV Angola infection. Heterologous DNA-GP/rAd5-GP prime-boost and single-modality rAd5-GP, as well as the DNA-GP-only vaccine, prevented death in all vaccinated subjects after challenge with a lethal dose of MARV Angola. The DNA/DNA vaccine induced humoral responses comparable to those induced by a single inoculation with rAd5-GP, as well as CD4⁺ and CD8⁺ cellular immune responses, with skewing toward CD4⁺ T-cell activity against MARV GP. Vaccine regimens containing rAd-GP, alone or as a boost, exhibited cellular responses with CD8⁺ T-cell dominance. Across vaccine groups, CD8⁺ T-cell subset dominance comprising cells exhibiting a tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) double-positive functional phenotype was associated with an absence or low frequency of clinical symptoms, suggesting that both the magnitude and functional phenotype of CD8⁺ T cells may determine vaccine efficacy against infection by MARV Angola.The filoviruses Marburgvirus (MARV) and Ebolavirus (EBOV) are endemic primarily to central Africa and cause a severe form of viral hemorrhagic fever. Of all the filovirus strains or species, the Angola strain of MARV is associated with the highest mortality rate (90%) in humans observed to date (26). An increase in natural filovirus outbreak frequency over the past decade and the potential for use to cause deliberate human mortality have focused attention on the need for therapeutics and vaccines against filoviruses. While regulatory pathways have been proposed to facilitate licensing of a preventive vaccine against potently lethal pathogens such as these, there is as yet no licensed vaccine for use in humans, and efforts remain targeted to the optimization of vaccine performance in nonhuman primates (NHP) since this animal model recapitulates many aspects of disease pathogenesis observed in humans.Genetic vaccines are a promising approach for immunization against pathogens that are rapidly changing due to natural evolution, cross-species transmission, or intentional modification. Gene-based vaccines are produced rapidly and can be delivered by a variety of vectors. DNA vectors are advantageous because they are inherently safe and stable and can be used repeatedly without inducing antivector immune responses. However, while filovirus DNA vaccines have demonstrated efficacy in small animal models, efforts to induce protective immunity by injection of plasmid DNA alone into NHP have yielded less encouraging results. EBOV DNA vectors generate immune protection in mice and guinea pigs, but this has not been demonstrated in NHP unless DNA immunization is boosted with a viral vector vaccine (23). MARV DNA fully protects mice and guinea pigs but provides only partial protection in NHP (17). The discordant results between rodent and primate species may be due to the use of slightly modified infectious challenge viruses in rodent models or may reflect underlying differences in vaccine performance and the mechanisms of immune protection between rodents and NHP.In the current study, we examined whether DNA plasmid-based vaccines could be improved to increase potency in NHP and compared immunogenicity of this vaccine modality with those of viral vector and prime-boost approaches. DNA-vectored vaccines were modified by codon optimizing gene target inserts for enhanced expression in primates. These vectors induced antigen-specific cellular and humoral immune responses similar to immunization using a recombinant adenoviral vector and provided protection after lethal challenge with MARV Angola. However, macaques vaccinated with DNA vectors exhibited clinical symptoms associated with MARV hemorrhagic fever (MHF) that were absent in NHP receiving a single inoculation with recombinant adenovirus (rAd) vectors, suggesting qualitative differences in the immune responses elicited by the different modalities.     

Role of the fourth intracellular loop of D1-like dopaminergic receptors in conferring subtype-specific signaling properties

We investigate whether the fourth intracellular loop (IL4) of D1 and D5 dopaminergic receptors (D1R, D5R) confers D1-like subtype-specific signaling properties. Using chimeric receptors (D1R-IL4B and D5R-IL4A), we show that swapping of IL4 leads to a switch in dopamine affinity and constitutive activity of D1R and D5R. Dopamine potency was reduced for both chimeras in comparison with wild-type receptors. Moreover, dopamine-mediated maximal activation was drastically increased in cells expressing D1R-IL4B when compared with those harboring D5R-IL4A or wild-type receptors. In conclusion, IL4 plays a pivotal role in imparting subtype-specific ligand binding and activation properties to highly homologous seven-transmembrane receptors.     

A Simple Geometrical Pattern for the Branching Distribution of the Bronchial Tree, Useful to Estimate Optimality Departures

The design of the bronchial tree has largely been proposed as a model of optimal design from a physical-functional perspective. However, the distributive function of the airway may be more related to a geometrical than a physical problem. The bronchial tree must distribute a three dimensional volume of inspired air on a two dimensional alveolar surface, included in a limited volume. It is thus valid to ask whether an optimal bronchial tree from a physical perspective is also optimum from a geometrical point of view. In this paper we generate a simple geometric model for the branching pattern of the bronchial tree, deducing relationships that permit estimation of the departures from the geometrical optimum of each bifurcation. We also, for comparative purposes, estimate the departures from the physical optimum. From the geometrical assumptions: i) a symmetrical dichotomic fractal design, ii) with minimum volume and iii) maximum dispersion of the terminal points; and several simulations we suggest that the optimality is characterized by a bifurcation angle theta approximately 60 degrees and a length reduction scale gamma = (1/2)(1/3) = 0.7937. We propose distances from the physical and geometrical optimality defined as Euclidean distances from the expected optima. We show how the advanced relationships and the distances can be used to estimate departures from the optimality in bronchographs of four species. We found lower physical and geometrical departures in the distal zone than those of the proximal zones, as well as lower physical than geometrical departures from optimality.     

An assessment of time-dependent effects of lead exposure in algerian mice (Mus spretus) using different methodological approaches

Time-dependent effects of lead (Pb) toxicity were studied in Algerian mice (Mus spretus) treated with Pb acetate via drinking water (1 g Pb acetate/L) for different periods of exposure (15, 45, and 90 d). End points included the determination of hepatic Pb concentration and the assessment of some morphophysiological, biochemical and cytogenetical parameters. A control group receiving distilled water was also monitored for comparative purposes. Hepatic Pb accumulation increased with the time of exposure and was significantly higher in treated mice when compared to controls. In association with significant body mass loss in Pb-exposed mice, for 15 and 45 d, a significant increase in the relative spleen mass was observed after 45 d of intoxication. Pb-exposed mice also showed significant decreases in red blood cells, hematocrit, and mean corpuscular hemoglobin. On the contrary, changes in plasma transferases (aspartate aminotransferase and alanine aminotransferase) and hepatic superoxide dismutase activities did not reach statistical significance. A significant increase in the frequency of micronucleated polychromatic bone marrow erythrocytes was also found in the 90-d-exposed mice, compared to nontreated mice and the other exposed groups. Exposure to Pb acetate resulted also in a slight time-dependent decrease of the polychromatic-normochromatic ratio. These results support the concept that a long-term chronic exposure to Pb induced alterations upon some morphophysiological and genetic paramaters in Algerian mice.