Javier García-Pérez

Allosteric Model of Maraviroc Binding to CC Chemokine Receptor 5 (CCR5)

Maraviroc is a nonpeptidic small molecule human immunodeficiency virus type 1 (HIV-1) entry inhibitor that has just entered the therapeutic arsenal for the treatment of patients. We recently demonstrated that maraviroc binding to the HIV-1 coreceptor, CC chemokine receptor 5 (CCR5), prevents it from binding the chemokine CCL3 and the viral envelope glycoprotein gp120 by an allosteric mechanism. However, incomplete knowledge of ligand-binding sites and the lack of CCR5 crystal structures have hampered an in-depth molecular understanding of how the inhibitor works. Here, we addressed these issues by combining site-directed mutagenesis (SDM) with homology modeling and docking. Six crystal structures of G-protein-coupled receptors were compared for their suitability for CCR5 modeling. All CCR5 models had equally good geometry, but that built from the recently reported dimeric structure of the other HIV-1 coreceptor CXCR4 bound to the peptide CVX15 (Protein Data Bank code 3OE0) best agreed with the SDM data and discriminated CCR5 from non-CCR5 binders in a virtual screening approach. SDM and automated docking predicted that maraviroc inserts deeply in CCR5 transmembrane cavity where it can occupy three different binding sites, whereas CCL3 and gp120 lie on distinct yet overlapped regions of the CCR5 extracellular loop 2. Data suggesting that the transmembrane cavity remains accessible for maraviroc in CCL3-bound and gp120-bound CCR5 help explain our previous observation that the inhibitor enhances dissociation of preformed ligand-CCR5 complexes. Finally, we identified residues in the predicted CCR5 dimer interface that are mandatory for gp120 binding, suggesting that receptor dimerization might represent a target for new CCR5 entry inhibitors.

G Protein-Dependent CCR5 Signaling Is Not Required for Efficient Infection of Primary T Lymphocytes and Macrophages by R5 Human Immunodeficiency Virus Type 1 Isolates

ABSTRACT The requirement of human immunodeficiency virus (HIV)-induced CCR5 activation for infection by R5 HIV type 1 (HIV-1) strains remains controversial. Ectopic CCR5 expression in CD4+-transformed cells or pharmacological inhibition of Gαi proteins coupled to CCR5 left unsolved whether CCR5-dependent cell activation is necessary for the HIV life cycle. In this study, we investigated the role played by HIV-induced CCR5-dependent cell signaling during infection of primary CD4-expressing leukocytes. Using lentiviral vectors, we restored CCR5 expression in T lymphocytes and macrophages from individuals carrying the homozygous 32-bp deletion of the CCR5 gene (ccr5 Δ32/Δ32). Expression of wild-type (wt) CCR5 in ccr5 Δ32/Δ32 cells permitted infection by R5 HIV isolates. We assessed the capacity of a CCR5 derivative carrying a mutated DRY motif (CCR5-R126N) in the second intracellular loop to work as an HIV-1 coreceptor. The R126N mutation is known to disable G protein coupling and agonist-induced signal transduction through CCR5 and other G protein-coupled receptors. Despite its inability to promote either intracellular calcium mobilization or cell chemotaxis, the inactive CCR5-R126N mutant provided full coreceptor function to several R5 HIV-1 isolates in primary cells as efficiently as wt CCR5. We conclude that in a primary, CCR5-reconstituted CD4+ cell environment, G protein signaling is dispensable for R5 HIV-1 isolates to actively infect primary CD4+ T lymphocytes or macrophages.

Mortality due to lung, laryngeal and bladder cancer in towns lying in the vicinity of combustion installations.

BACKGROUND Installations that burn fossil fuels to generate power may represent a health problem due to the toxic substances which they release into the environment. OBJECTIVES To investigate whether there might be excess mortality due to tumors of lung, larynx and bladder in the population residing near Spanish combustion installations included in the European Pollutant Emission Register. METHODS Ecologic study designed to model sex-specific standardized mortality ratios for the above three tumors in Spanish towns, over the period 1994-2003. Population exposure to pollution was estimated on the basis of distance from town of residence to pollution source. Using mixed Poisson regression models, we analyzed: risk of dying from cancer in a 5-kilometer zone around installations that commenced operations before 1990; effect of type of fuel used; and risk gradient within a 50-kilometer radius of such installations. RESULTS Excess mortality (relative risk, 95% confidence interval) was detected in the vicinity of pre-1990 installations for lung cancer (1.066, 1.041-1.091 in the overall population; 1.084, 1.057-1.111 in men), and laryngeal cancer among men (1.067, 0.992-1.148). Lung cancer displayed excess mortality for all types of fuel used, whereas in laryngeal and bladder cancer, the excess was associated with coal-fired industries. There was a risk gradient effect in the proximity of a number of installations. CONCLUSIONS Our results could support the hypothesis of an association between risk of lung, laryngeal and bladder cancer mortality and proximity to Spanish combustion installations.

A sensitive phenotypic assay for the determination of human immunodeficiency virus type 1 tropism

OBJECTIVES To develop a sensitive phenotypic assay based on recombinant viruses (RVs) for characterizing HIV-1 tropism. METHODS Viral tropism was assessed in 159 plasma samples. The env gene was amplified and ligated into pNL-lacZ/env-Ren, which carries a luciferase reporter gene. Resulting constructs were transfected into HEK293T cells to generate RVs. To assess co-receptor tropism, U87.CD4.CXCR4/CCR5 cells were infected and luciferase activity was measured. RESULTS RVs containing env from different HIV-1 subtypes were replication competent. Minor variants were detectable in 1% of the viral population. Tropism was determined in 65% of samples with a viral load of <1000 copies/mL. The phenotypic assay described here was validated with the Trofile™ and Trofile™ES assays. Considering the Trofile™ assay as a reference, the sensitivity for R5 and R5X4/X4 detection was 90% and 77%, and the specificity was 77% and 90%, respectively. Our assay was 86% concordant with Trofile™ (90% for R5 and 77% for R5X4/X4). When our system was compared with Trofile™ES, the concordance was 89% (86% for R5 and 92% for R5X4/X4), the sensitivity for R5 was 86% and for R5X4/X4 was 92%, and the specificity for R5 was 92% and for R5X4/X4 was 86%. The phenotypic results were compared with those obtained using the following V3 genotypic prediction tools: position-specific scoring matrix; geno2pheno[coreceptor]; C4.5; C4.5 using positions 8 and 12; PART; support vector machines; and the charge rule. CONCLUSIONS We describe a system to assess co-receptor tropism based on the generation of chimeric replication-competent viruses with high sensitivity in the detection of minor populations. A good correlation of our results with Trofile™ assays was found.

Description of industrial pollution in Spain

BackgroundToxic substances released into the environment (to both air and water) by many types of industries might be related with the occurrence of some malignant tumours and other diseases. The publication of the EPER (European Pollutant Emission Register) Spanish data allows to investigate the presence of geographical mortality patterns related to industrial pollution. The aim of this paper is to describe industrial air and water pollution in Spain in 2001, broken down by activity group and specific pollutant, and to plot maps depicting emissions of carcinogenic substances.MethodsAll information on industrial pollution discharge in 2001 was drawn from EPER-Spain public records provided by the European Commission server. We described the distribution of the number of industries and amounts discharged for each pollutant, as well as emission by pollutant group and the industrial activities associated with each pollutant. Maps of Spain were drawn up, with UTM coordinates being used to plot pollutant foci, and circles with an area proportional to the emission to depict pollution emission values.ResultsThe EPER-Spain contained information on 1,437 industrial installations. The industrial plants that discharge pollutant substances into air and water above the pollutant-specific EPER threshold were mainly situated in the Autonomous Regions of Aragon, Andalusia and Catalonia and in Catalonia, the Basque Country and Andalusia respectively. Pollution released in 2001 into air approached 158 million Mt. Emissions into water were over 8 million Mt.ConclusionA few single industrial plants are responsible for the highest percentage of emissions, thus rendering monitoring of their possible health impact on the surrounding population that much simpler. Among European countries Spain is the leading polluter in almost one third of all EPER-registered pollutant substances released into the air and ranks among the top three leading polluters in two-thirds of all such substances. Information obtained through publication of EPER data means that the possible consequences of reported pollutant foci on the health of neighbouring populations can now be studied.

The striking geographical pattern of gastric cancer mortality in Spain: environmental hypotheses revisited

BackgroundGastric cancer is decreasing in most countries. While socioeconomic development is the main factor to which this decline has been attributed, enormous differences among countries and within regions are still observed, with the main contributing factors remaining elusive. This study describes the geographic distribution of gastric cancer mortality at a municipal level in Spain, from 1994-2003.MethodsSmoothed relative risks of stomach cancer mortality were obtained, using the Besag-York-Molliè autoregressive spatial model. Maps depicting relative risk (RR) estimates and posterior probabilities of RR being greater than 1 were plotted.ResultsFrom 1994-2003, 62184 gastric cancer deaths were registered in Spain (7 percent of all deaths due to malignant tumors). The geographic pattern was similar for both sexes. RRs displayed a south-north and coast-inland gradient, with lower risks being observed in Andalusia, the Mediterranean coastline, the Balearic and Canary Islands and the Cantabrian seaboard. The highest risk was concentrated along the west coast of Galicia, broad areas of the Castile & Leon Autonomous community, the province of Cáceres in Extremadura, Lleida and other areas of Catalonia.ConclusionIn Spain, risk of gastric cancer mortality displays a striking geographic distribution. With some differences, this persistent and unique pattern is similar across the sexes, suggesting the implication of environmental exposures from sources, such as diet or ground water, which could affect both sexes and delimited geographic areas. Also, the higher sex-ratios found in some areas with high risk of smoking-related cancer mortality in males support the role of tobacco in gastric cancer etiology.

Air quality modeling and mortality impact of fine particles reduction policies in Spain.

BACKGROUND In recent years, Spain has implemented a number of air quality control measures that are expected to lead to a future reduction in fine particle concentrations and an ensuing positive impact on public health. OBJECTIVES We aimed to assess the impact on mortality attributable to a reduction in fine particle levels in Spain in 2014 in relation to the estimated level for 2007. METHODS To estimate exposure, we constructed fine particle distribution models for Spain for 2007 (reference scenario) and 2014 (projected scenario) with a spatial resolution of 16×16km(2). In a second step, we used the concentration-response functions proposed by cohort studies carried out in Europe (European Study of Cohorts for Air Pollution Effects and Rome longitudinal cohort) and North America (American Cancer Society cohort, Harvard Six Cities study and Canadian national cohort) to calculate the number of attributable annual deaths corresponding to all causes, all non-accidental causes, ischemic heart disease and lung cancer among persons aged over 25 years (2005-2007 mortality rate data). We examined the effect of the Spanish demographic shift in our analysis using 2007 and 2012 population figures. RESULTS Our model suggested that there would be a mean overall reduction in fine particle levels of 1µg/m(3) by 2014. Taking into account 2007 population data, between 8 and 15 all-cause deaths per 100,000 population could be postponed annually by the expected reduction in fine particle levels. For specific subgroups, estimates varied from 10 to 30 deaths for all non-accidental causes, from 1 to 5 for lung cancer, and from 2 to 6 for ischemic heart disease. The expected burden of preventable mortality would be even higher in the future due to the Spanish population growth. Taking into account the population older than 30 years in 2012, the absolute mortality impact estimate would increase approximately by 18%. CONCLUSIONS Effective implementation of air quality measures in Spain, in a scenario with a short-term projection, would amount to an appreciable decline in fine particle concentrations, and this, in turn, would lead to notable health-related benefits. Recent European cohort studies strengthen the evidence of an association between long-term exposure to fine particles and health effects, and could enhance the health impact quantification in Europe. Air quality models can contribute to improved assessment of air pollution health impact estimates, particularly in study areas without air pollution monitoring data.

IL-7 Induces SAMHD1 Phosphorylation in CD4+ T Lymphocytes, Improving Early Steps of HIV-1 Life Cycle

SUMMARY HIV-1 post-integration latency in CD4+ lymphocytes is responsible for viral persistence despite treatment, but mechanisms involved in the establishment of latent viral reservoirs are not fully understood. We determined that both interleukin 2 (IL-2) and IL-7 induced SAMHD1 phosphorylation in T592, abrogating its antiviral activity. However, IL-7 caused a much more profound stimulatory effect on HIV-1 reverse transcription and integration than IL-2 that required chemokine co-stimulation. Both cytokines barely induced transcription due to low NF-κB induction, favoring the establishment of latent reservoirs. Effect of IL-7 on SAMHD1 phosphorylation was confirmed in IL-7-treated patients (ACTG 5214 study). Dasatinib—a tyrosine-kinase inhibitor—blocked SAMHD1 phosphorylation induced by IL-2 and IL-7 and restored HIV-1 restriction. We propose that γc-cytokines play a major role in the reservoir establishment not only by driving homeostatic proliferation but also by increasing susceptibility of CD4+ lymphocytes to HIV-1 infection through SAMHD1 inactivation.

Spatial analysis of childhood cancer: a case/control study.

Background Childhood cancer was the leading cause of death among children aged 1-14 years for 2012 in Spain. Leukemia has the highest incidence, followed by tumors of the central nervous system (CNS) and lymphomas (Hodgkin lymphoma, HL, and Non-Hodgkin’s lymphoma, NHL). Spatial distribution of childhood cancer cases has been under concern with the aim of identifying potential risk factors. Objective The two objectives are to study overall spatial clustering and cluster detection of cases of the three main childhood cancer causes, looking to increase etiological knowledge. Methods We ran a case-control study. The cases were children aged 0 to 14 diagnosed with leukemia, lymphomas (HL and NHL) or CNS neoplasm in five Spanish regions for the period 1996-2011. As a control group, we used a sample from the Birth Registry matching every case by year of birth, autonomous region of residence and sex with six controls. We geocoded and validated the address of the cases and controls. For our two objectives we used two different methodologies. For the first, for overall spatial clustering detection, we used the differences of K functions from the spatial point patterns perspective proposed by Diggle and Chetwynd and the second, for cluster detection, we used the spatial scan statistic proposed by Kulldorff with a level for statistical significance of 0.05. Results We had 1062 cases of leukemia, 714 cases of CNS, 92 of HL and 246 of NHL. Accordingly we had 6 times the number of controls, 6372 controls for leukemia, 4284 controls for CNS, 552 controls for HL and 1476 controls for NHL. We found variations in the estimated empirical D(s) for the different regions and cancers, including some overall spatial clustering for specific regions and distances. We did not find statistically significant clusters. Conclusions The variations in the estimated empirical D(s) for the different regions and cancers could be partially explained by the differences in the spatial distribution of the population; however, according to the literature, we cannot discard environmental hazards or infections agents in the etiology of these cancers.

Modelling of municipal mortality due to haematological neoplasias in Spain

Objectives: To study the geographical pattern of mortality caused by haematological tumours in Spain at the municipal level using three Bayesian models and to compare their goodness of fit. Methods: The fitted Bayesian hierarchical models were: (1) the Besag York and Molliè (BYM) model; (2) a model based on zero-inflated Poisson (ZIP) distribution, which allowed a large number of event-free areas; and (3) a mixture of distributions that enabled discontinuities (jumps in the pattern) to be modelled. The three models allow smoothed relative risk maps to be obtained for the all countries. The goodness of fit was evaluated using the deviance information criteria. Results: The three models yielded similar results. The ZIP model plotted a pattern almost identical with the BYM model. The goodness-of-fit criteria indicate that the mixture model is the one that best fits our data. Haematological tumours display a geographical pattern that could be partly explained by environmental determinants, as many of the highest-risk towns are located in heavily industrialised areas. Conclusions: The choice of one or another model has scant practical consequences. The pattern of distribution supports the hypothesis that differences in lifestyles, air/industrial pollution and migratory phenomena may determine the pattern of urban mortality due to these tumours.