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1.  Dendritic cell based vaccines for HIV infection 
Human Vaccines & Immunotherapeutics  2013;9(11):2445-2452.
Dendritic cells have a central role in HIV infection. On one hand, they are essential to induce strong HIV-specific CD4+ helper T-cell responses that are crucial to achieve a sustained and effective HIV-specific CD8+ cytotoxic T-lymphocyte able to control HIV replication. On the other hand, DCs contribute to virus dissemination and HIV itself could avoid a correct antigen presentation. As the efficacy of immune therapy and therapeutic vaccines against HIV infection has been modest in the best of cases, it has been hypothesized that ex vivo generated DC therapeutic vaccines aimed to induce effective specific HIV immune responses might overcome some of these problems. In fact, DC-based vaccine clinical trials have yielded the best results in this field. However, despite these encouraging results, functional cure has not been reached with this strategy in any patient. In this Commentary, we discuss new approaches to improve the efficacy and feasibility of this type of therapeutic vaccine.
PMCID: PMC3981855  PMID: 23912672
therapeutic vaccine; HIV; dendritic cells; functional cure; T cell responses
2.  Protease inhibitor monotherapy is associated with a higher level of monocyte activation, bacterial translocation and inflammation 
Monotherapy with protease-inhibitors (MPI) may be an alternative to cART for HIV treatment. We assessed the impact of this strategy on immune activation, bacterial translocation and inflammation.
We performed a cross-sectional study comparing patients on successful MPI (n=40) with patients on cART (n=20). Activation, senescence, exhaustion and differentiation stage in CD4+ and CD8+ T lymphocyte subsets, markers of monocyte activation, microbial translocation, inflammation, coagulation and low-level viremia were assessed.
CD4+ or CD8+ T lymphocyte subset parameters were not significantly different between both groups. Conversely, as compared with triple cART, MPI patients showed a higher proportion of activated monocytes (CD14+ CD16−CD163+ cells, p=0.031), soluble markers of monocyte activation (sCD14 p=0.004, sCD163 p=0.002), microbial translocation (lipopolysaccharide (LPS)-binding protein; LBP p=0.07), inflammation (IL-6 p=0.04) and low-level viremia (p=0.035). In a multivariate model, a higher level of CD14+ CD16−CD163+ cells and sCD14, and presence of very low-level viremia were independently associated with MPI. Monocyte activation was independently associated with markers of inflammation (IL-6, p=0.006), microbial translocation (LBP, p=0.01) and low-level viremia (p=0.01).
Patients on MPI showed a higher level of monocyte activation than patients on standard therapy. Microbial translocation and low-level viremia were associated with the high level of monocyte activation observed in patients on MPI. The long-term clinical consequences of these findings should be assessed.
PMCID: PMC4185085  PMID: 25280865
protease inhibitor; monotherapy; immune activation; very low-level viremia; microbial translocation; monocyte
3.  Programmed cell death promotes male sterility in the functional dioecious Opuntia stenopetala (Cactaceae) 
Annals of Botany  2013;112(5):789-800.
Background and Aims
The sexual separation in dioecious species has interested biologists for decades; however, the cellular mechanism leading to unisexuality has been poorly understood. In this study, the cellular changes that lead to male sterility in the functionally dioecious cactus, Opuntia stenopetala, are described.
The spatial and temporal patterns of programmed cell death (PCD) were determined in the anthers of male and female flowers using scanning electron microscopy analysis and histological observations, focusing attention on the transition from bisexual to unisexual development. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assays were used as an indicator of DNA fragmentation to corroborate PCD.
Key results
PCD was detected in anthers of both female and male flowers, but their patterns differed in time and space. Functionally male individuals developed viable pollen, and normal development involved PCD on each layer of the anther wall, which occurred progressively from the inner (tapetum) to the outer layer (epidermis). Conversely, functional female individuals aborted anthers by premature and displaced PCD. In anthers of female flowers, the first signs of PCD, such as a nucleus with irregular shape, fragmented and condensed chromatin, high vacuolization and condensed cytoplasm, occurred at the microspore mother cell stage. Later these features were observed simultaneously in all anther wall layers, connective tissue and filament. Neither pollen formation nor anther dehiscence was detected in female flowers of O. stenopetala due to total anther disruption.
Temporal and spatial changes in the patterns of PCD are responsible for male sterility of female flowers in O. stenopetala. Male fertility requires the co-ordination of different events, which, when altered, can lead to male sterility and to functionally unisexual individuals. PCD could be a widespread mechanism in the determination of functionally dioecious species.
PMCID: PMC3747795  PMID: 23877075
Opuntia stenopetala; programmed cell death; PCD; male sterility; dioecy; unisexuality; TUNEL; Cactaceae; reproductive systems; microsporogenesis
4.  Rate and Predictors of Non-AIDS Events in a Cohort of HIV-Infected Patients with a CD4 T Cell Count Above 500 Cells/mm3 
AIDS Research and Human Retroviruses  2013;29(8):1161-1167.
The reduction of risk of non-AIDS events after combined antiretroviral therapy (cART) initiation and the crude incidence rate (CIR) of these events in patients who control the viral load without cART (controllers) in a cohort of 574 antiretroviral-naive patients with a baseline CD4 T cell count above 500 cells/mm3 were assessed. Non-AIDS severe events were defined as a first admission to the hospital due to non-AIDS-defining malignancies, cardiovascular, neuropsychiatric, liver-related, or end-stage renal disease events. Potential determinants of non-AIDS/death events were studied using Cox regression models. Eighty-five non-AIDS/death events occurred during 6,062 persons-years of follow-up (PYFU) with a CIR of 1.4 per 100 PYFU. Factors associated with non-AIDS/death event were age (HR 3.4; 95% CI: 1.6–6.9), nadir CD4 below 350 cells/mm3 (HR 2.5; 95% CI: 1.4–4.6), and a last determination of viral load above the median (HR 1.9; 95% CI: 1.0–3.3). The CIR of non-AIDS/death events was 2.1 and 1.8 per 100 PYFU before and after cART in patients who started cART (n=446). A reduction of CIR of non-AIDS events after cART initiation was observed only in patients with a nadir of CD4 above 350 cells/mm3 (2.5 vs. 0.6 per 100 PYFU, p=0.004, and remained stable after cART in patients with a median nadir of CD4 below 350 cells/mm3. CIR was similar in elite, viremic, and noncontrollers (1.1, 1.0, and 1.5 per 100 PYFU, respectively, p=0.25). Reduction of CIR of non-AIDS events after cART initiation depends on nadir CD4 T cell count. Most of the controllers patients had a CIR similar to noncontrollers. These data support the early initiation of cART in HIV-infected patients.
PMCID: PMC3715811  PMID: 23530980
5.  A novel motif in the NaTrxh N-terminus promotes its secretion, whereas the C-terminus participates in its interaction with S-RNase in vitro 
BMC Plant Biology  2014;14:147.
NaTrxh, a thioredoxin type h, shows differential expression between self-incompatible and self-compatible Nicotiana species. NaTrxh interacts in vitro with S-RNase and co-localizes with it in the extracellular matrix of the stylar transmitting tissue. NaTrxh contains N- and C-terminal extensions, a feature shared by thioredoxin h proteins of subgroup 2. To ascertain the function of these extensions in NaTrxh secretion and protein-protein interaction, we performed a deletion analysis on NaTrxh and fused the resulting variants to GFP.
We found an internal domain in the N-terminal extension, called Nβ, that is essential for NaTrxh secretion but is not hydrophobic, a canonical feature of a signal peptide. The lack of hydrophobicity as well as the location of the secretion signal within the NaTrxh primary structure, suggest an unorthodox secretion route for NaTrxh. Notably, we found that the fusion protein NaTrxh-GFP(KDEL) is retained in the endoplasmic reticulum and that treatment of NaTrxh-GFP-expressing cells with Brefeldin A leads to its retention in the Golgi, which indicates that NaTrxh uses, to some extent, the endoplasmic reticulum and Golgi apparatus for secretion. Furthermore, we found that Nβ contributes to NaTrxh tertiary structure stabilization and that the C-terminus functions in the protein-protein interaction with S-RNase.
The extensions contained in NaTrxh sequence have specific functions on the protein. While the C-terminus directly participates in protein-protein interaction, particularly on its interaction with S-RNase in vitro; the N-terminal extension contains two structurally different motifs: Nα and Nβ. Nβ, the inner domain (Ala-17 to Pro-27), is essential and enough to target NaTrxh towards the apoplast. Interestingly, when it was fused to GFP, this protein was also found in the cell wall of the onion cells. Although the biochemical features of the N-terminus suggested a non-classical secretion pathway, our results provided evidence that NaTrxh at least uses the endoplasmic reticulum, Golgi apparatus and also vesicles for secretion. Therefore, the Nβ domain sequence is suggested to be a novel signal peptide.
PMCID: PMC4065587  PMID: 24886483
Thioredoxin; Secretion; Self-incompatibility; Nicotiana alata; Gametophytic; S-RNase
6.  Influence of Episodes of Intermittent Viremia (“Blips”) on Immune Responses and Viral Load Rebound in Successfully Treated HIV-Infected Patients 
Presenting episodes of intermittent viremia (EIV) under combination antiretroviral therapy (cART) is frequent, but there exists some controversy about their consequences. They have been described as inducing changes in immune responses potentially associated with a better control of HIV infection. Conversely, it has been suggested that EIV increases the risk of virological failure. A retrospective analysis of a prospective, randomized double-blinded placebo-controlled study was performed. Twenty-six successfully treated HIV-infected adults were randomized to receive an immunization schedule or placebo, and after 1 year of follow-up cART was discontinued. The influence of EIV on T cell subsets, HIV-1-specific T cell immune responses, and viral load rebound, and the risk of developing genotypic mutations were evaluated, taking into account the immunization received. Patients with EIV above 200 copies/ml under cART had a lower proportion of CD4+ and CD4+CD45RA+RO− T cells, a higher proportion of CD8+ and CD4+CD38+HLADR+ T cells, and higher HIV-specific CD8+ T cell responses compared to persistently undetectable patients. After cART interruption, patients with EIV presented a significantly higher viral rebound (p=0.007), associated with greater increases in HIV-specific lymphoproliferative responses and T cell populations with activation markers. When patients with EIV between 20 and 200 copies/ml were included, most of the differences disappeared. Patients who present EIV above 200 copies/ml showed a lower CD4+ T cell count and higher activation markers under cART. After treatment interruption, they showed greater specific immune responses against HIV, which did not prevent a higher virological rebound. EIV between 20 and 200 copies/ml did not have this deleterious effect.
PMCID: PMC3537296  PMID: 23121249
7.  Use of RT-Defective HIV Virions: New Tool to Evaluate Specific Response in Chronic Asymptomatic HIV-Infected Individuals 
PLoS ONE  2013;8(3):e58927.
Generation of new reagents that can be used to screen or monitor HIV-1-specific responses constituted an interesting field in the development of HIV vaccines to improve their efficacy.
We have evaluated the specific T cell response against different types of NL4-3 virions (including NL4-3 aldrithiol-2 treated, NL4-3/ΔRT and R5 envelopes: NL4-3/ΔRT/ΔEnv[AC10] and NL4-3/ΔRT/ΔEnv[Bal]) and against pools of overlapping peptides (15 mer) encompassing the HIV-1 Gag and Nef regions. Cryopreserved PBMC from a subset of 69 chronic asymptomatic HIV positive individuals have been employed using different techniques including IFN-γ ELISPOT assay, surface activation markers and intracellular cytokine staining (ICS) by flow cytometry.
The differential response obtained against NL4-3 aldrithiol-2 treated and NL4-3/ΔRT virions (25% vs 55%, respectively) allow us to divide the population in three groups: “full-responders” (positive response against both viral particles), “partial-responders” (positive response only against NL4-3/ΔRT virions) and “non-responders” (negative responses). There was no difference between X4 and R5 envelopes. The magnitude of the total responses was higher against NL4-3/ΔRT and was positively correlated with gender and inverse correlated with viral load. On the contrary CD4+ T cell count was not associated with this response. In any case responses to the viruses tended to be lower in magnitude than those detected by the overlapping peptides tested. Finally we have found an increased frequency of HLA-B27 allele (23% vs 9%) and a significant reduction in some activation markers (CD69 and CD38) on T cells surface in responders vs non-responders individuals.
In summary these virions could be considered as alternative and useful reagents for screening HIV-1-specific T cell responses in HIV exposed uninfected people, HIV infected patients and to assess immunogenicity of new prototypes both in vitro and in vaccine trials, by a feasible, simply, effective and low cost assay.
PMCID: PMC3597525  PMID: 23516578
8.  Effects on Innate Immunity of a Therapeutic Dendritic Cell-Based Vaccine for HIV-1 Infection 
Viral Immunology  2012;25(1):37-44.
Changes in natural killer (NK) cells according to their phenotype and expression of certain regulatory receptors were analyzed in a double-blind, controlled study of antiretroviral therapy (ART)-untreated HIV-seropositive patients, who had been vaccinated with monocyte-derived dendritic cells pulsed with inactivated HIV-1 autologous virus. This work extends other recently published studies of the same group of HIV-1+ vaccinated patients, which demonstrated that the viral load significantly decreases and correlates inversely with an increase in HIV-specific T-cell responses in vaccinated patients, but not in controls who received placebo. Our results indicate that this vaccine raises the level of the NK CD56neg cell subpopulation, while levels of the NK CD56dim and NK CD56bright cells expressing the inhibitory receptor CD85j/ILT-2 fell in vaccinated patients. Taken together, these results suggest that this vaccine might enhance innate immunity by amplifying the inflammatory and cytolytic capacity.
PMCID: PMC3271373  PMID: 22233253
9.  Adenosine Deaminase Enhances the Immunogenicity of Human Dendritic Cells from Healthy and HIV-Infected Individuals 
PLoS ONE  2012;7(12):e51287.
ADA is an enzyme implicated in purine metabolism, and is critical to ensure normal immune function. Its congenital deficit leads to severe combined immunodeficiency (SCID). ADA binding to adenosine receptors on dendritic cell surface enables T-cell costimulation through CD26 crosslinking, which enhances T-cell activation and proliferation. Despite a large body of work on the actions of the ecto-enzyme ADA on T-cell activation, questions arise on whether ADA can also modulate dendritic cell maturation. To this end we investigated the effects of ADA on human monocyte derived dendritic cell biology. Our results show that both the enzymatic and non-enzymatic activities of ADA are implicated in the enhancement of CD80, CD83, CD86, CD40 and CCR7 expression on immature dendritic cells from healthy and HIV-infected individuals. These ADA-mediated increases in CD83 and costimulatory molecule expression is concomitant to an enhanced IL-12, IL-6, TNF-α, CXCL8(IL-8), CCL3(MIP1-α), CCL4(MIP-1β) and CCL5(RANTES) cytokine/chemokine secretion both in healthy and HIV-infected individuals and to an altered apoptotic death in cells from HIV-infected individuals. Consistently, ADA-mediated actions on iDCs are able to enhance allogeneic CD4 and CD8-T-cell proliferation, globally yielding increased iDC immunogenicity. Taken together, these findings suggest that ADA would promote enhanced and correctly polarized T-cell responses in strategies targeting asymptomatic HIV-infected individuals.
PMCID: PMC3519778  PMID: 23240012
10.  Generation and Characterization of a Defective HIV-1 Virus as an Immunogen for a Therapeutic Vaccine 
PLoS ONE  2012;7(11):e48848.
The generation of new immunogens able to elicit strong specific immune responses remains a major challenge in the attempts to obtain a prophylactic or therapeutic vaccine against HIV/AIDS. We designed and constructed a defective recombinant virus based on the HIV-1 genome generating infective but non-replicative virions able to elicit broad and strong cellular immune responses in HIV-1 seropositive individuals.
Viral particles were generated through transient transfection in producer cells (293-T) of a full length HIV-1 DNA carrying a deletion of 892 base pairs (bp) in the pol gene encompassing the sequence that codes for the reverse transcriptase (NL4-3/ΔRT clone). The viral particles generated were able to enter target cells, but due to the absence of reverse transcriptase no replication was detected. The immunogenic capacity of these particles was assessed by ELISPOT to determine γ-interferon production in a cohort of 69 chronic asymptomatic HIV-1 seropositive individuals. Surprisingly, defective particles produced from NL4-3/ΔRT triggered stronger cellular responses than wild-type HIV-1 viruses inactivated with Aldrithiol-2 (AT-2) and in a larger proportion of individuals (55% versus 23% seropositive individuals tested). Electron microscopy showed that NL4-3/ΔRT virions display immature morphology. Interestingly, wild-type viruses treated with Amprenavir (APV) to induce defective core maturation also induced stronger responses than the same viral particles generated in the absence of protease inhibitors.
We propose that immature HIV-1 virions generated from NL4-3/ΔRT viral clones may represent new prototypes of immunogens with a safer profile and stronger capacity to induce cellular immune responses than wild-type inactivated viral particles.
PMCID: PMC3492255  PMID: 23144996
11.  Compatibility and incompatibility in S-RNase-based systems 
Annals of Botany  2011;108(4):647-658.
S-RNase-based self-incompatibility (SI) occurs in the Solanaceae, Rosaceae and Plantaginaceae. In all three families, compatibility is controlled by a polymorphic S-locus encoding at least two genes. S-RNases determine the specificity of pollen rejection in the pistil, and S-locus F-box proteins fulfill this function in pollen. S-RNases are thought to function as S-specific cytotoxins as well as recognition proteins. Thus, incompatibility results from the cytotoxic activity of S-RNase, while compatible pollen tubes evade S-RNase cytotoxicity.
The S-specificity determinants are known, but many questions remain. In this review, the genetics of SI are introduced and the characteristics of S-RNases and pollen F-box proteins are briefly described. A variety of modifier genes also required for SI are also reviewed. Mutations affecting compatibility in pollen are especially important for defining models of compatibility and incompatibility. In Solanaceae, pollen-side mutations causing breakdown in SI have been attributed to the heteroallelic pollen effect, but a mutation in Solanum chacoense may be an exception. This has been interpreted to mean that pollen incompatibility is the default condition unless the S-locus F-box protein confers resistance to S-RNase. In Prunus, however, S-locus F-box protein gene mutations clearly cause compatibility.
Two alternative mechanisms have been proposed to explain compatibility and incompatibility: compatibility is explained either as a result of either degradation of non-self S-RNase or by its compartmentalization so that it does not have access to the pollen tube cytoplasm. These models are not necessarily mutually exclusive, but each makes different predictions about whether pollen compatibility or incompatibility is the default. As more factors required for SI are identified and characterized, it will be possible to determine the role each process plays in S-RNase-based SI.
PMCID: PMC3170157  PMID: 21803740
S-RNase; self-incompatibility; Solanaceae; Rosaceae; Nicotiana; Prunus
12.  The HIV/AIDS Vaccine Candidate MVA-B Administered as a Single Immunogen in Humans Triggers Robust, Polyfunctional, and Selective Effector Memory T Cell Responses to HIV-1 Antigens ▿ ‡  
Journal of Virology  2011;85(21):11468-11478.
Attenuated poxvirus vectors expressing human immunodeficiency virus type 1 (HIV-1) antigens are considered promising HIV/AIDS vaccine candidates. Here, we describe the nature of T cell immune responses induced in healthy volunteers participating in a phase I clinical trial in Spain after intramuscular administration of three doses of the recombinant MVA-B-expressing monomeric gp120 and the fused Gag-Pol-Nef (GPN) polyprotein of clade B. The majority (92.3%) of the volunteers immunized had a positive specific T cell response at any time postvaccination as detected by gamma interferon (IFN-γ) intracellular cytokine staining (ICS) assay. The CD4+ T cell responses were predominantly Env directed, whereas the CD8+ T cell responses were similarly distributed against Env, Gag, and GPN. The proportion of responders after two doses of MVA-B was similar to that obtained after the third dose of MVA-B vaccination, and the responses were sustained (84.6% at week 48). Vaccine-induced CD8+ T cells to HIV-1 antigens after 1 year were polyfunctional and distributed mainly within the effector memory (TEM) and terminally differentiated effector memory (TEMRA) T cell populations. Antivector T cell responses were mostly induced by CD8+ T cells, highly polyfunctional, and of TEMRA phenotype. These findings demonstrate that the poxvirus MVA-B vaccine candidate given alone is highly immunogenic, inducing broad, polyfunctional, and long-lasting CD4 and CD8 T cell responses to HIV-1 antigens, with preference for TEM. Thus, on the basis of the immune profile of MVA-B in humans, this immunogen can be considered a promising HIV/AIDS vaccine candidate.
PMCID: PMC3194965  PMID: 21865377
13.  Lymphoid Tissue Collagen Deposition in HIV-Infected Patients Correlates With the Imbalance Between Matrix Metalloproteinases and Their Inhibitors 
The Journal of Infectious Diseases  2011;203(6):810-813.
We studied the lymphoid tissue biopsies of 20 patients with chronic human immunodeficiency virus (HIV) infection by analyzing collagen deposition, CD4+ cell number, and gene expression of metalloproteinases (MMPs; MMP-2, MMP-9) and tissue inhibitors of MMPs (TIMPs; TIMP-1, TIMP-2). HIV-infected patients had significantly increased collagen deposition (P = .001), fewer CD4+ T cells (P = .05), and decreased MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios (P = .01), compared with HIV-negative control patients. Moreover, we found a significant negative correlation between collagen deposition and the MMP-9/TIMP-1 ratio (ρ = −0.50; P = .047). To our knowledge, this is the first time that MMP/TIMP imbalance has been correlated with lymphoid tissue collagen deposition and incomplete immune recovery in HIV-infected patients, even after long-term antiretroviral treatment.
PMCID: PMC3071137  PMID: 21343147
14.  A Therapeutic Dendritic Cell-Based Vaccine for HIV-1 Infection 
The Journal of Infectious Diseases  2011;203(4):473-478.
A double-blinded, controlled study of vaccination of untreated patients with chronic human immunodeficiency virus type 1 (HIV-1) infection with 3 doses of autologous monocyte-derived dendritic cells (MD-DCs) pulsed with heat inactivated autologous HIV-1 was performed. Therapeutic vaccinations were feasible, safe, and well tolerated. At week 24 after first vaccination (primary end point), a modest significant decrease in plasma viral load was observed in vaccine recipients, compared with control subjects (P = .03). In addition, the change in plasma viral load after vaccination tended to be inversely associated with the increase in HIV-specific T cell responses in vaccinated patients but tended to be directly correlated with HIV-specific T cell responses in control subjects.
Clinical NCT00402142
PMCID: PMC3071229  PMID: 21233310
15.  The human genome: a multifractal analysis 
BMC Genomics  2011;12:506.
Several studies have shown that genomes can be studied via a multifractal formalism. Recently, we used a multifractal approach to study the genetic information content of the Caenorhabditis elegans genome. Here we investigate the possibility that the human genome shows a similar behavior to that observed in the nematode.
We report here multifractality in the human genome sequence. This behavior correlates strongly on the presence of Alu elements and to a lesser extent on CpG islands and (G+C) content. In contrast, no or low relationship was found for LINE, MIR, MER, LTRs elements and DNA regions poor in genetic information. Gene function, cluster of orthologous genes, metabolic pathways, and exons tended to increase their frequencies with ranges of multifractality and large gene families were located in genomic regions with varied multifractality. Additionally, a multifractal map and classification for human chromosomes are proposed.
Based on these findings, we propose a descriptive non-linear model for the structure of the human genome, with some biological implications. This model reveals 1) a multifractal regionalization where many regions coexist that are far from equilibrium and 2) this non-linear organization has significant molecular and medical genetic implications for understanding the role of Alu elements in genome stability and structure of the human genome. Given the role of Alu sequences in gene regulation, genetic diseases, human genetic diversity, adaptation and phylogenetic analyses, these quantifications are especially useful.
PMCID: PMC3277318  PMID: 21999602
16.  Dendritic Cells Exposed to MVA-Based HIV-1 Vaccine Induce Highly Functional HIV-1-Specific CD8+ T Cell Responses in HIV-1-Infected Individuals 
PLoS ONE  2011;6(5):e19644.
Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8+ T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8+ T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4+ T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.
PMCID: PMC3097254  PMID: 21625608
17.  Selective Induction of Host Genes by MVA-B, a Candidate Vaccine against HIV/AIDS▿ †  
Journal of Virology  2010;84(16):8141-8152.
The aim of this study was to define the effects on antigen-presenting cells of the expression of HIV antigens from an attenuated poxvirus vector. We have analyzed the transcriptional changes in gene expression following infection of human immature monocyte-derived dendritic cells (DC) with recombinant modified vaccinia virus Ankara (MVA) expressing the genes encoding the gp120 and Gag-Pol-Nef antigens of HIV type 1 clade B (referred to as MVA-B) versus parental MVA infection. Using microarray technology and real-time reverse transcription-PCR, we demonstrated that the HIV proteins induced the expression of cytokines, cytokine receptors, chemokines, chemokine receptors, and molecules involved in antigen uptake and processing, including major histocompatibility complex (MHC) genes. Levels of mRNAs for interleukin-1, beta interferon, CCR8, and SCYA20 were higher after HIV antigen production. MVA-B infection also modulated the expression of antigen processing and presentation genes: the gene for MICA was upregulated, whereas those for HLA-DRA and HSPA5 were downregulated. Indeed, the increased expression of the gene for MICA, a glycoprotein related to major histocompatibility complex class I molecules, was shown to enhance the interaction between MVA-B-infected target cells and cytotoxic lymphocytes. The expression profiles of the genes for protein kinases such as JAK1 and IRAK2 were activated after HIV antigen expression. Several genes included in the JAK-STAT and mitogen-activated protein kinase signaling pathways were regulated after HIV antigen expression. Our findings provide the first gene signatures in DC of a candidate MVA-B vaccine expressing four HIV antigens and identified the biological roles of some of the regulatory genes, like that for MICA, which will help in the design of more effective MVA-derived vaccines.
PMCID: PMC2916545  PMID: 20534857
18.  A New Multidisciplinary Home Care Telemedicine System to Monitor Stable Chronic Human Immunodeficiency Virus-Infected Patients: A Randomized Study 
PLoS ONE  2011;6(1):e14515.
Antiretroviral therapy has changed the natural history of human immunodeficiency virus (HIV) infection in developed countries, where it has become a chronic disease. This clinical scenario requires a new approach to simplify follow-up appointments and facilitate access to healthcare professionals.
We developed a new internet-based home care model covering the entire management of chronic HIV-infected patients. This was called Virtual Hospital. We report the results of a prospective randomised study performed over two years, comparing standard care received by HIV-infected patients with Virtual Hospital care. HIV-infected patients with access to a computer and broadband were randomised to be monitored either through Virtual Hospital (Arm I) or through standard care at the day hospital (Arm II). After one year of follow up, patients switched their care to the other arm. Virtual Hospital offered four main services: Virtual Consultations, Telepharmacy, Virtual Library and Virtual Community. A technical and clinical evaluation of Virtual Hospital was carried out.
Of the 83 randomised patients, 42 were monitored during the first year through Virtual Hospital (Arm I) and 41 through standard care (Arm II). Baseline characteristics of patients were similar in the two arms. The level of technical satisfaction with the virtual system was high: 85% of patients considered that Virtual Hospital improved their access to clinical data and they felt comfortable with the videoconference system. Neither clinical parameters [level of CD4+ T lymphocytes, proportion of patients with an undetectable level of viral load (p = 0.21) and compliance levels >90% (p = 0.58)] nor the evaluation of quality of life or psychological questionnaires changed significantly between the two types of care.
Virtual Hospital is a feasible and safe tool for the multidisciplinary home care of chronic HIV patients. Telemedicine should be considered as an appropriate support service for the management of chronic HIV infection.
Trial Registration NCT01117675.
PMCID: PMC3024968  PMID: 21283736
19.  Effect of TNF-α genetic variants and CCR5Δ32 on the vulnerability to HIV-1 infection and disease progression in Caucasian Spaniards 
BMC Medical Genetics  2010;11:63.
Tumor necrosis factor alpha (TNF-α) is thought to be involved in the various immunogenetic events that influence HIV-1 infection.
We aimed to determine whether carriage of the TNF-α-238G>A, -308G>A and -863 C>A gene promoter single nucleotide polymorphisms (SNP) and the CCR5Δ32 variant allele influence the risk of HIV-1 infection and disease progression in Caucasian Spaniards. The study group consisted of 423 individuals. Of these, 239 were uninfected (36 heavily exposed but uninfected [EU] and 203 healthy controls [HC]) and 184 were HIV-1-infected (109 typical progressors [TP] and 75 long-term nonprogressors [LTNP] of over 16 years' duration). TNF-α SNP and the CCR5Δ32 allele were assessed using PCR-RFLP and automatic sequencing analysis methods on white blood cell DNA. Genotype and allele frequencies were compared using the χ 2 test and the Fisher exact test. Haplotypes were compared by logistic regression analysis.
The distribution of TNF-α-238G>A, -308G>A and -863 C>A genetic variants was non-significantly different in HIV-1-infected patients compared with uninfected individuals: -238G>A, p = 0.7 and p = 0.3; -308G>A, p = 0.05 and p = 0.07; -863 C>A, p = 0.7 and p = 0.4, for genotype and allele comparisons, respectively. Haplotype analyses, however, indicated that carriers of the haplotype H3 were significantly more common among uninfected subjects (p = 0.04). Among the infected patients, the distribution of the three TNF-α genetic variants assessed was non-significantly different between TP and LTNP: -238G>A, p = 0.35 and p = 0.7; -308G>A, p = 0.7 and p = 0.6: -863 C>A, p = 0.2 and p = 0.2, for genotype and allele comparisons, respectively. Haplotype analyses also indicated non-significant associations. Subanalyses in the LTNP subset indicated that the TNF-α-238A variant allele was significantly overrepresented in patients who spontaneously controlled plasma viremia compared with those who had a detectable plasma viral load (genotype comparisons, p = 0.02; allele comparisons, p = 0.03). The CCR5Δ32 distribution was non-significantly different in HIV-1-infected patients with respect to the uninfected population (p = 0.15 and p = 0.2 for genotype and allele comparisons, respectively) and in LTNP vs TP (p = 0.4 and p = 0.5 for genotype and allele comparisons, respectively).
In our cohort of Caucasian Spaniards, TNF-α genetic variants could be involved in the vulnerability to HIV-1 infection. TNF-α genetic variants were unrelated to disease progression in infected subjects. The -238G>A SNP may modulate the control of viremia in LTNP. Carriage of the CCR5Δ32 variant allele had no effect on the risk of infection and disease progression.
PMCID: PMC2877017  PMID: 20420684
20.  Increased α-Defensins 1-3 Production by Dendritic Cells in HIV-Infected Individuals Is Associated with Slower Disease Progression 
PLoS ONE  2010;5(2):e9436.
Defensins are natural endogenous antimicrobial peptides with potent anti-HIV activity and immuno-modulatory effects. We recently demonstrated that immature dendritic cells (DC) produce α-defensins1-3 and that α-defensins1-3 modulate DC generation and maturation. Since DC-HIV interaction plays a critical role during the first steps of HIV infection, we investigated the possible impact of α-defensins1-3 production by DC on disease progression.
Methodology/Principal Findings
Monocyte-derived DC (MDDC) were analyzed comparatively in healthy controls (HC) and HIV-infected patients, including untreated “elite” and “viremic” controllers, untreated viremic non-controllers and antiretroviral-treated patients. We found that production of α-defensins1-3 was significantly increased in MDDC from HIV-infected patients versus HC, and this increase was mainly due to that observed in controllers, while in non-controllers the increase was not statistically significant (controllers vs. HC, p<0.005; controllers vs. non-controllers p<0.05). Secreted α-defensins1-3 by immature MDDC positively correlated with CD4 T cell counts in controllers, but not in non-controllers. Moreover, independently of their clinical classification, HIV-infected patients with higher α-defensins1-3 secretion by immature MDDC showed slower disease progression, measured as no decrease in the number of CD4+ T-cells below 350 cell/mm3, lower increase of plasma viral load and no initiation of treatment over time. Plasma alpha-defensins1-3 levels lacked any relationship with immunologic and virologic parameters.
High production of α-defensins1-3 by immature DCs appears as a host protective factor against progression of HIV-1infection, suggesting potential diagnostic, therapeutic and preventive implications. This protective effect may arise from the activity of α-defensins1-3 to damage the virions prior and/or after their internalization by immature DC, and hence favoring a more efficient viral processing and presentation to HIV-specific CD4+ T cells, without or with a minor rate of transmission of infectious HIV-1 virions.
PMCID: PMC2828484  PMID: 20195543
21.  Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue 
Journal of Experimental Botany  2008;59(11):3187-3201.
After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen–stigma interactions that regulate pollen tube growth in Nicotiana.
PMCID: PMC2504342  PMID: 18689443
Kunitz proteinase inhibitor homologue; Nicotiana; pollen–pistil interactions; pollination
22.  Monocytes from Cystic Fibrosis Patients Are Locked in an LPS Tolerance State: Down-Regulation of TREM-1 as Putative Underlying Mechanism 
PLoS ONE  2008;3(7):e2667.
Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene that codes for the chloride channel, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). CF patients are frequently colonized by several pathogens, but the mechanisms that allow colonization in spite of apparently functional immune systems are incompletely understood. In this paper we show that blood peripheral monocytes isolated from CF patients are found in an endotoxin tolerance state, yet this is not due to a deficient TLR activation. On the other hand, levels of the amplifier of inflammatory responses, TREM-1 (Triggering Receptor Expressed on Myeloid cells), are notably down-regulated in monocytes from patients, in comparison to those extracted from healthy volunteers. Furthermore, the soluble form of TREM-1 (sTREM-1) was not detected in the sera of patients. Additionally, and in strict contrast to patients who suffer from Chronic Obstructive Pulmonary Disease (COPD), CF monocytes challenged ex vivo with LPS neither up-regulated membrane-anchored TREM-1 nor sTREM-1. Finally, similar levels of PGE2 expression and p65 translocation into the nucleus were found in both patients and healthy volunteers, thus suggesting that TREM-1 regulation is neither controlled by PGE2 levels nor by p65 activation in this case. However, PU.1 translocation into the nucleus was significantly higher in CF monocytes than in controls, suggesting a role for this transcription factor in the control of TREM-1 expression. We conclude that down-regulation of TREM-1 expression in cystic fibrosis patients is at least partly responsible for the endotoxin tolerance state in which their monocytes are locked.
PMCID: PMC2442190  PMID: 18628981
23.  Inflammatory Responses in Blood Samples of Human Immunodeficiency Virus-Infected Patients with Pulmonary Infections 
We analyzed the characteristics of the inflammatory response occurring in blood during pulmonary infections in human immunodeficiency virus (HIV)-infected patients. A prospective study of consecutive hospital admissions of HIV-infected patients with new-onset radiologic pulmonary infiltrates was carried out in a tertiary university hospital from April 1998 to May 2001. Plasma cyclic AMP receptor protein (CRP), interleukin 1β (IL-1β), IL-6, IL-8, IL-10, and tumor necrosis factor alpha (TNF-α) levels were determined at the time of admission and 4, 5, and 6 days later. Patients were included in a protocol addressed to study etiology and outcome of disease. A total of 249 episodes of infection were included, with the main diagnoses being bacterial pneumonia (BP) (118 episodes), Pneumocystis carinii pneumonia (PCP) (41 episodes), and mycobacteriosis (36 episodes). For these three patient groups, at the time of admission the median CRP and cytokine levels were as follows: CRP, 10.2, 3.8 and 5 mg/dl, respectively (P = 0.0001); IL-8, 19, 3, and 2.9 pg/ml (P = 0.045); and TNF-α, 46.4, 44, and 75 pg/ml, respectively (P = 0.029). There were no significant differences in levels of IL-1β, IL-6, or IL-10 among the patient groups. A total of 23 patients died. At the time of admission, HIV-infected patients with BP had higher plasma CRP and IL-8 levels than did PCP and mycobacteriosis patients. TNF-α levels were higher in patients with mycobacteriosis. An elevated IL-8 level (>61 pg/ml) at the time of admission was an independent factor associated with higher mortality (odds ratio, 12; 95% confidence interval, 1.2 to 235.5).
PMCID: PMC404570  PMID: 15138189

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