Bone turnover markers in HIV-infected patients before starting antiretroviral therapy.

Purpose: Bone turnover markers (BTM) - aminoterminal propeptide of type 1 collagen (P1NP) and C-terminal telopeptide of type 1 collagen (b-CTX) - are related to bone density and fracture risk. A high prevalence of osteopenia/osteoporosis and hypovitaminosis D has been reported in HIV patients, however there are few data about BTM in this population. Our aim was to analyse the prevalence of elevated serum levels of BTM in HIV patients before starting antiretroviral therapy (ART), and related factors. Methods: Cross-sectional study of a series of HIV-patients who started ART during June/11-June/12 in our hospital. Patients with presence of diseases or treatments known to affect bone metabolism were excluded. Epidemiological, clinical, and immunovirological data in addition to serum fasting levels of glucose, lipid profile, calcium, phosphate, alkaline phosphatase, 25-hydroxyvitamin D3 (25OHD), parathyroid hormone (PTH), P1NP, and β-CTX were collected. Definitions: hypovitaminosis D if 25OHD<30 ng/ml, vitamin D deficiency if 25OHD<20 ng/ml; elevated levels of BTM if β-CTX (ng/ml) >0.64 (men<70 years),>0.85 (men>70 years),>0.58 (pre-menopause women), >0.99 (post-menopause women), or P1NP (ng/mL)>69.4 (men<60 years), >71.1 (men>60 years), >55.7 (pre-menopause women), >61.2 (post-menopause women). Results: 47 patients were included, 91.5% men, median age 37.1 years (30.0-44.3), and 93.6% sexual transmission of HIV (34 HMX, 10 HTX). Median time since the diagnosis of HIV was 3.4 months (1.4- 31.7); there were 7 (14.9%) Aids cases, median CD4 count was 277/ mm3 (155-433), and HIV-VL 4.8 log10 (4.1-5.2). Median serum 25OHD was 29 mg/L (21.9-41.1), with a prevalence of hypovitaminosis of 52.2%, and deficiency of 17.4%. PTH was in range in all cases. Median serum P1NP was 33.3 ng/mL (24.5-52.5) and β-CTX 0.25 ng/mL (0.20-0.45); five (11.4%) patients presented high levels of BTM: 4 men, median age 37.1 years, median CD4 count 247/mm3, median HIV-VL 5.18 log10, and one with hypovitaminosis D. Elevated BTM were related with no clinical, analytical, immunovirological parameters nor with serum levels of 25OHD nor PTH. Conclusions: The prevalence of elevated BTM was high in this series of HIV-patients, mostly young men, with short time of HIV infection and with no immunovirologic control. BTM were related with no clinical nor analytical data.

Long-term efficacy and safety of atazanavir/ritonavir treatment in a cohort of treatment-naïve HIV patients: An interim analysis of the REMAIN study.

Purpose: Combined antiretroviral therapy has dramatically improved HIV-infected individuals survival. Long-term strategies are currently needed to achieve the goal of durable virologic suppression. However, long-term available data for specific antiretrovirals (ARV) are limited. In clinical trials, boosted atazanavir (ATV/r) regimens has shown good efficacy and tolerability in ARV-naïve patients for up to 4 years. The REMAIN study aimed to evaluate the long-term outcomes of ATV/r regimens in ARV-naïve patients in a real life setting. Methods: Non-comparative, observational study conducted in Germany, Portugal and Spain. Historical and longitudinal follow-up data was extracted six monthly from the medical record of HIV-infected, treatment-naïve patients, who initiated an ATV/r-regimen between 2008 and 2010. The primary endpoint was the proportion of patients remaining on ATV treatment over time. Secondary endpoints included virologic response (HIV-1 RNA <50 c/mL and <500 c/mL), reasons for discontinuation and long-term safety. The duration of treatment and time to virologic failure (VF) were analyzed using the Kaplan- Meier method. Data from an interim analysis including patients with at least one year of follow-up are reported here. Results: A total of 411 patients were included in this interim analysis [median (Q1, Q3) follow-up: 23.42 (16.25, 32.24) months≥: 77% male; median age 40 years [min, max: 19, 78≥; 16% IDUs; 18% CDC C; 18% hepatitis C. TDF/FTC was the most common backbone (85%). At baseline, median (Q1, Q3) HIV-RNA and CD4 cell count were 4.91 (4.34, 5.34) log10 c/mL and 256 (139, 353) cells/mm3, respectively. The probability of remaining on treatment was 0.84 (95% CI: 0.80, 0.87) and 0.72 (95% CI: 0.67, 0.76) for the first and second year, respectively. After 2 years of follow-up, 84% (95% CI: 0.79, 0.88) of patients were virologically suppressed (<50 c/mL). No major protease inhibitors mutations were observed at VF. Overall, 125 patients (30%) discontinued ATV therapy [median (Q1, Q3) time to discontinuation: 11.14 (6.24, 19.35) months]. Adverse events (AEs) were the main reason for discontinuation (n =47, 11%). Hyperbilirubinaemia was the most common AE leading to discontinuation (14 patients). No unexpected AEs were reported. Conclusions: In a real life clinical setting, ATV/r regimens showed durable virologic efficacy with good tolerability in an ARV-naïve population. Data from longer follow-up will provide additional valuable information.

Neurofilament triplet proteins are restricted to a subset of neurons in the rat neocortex.

The cellular localisation of neurofilament triplet subunits was investigated in the rat neocortex. A subset of mainly pyramidal neurons showed colocalisation of subunit immunolabelling throughout the neocortex, including labelling with the antibody SMI32, which has been used extensively in other studies of the primate cortex as a selective cellular marker. Neurofilament-labelled neurons were principally localised to two or three cell layers in most cortical regions, but dramatically reduced labelling was present in areas such as the perirhinal cortex, anterior cingulate and a strip of cortex extending from caudal motor regions through the medial parietal region to secondary visual areas. However, quantitative analysis demonstrated a similar proportion (10-20%) of cells with neurofilament triplet labelling in regions of high or low labelling. Combining retrograde tracing with immunolabelling showed that cellular content of the neurofilament proteins was not correlated with the length of projection. Double labelling immunohistochemistry demonstrated that neurofilament content in axons was closely associated with myelination. Analysis of SMI32 labelling in development indicated that content of this epitope within cell bodies was associated with relatively late maturation, between postnatal days 14 and 21. This study is further evidence of a cell type-specific regulation of neurofilament proteins within neocortical neurons. Neurofilament triplet content may be more closely related to the degree of myelination, rather than the absolute length, of the projecting axon.

Metabolomics in the fight against malaria

Metabolomics uses high-resolution mass spectrometry to provide a chemical fingerprint of thousands of metabolites present in cells, tissues or body fluids. Such metabolic phenotyping has been successfully used to study various biologic processes and disease states. High-resolution metabolomics can shed new light on the intricacies of host-parasite interactions in each stage of the Plasmodium life cycle and the downstream ramifications on the host’s metabolism, pathogenesis and disease. Such data can become integrated with other large datasets generated using top-down systems biology approaches and be utilised by computational biologists to develop and enhance models of malaria pathogenesis relevant for identifying new drug targets or intervention strategies. Here, we focus on the promise of metabolomics to complement systems biology approaches in the quest for novel interventions in the fight against malaria. We introduce the Malaria Host-Pathogen Interaction Center (MaHPIC), a new systems biology research coalition. A primary goal of the MaHPIC is to generate systems biology datasets relating to human and non-human primate (NHP) malaria parasites and their hosts making these openly available from an online relational database. Metabolomic data from NHP infections and clinical malaria infections from around the world will comprise a unique global resource.

Quantitative analysis of postnatal neurogenesis and neuron number in the macaque monkey dentate gyrus

The dentate gyrus is one of only two regions of the mammalian brain where substantial neurogenesis occurs postnatally. However, detailed quantitative information about the postnatal structural maturation of the primate dentate gyrus is meager. We performed design-based, stereological studies of neuron number and size, and volume of the dentate gyrus layers in rhesus macaque monkeys (Macaca mulatta) of different postnatal ages. We found that about 40% of the total number of granule cells observed in mature 5-10-year-old macaque monkeys are added to the granule cell layer postnatally; 25% of these neurons are added within the first three postnatal months. Accordingly, cell proliferation and neurogenesis within the dentate gyrus peak within the first 3 months after birth and remain at an intermediate level between 3 months and at least 1 year of age. Although granule cell bodies undergo their largest increase in size during the first year of life, cell size and the volume of the three layers of the dentate gyrus (i.e. the molecular, granule cell and polymorphic layers) continue to increase beyond 1 year of age. Moreover, the different layers of the dentate gyrus exhibit distinct volumetric changes during postnatal development. Finally, we observe significant levels of cell proliferation, neurogenesis and cell death in the context of an overall stable number of granule cells in mature 5-10-year-old monkeys. These data identify an extended developmental period during which neurogenesis might be modulated to significantly impact the structure and function of the dentate gyrus in adulthood.

Incremento de la participación de Atención Primaria en la asistencia al virus de la inmunodeficiencia humana: opinan los profesionales de las unidades hospitalarias.

AIM To determine the opinions of infectious diseases professionals on the possibilities of monitoring patients with HIV in Primary Care. DESIGN Qualitative study using in-depth interviews. LOCATION Infectious Diseases Unit in the University Hospital "Virgen de la Victoria" in Málaga. PARTICIPANTS Health professionals with more than one year experience working in infectious diseases. A total of 25 respondents: 5 doctors, 15 nurses and 5 nursing assistants. METHOD Convenience sample. Semi-structured interviews were used that were later transcribed verbatim. Content analysis was performed according to the Taylor and Bogdan approach with computer support. Validation of information was made through additional analysis, expert participation, and feedback of part of the results to the participants. RESULTS Hospital care professionals considered the disease-related complexity of HIV, treatment and social aspects that may have an effect on the organizational level of care. Professionals highlighted the benefits of specialized care, although opinions differed between doctors and nurses as regards follow up in Primary Care. Some concerns emerged about the level of training, confidentiality and workload in Primary Care, although they mentioned potential advantages related to accessibility of patients. CONCLUSIONS Physicians perceive difficulties in following up HIV patients in Primary Care, even for those patients with a good control of their disease. Nurses and nursing assistants are more open to this possibility due to the proximity to home and health promotion in Primary Care.

Yellow fever impact on brown howler monkeys (Alouatta guariba clamitans) in Argentina: a metamodelling approach based on population viability analysis and epidemiological dynamics

In South America, yellow fever (YF) is an established infectious disease that has been identified outside of its traditional endemic areas, affecting human and nonhuman primate (NHP) populations. In the epidemics that occurred in Argentina between 2007-2009, several outbreaks affecting humans and howler monkeys (Alouatta spp) were reported, highlighting the importance of this disease in the context of conservation medicine and public health policies. Considering the lack of information about YF dynamics in New World NHP, our main goal was to apply modelling tools to better understand YF transmission dynamics among endangered brown howler monkey (Alouatta guariba clamitans) populations in northeastern Argentina. Two complementary modelling tools were used to evaluate brown howler population dynamics in the presence of the disease: Vortex, a stochastic demographic simulation model, and Outbreak, a stochastic disease epidemiology simulation. The baseline model of YF disease epidemiology predicted a very high probability of population decline over the next 100 years. We believe the modelling approach discussed here is a reasonable description of the disease and its effects on the howler monkey population and can be useful to support evidence-based decision-making to guide actions at a regional level.

Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif.

Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high α-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the α-helical coiled coil structures. In addition, ex vivo production of IFN-γ by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the α-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes.

Patterns of evolution of host proteins involved in retroviral pathogenesis.

BACKGROUND: Evolutionary analysis may serve as a useful approach to identify and characterize host defense and viral proteins involved in genetic conflicts. We analyzed patterns of coding sequence evolution of genes with known (TRIM5alpha and APOBEC3G) or suspected (TRIM19/PML) roles in virus restriction, or in viral pathogenesis (PPIA, encoding Cyclophilin A), in the same set of human and non-human primate species. RESULTS AND CONCLUSION: This analysis revealed previously unidentified clusters of positively selected sites in APOBEC3G and TRIM5alpha that may delineate new virus-interaction domains. In contrast, our evolutionary analyses suggest that PPIA is not under diversifying selection in primates, consistent with the interaction of Cyclophilin A being limited to the HIV-1M/SIVcpz lineage. The strong sequence conservation of the TRIM19/PML sequences among primates suggests that this gene does not play a role in antiretroviral defense.

Structure-function analyses point to a polynucleotide-accommodating groove essential for APOBEC3A restriction activities.

Members of the human APOBEC3 family of editing enzymes can inhibit various mobile genetic elements. APOBEC3A (A3A) can block the retrotransposon LINE-1 and the parvovirus adeno-associated virus type 2 (AAV-2) but does not inhibit retroviruses. In contrast, APOBEC3G (A3G) can block retroviruses but has only limited effects on AAV-2 or LINE-1. What dictates this differential target specificity remains largely undefined. Here, we modeled the structure of A3A based on its homology with the C-terminal domain of A3G and further compared the sequence of human A3A to those of 11 nonhuman primate orthologues. We then used these data to perform a mutational analysis of A3A, examining its ability to restrict LINE-1, AAV-2, and foreign plasmid DNA and to edit a single-stranded DNA substrate. The results revealed an essential functional role for the predicted single-stranded DNA-docking groove located around the A3A catalytic site. Within this region, amino acid differences between A3A and A3G are predicted to affect the shape of the polynucleotide-binding groove. Correspondingly, transferring some of these A3A residues to A3G endows the latter protein with the ability to block LINE-1 and AAV-2. These results suggest that the target specificity of APOBEC3 family members is partly defined by structural features influencing their interaction with polynucleotide substrates.

Evolutionary and Functional Analyses of the Interaction between the Myeloid Restriction Factor SAMHD1 and the Lentiviral Vpx Protein.

SAMHD1 has recently been identified as an HIV-1 restriction factor operating in myeloid cells. As a countermeasure, the Vpx accessory protein from HIV-2 and certain lineages of SIV have evolved to antagonize SAMHD1 by inducing its ubiquitin-proteasome-dependent degradation. Here, we show that SAMHD1 experienced strong positive selection episodes during primate evolution that occurred in the Catarrhini ancestral branch prior to the separation between hominoids (gibbons and great apes) and Old World monkeys. The identification of SAMHD1 residues under positive selection led to mapping the Vpx-interaction domain of SAMHD1 to its C-terminal region. Importantly, we found that while SAMHD1 restriction activity toward HIV-1 is evolutionarily maintained, antagonism of SAMHD1 by Vpx is species-specific. The distinct evolutionary signature of SAMHD1 sheds light on the development of its antiviral specificity.

Autologous adult cortical cell transplantation enhances functional recovery following unilateral lesion of motor cortex in primates: a pilot study.

BACKGROUND:: Although cell therapy is a promising approach after cerebral cortex lesion, few studies assess quantitatively its behavioral gain in non-human primates. Furthermore, implantations of fetal grafts of exogenous stem cells are limited by safety and ethical issues. OBJECTIVE:: To test in non-human primates the transplantation of autologous adult neural progenitor cortical cells with assessment of functional outcome. METHODS:: Seven adult macaque monkeys were trained to perform a manual dexterity task, before the hand representation in motor cortex was chemically lesioned unilaterally. Five monkeys were used as control, compared to two monkeys subjected to different autologous cells transplantation protocols performed at different time intervals. RESULTS:: After lesion, there was a complete loss of manual dexterity in the contralesional hand. The five "control" monkeys recovered progressively and spontaneously part of their manual dexterity, reaching a unique and definitive plateau of recovery, ranging from 38% to 98% of pre-lesion score after 10 to 120 days. The two "treated" monkeys reached a first spontaneous recovery plateau at about 25 and 40 days post-lesion, representing 35% and 61% of the pre-lesion performance, respectively. In contrast to the controls, a second recovery plateau took place 2-3 months after cell transplantation, corresponding to an additional enhancement of functional recovery, representing 24 and 37% improvement, respectively. CONCLUSIONS:: These pilot data, derived from two monkeys treated differently, suggest that, in the present experimental conditions, autologous adult brain progenitor cell transplantation in non-human primate is safe and promotes enhancement of functional recovery.

CAV2 vector gene transfer into central nervious System

Estudi realitzat a partir d’una estada al Institut de Génétique Moléculaire de Montpellier, França, entre 2010 i 2012. En aquest projecte s’ha avaluat les avantatges dels vectors adenovirals canins tipus 2 (CAV2) com a vectors de transferència gènica al sistema nerviós central (SNC) en un model primat no-humà i en un model caní del síndrome de Sly (mucopolisacaridosis tipus 7, MPS VII), malaltia monogènica que cursa amb neurodegeneració. En una primera part del projecte s’ha avaluat la biodistribució, l’eficàcia i la durada de l’expressió del transgen en un model primat no humà, (Microcebus murinus). Com ha vector s’ha utilitzat un CAV2 de primera generació que expressa la proteïna verda fluorescent (CAVGFP). Els resultats aportats en aquesta memòria demostren que en primats no humans, com en d’altres espècies testades anteriorment per l’equip de l’EJ Kremer, la injecció intracerebral de CAV2 resulta en una extensa transducció del SNC, siguent les neurones i els precursors neuronals les cèl•lules preferencialment transduïdes. Els vectors canins, servint-se de vesícules intracel•lulars són transportats, majoritàriament, des de les sinapsis cap al soma neuronal, aquest transport intracel•lular permet una extensa transducció del SNC a partir d’una única injecció intracerebral dels vectors virals. En una segona part d’aquest projecte s’ha avaluat l’ús terapèutic dels CAV2. S’ha injectat un vector helper-dependent que expressa el gen la b-glucuronidasa i el gen de la proteïna verda fluorescent (HD-RIGIE), en el SNC del model caní del síndrome de Sly (MPS VII). La biodistribució i la eficàcia terapèutica han estat avaluades. Els nivells d’activitat enzimàtica en animals malalts injectats amb el vector terapèutic va arribar a valors similars als dels animals no afectes. A més a més s’ha observat una reducció en la quantitat dels GAGs acumulats en les cèl•lules dels animals malalts tractats amb el vector terapèutic, demostrant la potencialitat terapèutica dels CAV2 per a malalties que afecten al SNC. Els resultats aportats en aquest treball ens permeten dir que els CAV2 són unes bones eines terapèutiques per al tractament de malalties que afecten al SNC.

The evolutionary history of the CD209 (DC-SIGN) family in humans and non-human primates

The CD209 gene family that encodes C-type lectins in primates includes CD209 (DC-SIGN), CD209L (L-SIGN) and CD209L2. Understanding the evolution of these genes can help understand the duplication events generating this family, the process leading to the repeated neck region and identify protein domains under selective pressure. We compiled sequences from 14 primates representing 40 million years of evolution and from three non-primate mammal species. Phylogenetic analyses used Bayesian inference, and nucleotide substitutional patterns were assessed by codon-based maximum likelihood. Analyses suggest that CD209 genes emerged from a first duplication event in the common ancestor of anthropoids, yielding CD209L2 and an ancestral CD209 gene, which, in turn, duplicated in the common Old World primate ancestor, giving rise to CD209L and CD209. K(A)/K(S) values averaged over the entire tree were 0.43 (CD209), 0.52 (CD209L) and 0.35 (CD209L2), consistent with overall signatures of purifying selection. We also assessed the Toll-like receptor (TLR) gene family, which shares with CD209 genes a common profile of evolutionary constraint. The general feature of purifying selection of CD209 genes, despite an apparent redundancy (gene absence and gene loss), may reflect the need to faithfully recognize a multiplicity of pathogen motifs, commensals and a number of self-antigens

Pseudogenes in the ENCODE regions: consensus annotation, analysis of transcription, and evolution

Arising from either retrotransposition or genomic duplication of functional genes, pseudogenes are “genomic fossils” valuable for exploring the dynamics and evolution of genes and genomes. Pseudogene identification is an important problem in computational genomics, and is also critical for obtaining an accurate picture of a genome’s structure and function. However, no consensus computational scheme for defining and detecting pseudogenes has been developed thus far. As part of the ENCyclopedia Of DNA Elements (ENCODE) project, we have compared several distinct pseudogene annotation strategies and found that different approaches and parameters often resulted in rather distinct sets of pseudogenes. We subsequently developed a consensus approach for annotating pseudogenes (derived from protein coding genes) in the ENCODE regions, resulting in 201 pseudogenes, two-thirds of which originated from retrotransposition. A survey of orthologs for these pseudogenes in 28 vertebrate genomes showed that a significant fraction (∼80%) of the processed pseudogenes are primate-specific sequences, highlighting the increasing retrotransposition activity in primates. Analysis of sequence conservation and variation also demonstrated that most pseudogenes evolve neutrally, and processed pseudogenes appear to have lost their coding potential immediately or soon after their emergence. In order to explore the functional implication of pseudogene prevalence, we have extensively examined the transcriptional activity of the ENCODE pseudogenes. We performed systematic series of pseudogene-specific RACE analyses. These, together with complementary evidence derived from tiling microarrays and high throughput sequencing, demonstrated that at least a fifth of the 201 pseudogenes are transcribed in one or more cell lines or tissues.