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Under non-pathological conditions, human γδ T cells represent a small fraction of CD3+ T cells in peripheral blood (1-10%). They constitute a unique subset of T lymphocytes that recognize stress ligands or non-peptide antigens through MHC-independent presentation. Major human γδ T cell subsets, Vδ1 and Vδ2, expand in response to microbial infection or malignancy, but possess distinct tissue localization, antigen recognition, and effector responses. We hypothesized that differences at the gene, phenotypic, and functional level would provide evidence that γδ T cell subpopulations belong to distinct lineages. Comparisons between each subset and the identification of the molecular determinants that underpin their differences has been hampered by experimental challenges in obtaining sufficient numbers of purified cells. By utilizing a stringent FACS-based isolation method, we compared highly purified human Vδ1 and Vδ2 cells in terms of phenotype, gene expression profile, and functional responses. We found distinct genetic and phenotypic signatures that define functional differences in γδ T cell populations. Differences in TCR components, repertoire, and responses to calcium-dependent pathways suggest that Vδ1 and Vδ2 T cells are different lineages. These findings will facilitate further investigation into the ligand specificity and unique role of Vδ1 and Vδ2 cells in early immune responses.

Despite the considerable progress that has been made in identifying cellular factors and pathways that contribute to establishment and maintenance of the latent HIV reservoir, it remains the major obstacle to eradicating this virus. Most recently, noncoding genes have been implicated in regulation of HIV expression. In this study, small RNA sequencing was used to profile expression of microRNAs (miRNAs) in a primary CD4+ T cell in vitro model of HIV latency. Previously, we have shown that protein-coding genes dysregulated in this model were enriched for the p53 signaling pathway, which was confirmed experimentally. We further found a link between p53 signaling and dysregulated long noncoding RNAs. In this study, we hypothesized that miRNAs may provide an additional level of regulation of the p53 signaling pathway during HIV latency. Twenty-six miRNAs were identified to be dysregulated in our latency model. A subset of these miRNAs was validated by real-time quantitative polymerase chain reaction. Predicted messenger RNA (mRNA) targets and cellular pathways enriched for mRNA targets were identified using several analytical methods. Our analyses showed that many protein-coding genes and pathways targeted by dysregulated miRNAs have relevance to regulation of HIV expression or establishment of HIV latency. The p53 signaling pathway was found among pathways that were targeted by dysregulated miRNAs at a greater level than expected by chance. This study provides a mechanistic insight into regulation of the p53 pathway through miRNAs that may contribute to the establishment of latency.

The problem of Static Loads issuing probability requirements for system failure cases, from the point of view of interaction of Systems and Structure, has become more relevant in recent years, since the complexity of aircraft systems has increased. In this paper, we analyze flight control system failure cases for the Clean Sky 2 FTB#2 aircraft, by performing simulations with the latest loads calculation software at Airbus Defence and Space. The results indicate that Static Loads is complementary to System Safety Assessments in terms of failure probability targets in failure cases related to aileron runaway.

The latent cellular reservoir of HIV is recognized as the major barrier to cure from HIV infection. Long non-coding RNAs (lncRNAs) are more tissue and cell type-specific than protein coding genes, and may represent targets of choice for HIV latency reversal. Using two in vitro primary T-cell models, we identified lncRNAs dysregulated in latency. PVT1 and RP11-347C18.3 were up-regulated in common between the two models, and RP11-539L10.2 was down-regulated. The major component of the latent HIV reservoir, memory CD4+ T-cells, had higher expression of these lncRNAs, compared to naïve T-cells. Guilt-by-association analysis demonstrated that lncRNAs dysregulated in latency were associated with several cellular pathways implicated in HIV latency establishment and maintenance: proteasome, spliceosome, p53 signaling, and mammalian target of rapamycin (MTOR). PVT1, RP11-347C18.3, and RP11-539L10.2 were down-regulated by latency reversing agents, suberoylanilide hydroxamic acid and Romidepsin, suggesting that modulation of lncRNAs is a possible secondary mechanism of action of these compounds. These results will facilitate prioritization of lncRNAs for evaluation as targets for HIV latency reversal. Importantly, our study provides insights into regulatory function of lncRNA during latent HIV infection.

Combination antiretroviral therapy (ART) is able to suppress HIV-1 replication to undetectable levels. However, the persistence of latent viral reservoirs allows for a rebound of viral load upon cessation of therapy. Thus, therapeutic strategies to eradicate the viral latent reservoir are critically needed. Employing a targeted RNAi screen, we identified the ubiquitin ligase BIRC2 (cIAP1), a repressor of the noncanonical NF-κB pathway, as a potent negative regulator of LTR-dependent HIV-1 transcription. Depletion of BIRC2 through treatment with small molecule antagonists known as Smac mimetics enhanced HIV-1 transcription, leading to a reversal of latency in a JLat latency model system. Critically, treatment of resting CD4+ T cells isolated from ART-suppressed patients with the histone deacetylase inhibitor (HDACi) panobinostat together with Smac mimetics resulted in synergistic activation of the latent reservoir. These data implicate Smac mimetics as useful agents for shock-and-kill strategies to eliminate the latent HIV reservoir.

La tuberculosis, cuyo agente etiológico es M. tuberculosis, es un problema de salud pública mundial. El incremento de prevalencia de cepas resistentes (multirresistentes, extensamente resistentes y totalmente resistentes) y la alta tasa de coinfección con VIH/AIDS dificultan su tratamiento y control. La emergencia y propagación de cepas resistentes disminuyen las opciones terapéuticas conocidas y su eficacia, siendo necesario disponer de nuevos fármacos. Estos deben permitir amplios intervalos de dosificación, erradicar las formas latentes y de crecimiento lento, tener actividad antimicobacteriana potente y precoz, así como carecer de interacciones con otros antibióticos y antirretrovirales. Se han descrito en los últimos años cambios en la microbiota intestinal y del esputo en enfermos con tuberculosis, así como en la microbiota pulmonar recientemente descubierta. Las comunidades bacterianas, víricas y fúngicas que habitan el cuerpo humano pueden influir en la salud del hospedador y contribuir al desarrollo de enfermedades y al aumento de resistencias. En este Trabajo de Fin de Grado se resumen los principales regímenes de tratamiento utilizados en tuberculosis sensible y resistente a antibióticos, los antibióticos en investigación y desarrollo, base para el tratamiento futuro, así como la microbiota intestinal, pulmonar y del esputo en condiciones fisiológicas y en enfermos de tuberculosis.

The search for an HIV-1 cure has been greatly hindered by the presence of a viral reservoir that persists despite antiretroviral therapy (ART). Studies of HIV-1 latency in vivo are also complicated by the low proportion of latently infected cells in HIV-1 infected individuals. A number of models of HIV-1 latency have been developed to examine the signaling pathways and viral determinants of latency and reactivation. A primary cell model of HIV-1 latency, which incorporates the generation of primary central memory CD4 T cells (TCM), full-length virus infection (HIVNL4-3) and ART to suppress virus replication, was used to investigate the establishment of HIV latency using RNA-Seq. Initially, an investigation of host and viral gene expression in the resting and activated states of this model indicated that the resting condition was reflective of a latent state. Then, a comparison of the host transcriptome between the uninfected and latently infected conditions of this model identified 826 differentially expressed genes, many of which were related to p53 signaling. Inhibition of the transcriptional activity of p53 by pifithrin-α during HIV-1 infection reduced the ability of HIV-1 to be reactivated from its latent state by an unknown mechanism. In conclusion, this model may be used to screen latency reversing agents utilized in shock and kill approaches to cure HIV, to search for cellular markers of latency, and to understand the mechanisms by which HIV-1 establishes latency.

The presence of latent HIV-1 in infected individuals represents a major barrier preventing viral eradication. For that reason, reactivation of latent viruses in the presence of antiretroviral regimens has been proposed as a therapeutic strategy to achieve remission. We screened for small molecules and identified several benzotriazole derivatives with the ability to reactivate latent HIV-1. In the presence of IL-2, benzotriazoles reactivated and reduced the latent reservoir in primary cells, and, remarkably, viral reactivation was achieved without inducing cell proliferation, T cell activation, or cytokine release. Mechanistic studies showed that benzotriazoles block SUMOylation of phosphorylated STAT5, increasing STAT5's activity and occupancy of the HIV-1 LTR. Our results identify benzotriazoles as latency reversing agents and STAT5 signaling and SUMOylation as targets for HIV-1 eradication strategies. These compounds represent a different direction in the search for "shock and kill" therapies.

Additional file 2: Figure 1. Ingenol-3,20-dibenzoate (Ingenol DB) and CD3/CD28 antibody-induced fold changes in TNFα and IFNγ among peripheral blood cell subsets. Ingenol DB and CD3/28 antibodies increased the number of TNFα and IFNγ positive CD8+ and CD56+ cells compared to media alone at 72 h post-stimulation in two donors. CD3/CD28 antibody treatment increased the number of TNFα and IFNγ positive CD4+ cells, whereas ingenol DB treatment decreased the number of cytokine positive cells in both donors, likely due to a down-regulation of CD4 upon ingenol DB treatment [30]. TNFα and IFNγ positive CD11b+ cells varied between donors.

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.

Additional file 3: Figure 2. Ingenols do not induce pro-inflammatory cytokine release in purified resting CD4+ T cell cultures. Concentrations of pro-inflammatory cytokines TNFα, IFNγ, IL-1β and IL-6 were not significantly elevated in the presence of ingenol-3,20-dibenzoate (ingenol DB) or ingenol B compared to media alone control at 72 h in purified resting CD4+ T cell cultures. Mean values and standard deviation of seven independent experiments using resting CD4+ T cells from aviremic ART-treated HIV positive participants are shown. Pro-inflammatory cytokine concentrations were significantly elevated in positive control cultures (T cell receptor stimulation via CD3 and CD28 antibodies). ** P value

HIV-specific CD8+ T-cell responses limit viral replication in untreated infection. After the initiation of antiretroviral therapy (ART), these responses decay and the infected cell population that remains is commonly considered to be invisible to T-cells. We hypothesized that HIV antigen recognition may persist in ART-treated individuals due to low-level or episodic protein expression. We posited that if persistent recognition were occurring it would be preferentially directed against the early HIV gene products Nef, Tat, and Rev as compared to late gene products, such as Gag, Pol, and Env, which have higher barriers to expression. Using a primary cell model of latency, we observed that a Nef-specific CD8+ T-cell clone exhibited low-level recognition of infected cells prior to reactivation and robust recognition shortly thereafter. A Gag-specific CD8+ T-cell clone failed to recognized infected cells under these conditions, corresponding with a lack of detectable Gag expression. We measured HIV-specific T-cell responses in 96 individuals who had been suppressed on ART for a median of 7 years, and observed a significant, direct correlation between cell-associated HIV DNA levels and magnitudes of IFN-γ-producing Nef/Tat/Rev-specific T-cell responses. This correlation was confirmed in an independent cohort (n = 18). Correlations were not detected between measures of HIV persistence and T-cell responses to other HIV antigens. The correlation with Nef/Tat/Rev-specific T-cells was attributable to Nef-specific responses, the breadth of which also correlated with HIV DNA levels. These results suggest that ongoing Nef expression in ART-treated individuals drives preferential maintenance and/or expansion of T-cells reactive to this protein, implying sensing of infected cells by the immune system. The direct correlation, however, suggests that recognition does not result in efficient elimination of infected cells. These results raise the possibility that enhancing the cytolytic activity of Nef-specific T-cells may lead to reductions in infected cell frequencies, even in the absence of therapeutic latency reversal.

Additional file 2: Figure 1. Ingenol-3,20-dibenzoate (Ingenol DB) and CD3/CD28 antibody-induced fold changes in TNFα and IFNγ among peripheral blood cell subsets. Ingenol DB and CD3/28 antibodies increased the number of TNFα and IFNγ positive CD8+ and CD56+ cells compared to media alone at 72 h post-stimulation in two donors. CD3/CD28 antibody treatment increased the number of TNFα and IFNγ positive CD4+ cells, whereas ingenol DB treatment decreased the number of cytokine positive cells in both donors, likely due to a down-regulation of CD4 upon ingenol DB treatment [30]. TNFα and IFNγ positive CD11b+ cells varied between donors.

Additional file 5: Figure 4. Ruxolitinib-induced cytokine changes in PBMCs from aviremic HIV-1 infected donors. CD25 (IL2r), IL-5, and IL-13 concentrations were significantly decreased in PBMC cultures exposed to ruxolitinib + ingenol-3,20-dibenzoate compared to ingenol-3,20-dibenzoate alone. Ruxolitinib significantly reduced these cytokine concentrations in CD3/28 antibody-treated PBMC cultures as well, along with reduction in IL-10. Ruxolitinib did not decrease pro-inflammatory cytokines induced by CD3/28 antibody stimulation to the same degree as ingenol-stimulated cells (shown in Fig. 4).

Additional file 5: Figure 4. Ruxolitinib-induced cytokine changes in PBMCs from aviremic HIV-1 infected donors. CD25 (IL2r), IL-5, and IL-13 concentrations were significantly decreased in PBMC cultures exposed to ruxolitinib + ingenol-3,20-dibenzoate compared to ingenol-3,20-dibenzoate alone. Ruxolitinib significantly reduced these cytokine concentrations in CD3/28 antibody-treated PBMC cultures as well, along with reduction in IL-10. Ruxolitinib did not decrease pro-inflammatory cytokines induced by CD3/28 antibody stimulation to the same degree as ingenol-stimulated cells (shown in Fig. 4).

Additional file 3: Figure 2. Ingenols do not induce pro-inflammatory cytokine release in purified resting CD4+ T cell cultures. Concentrations of pro-inflammatory cytokines TNFα, IFNγ, IL-1β and IL-6 were not significantly elevated in the presence of ingenol-3,20-dibenzoate (ingenol DB) or ingenol B compared to media alone control at 72 h in purified resting CD4+ T cell cultures. Mean values and standard deviation of seven independent experiments using resting CD4+ T cells from aviremic ART-treated HIV positive participants are shown. Pro-inflammatory cytokine concentrations were significantly elevated in positive control cultures (T cell receptor stimulation via CD3 and CD28 antibodies). ** P value

Additional file 4: Figure 3. PKC agonists induce pro-inflammatory cytokines in a dose-dependent manner. PBMCs from aviremic, HIV-1-infected participants (n = 4) were exposed to PKC agonists ingenol-3,20-dibenzoate, prostratin and bryostatin-1 at concentrations shown to reverse latency as well as tenfold higher and lower concentrations. Pro-inflammatory cytokine concentrations were measured in culture supernatant after 72 h in culture. Bars represent mean fold-change above cytokine concentrations from media-alone PBMC cultures (negative control) with one standard deviation shown. When comparing concentrations of these PKC agonists previously shown to reverse viral latency, ingenol-3,20-dibenzoate demonstrated the highest pro-inflammatory cytokine induction.

Additional file 4: Figure 3. PKC agonists induce pro-inflammatory cytokines in a dose-dependent manner. PBMCs from aviremic, HIV-1-infected participants (n = 4) were exposed to PKC agonists ingenol-3,20-dibenzoate, prostratin and bryostatin-1 at concentrations shown to reverse latency as well as tenfold higher and lower concentrations. Pro-inflammatory cytokine concentrations were measured in culture supernatant after 72 h in culture. Bars represent mean fold-change above cytokine concentrations from media-alone PBMC cultures (negative control) with one standard deviation shown. When comparing concentrations of these PKC agonists previously shown to reverse viral latency, ingenol-3,20-dibenzoate demonstrated the highest pro-inflammatory cytokine induction.