Paediatric HIV infection in the 'omics era: defining transcriptional signatures of viral control and vaccine responses

Modern technologies and their increased accessibility have shifted 'benchtop' medical research to the larger dimension of 'omics. The huge amount of data derived from gene expression and sequencing experiments has propelled physicians, basic scientists and bioinformaticians towards a common goal to transform 'big data' into predictive constructs that are readily available and will offer clinical utility. Although most of the studies available in the literature have been performed on healthy subjects and in peripheral blood mononuclear cells (PBMC), which are a heterogenous and extremely variable pool of cells, scientists are now trying to address mechanistic questions in purified cell subsets in pathological conditions. In the field of HIV, few attempts have been made to comprehensively evaluate gene-expression profiles of infected patients with different disease status. With the view of discovering a path towards remission or viral eradication, perinatally HIV-infected children represent a unique model. In fact the well-defined time of infection and the resulting opportunity to start early treatment, thereby generating a smaller size of viral reservoir and a more intact immune system, allow for investigation of therapeutic strategies to defeat the virus. In this scenario, 'transcriptomic' or gene expression technologies and supporting bioinformatics applications need to be strategically integrated to provide novel information about immune correlates of virus control following treatment interruption. Here we review modern techniques for gene expression analysis and discuss the best transcriptomic strategies applicable to the field of functional cure in paediatric HIV infection

Cyclic and Acyclic Defensins Inhibit Human Immunodeficiency Virus Type-1 Replication by Different Mechanisms

Defensins are antimicrobial peptides expressed by plants and animals. In mammals there are three subfamilies of defensins, distinguished by structural features: α, β and θ. Alpha and β-defensins are linear peptides with broad anti-microbial activity that are expressed by many mammals including humans. In contrast, θ-defensins are cyclic anti-microbial peptides made by several non-human primates but not humans. All three defensin types have anti-HIV-1 activity, but their mechanisms of action differ. We studied the anti-HIV-1 activity of one defensin from each group, HNP-1 (α), HBD-2 (β) and RTD-1 (θ). We examined how each defensin affected HIV-1 infection and demonstrated that the cyclic defensin RTD-1 inhibited HIV-1 entry, while acyclic HNP-1 and HBD-2 inhibited HIV-1 replication even when added 12 hours post-infection and blocked viral replication after HIV-1 cDNA formation. We further found that all three defensins downmodulated CXCR4. Moreover, RTD-1 inactivated X4 HIV-1, while HNP-1 and HBD-2 inactivated both X4 and R5 HIV-1. The data presented here show that acyclic and cyclic defensins block HIV-1 replication by shared and diverse mechanisms. Moreover, we found that HNP-1 and RTD-1 directly inhibited firefly luciferase enzymatic activity, which may affect the interpretation of previously published data

Chapter Sixteen - Natural killer cytolytic activity

Natural killer (NK)–mediated cellular killing has been extensively studied over the past 35 years and the current “granule exocytosis” model integrates work from many investigators to outline the primary means of NK cytotoxicity. A secondary pathway is mediated through so-called death-receptors, and exhibits less relevance for host defense against pathogens in vivo but is important for the elimination of auto-reactive lymphoid cells and also homeostasis. NK cells directly execute the destruction of both autologous and allogeneic cells. NK cytotoxicity has a direct role in host defense and immunopathology in response to viral infection and malignant transformation. NK cells are heralded as the first wave of defense against a variety of pathogens in which they employ potent weaponry to kill infected and malignant cells. In addition to killing these “stressed” cells, NK are implicated in the elimination of autologous activated immune cells (T cells and macrophages) following an inflammatory response. Furthermore, NK are critical producers of cytokines, most notably the pro-inflammatory interferon-γ (IFN-γ), which has pleiotropic effects on cells that modulate the adaptive immune response. NK cells are the innate constituents of cytotoxic lymphocytes and they utilize identical cytolytic pathways as the CD8+ cytotoxic T lymphocytes (CTL), namely via granule exocytosis and death-receptor mediated killing. NK cells are considered part of the innate immune system due to their germ-line encoded, early and rapid cytolytic response to a diverse array of pathogens, although they do not kill “non-specifically”. The simple fact that NK cytotoxicity can cause cell death demands strict regulation upon the activation of NK by potential target cells

Implications of Immune Checkpoint Expression During Aging in HIV-Infected People on Antiretroviral Therapy

Immune checkpoint molecules (ICMs) regulate T cell responses. In chronic viral infections and cancer, where antigens can persistently stimulate the immune system, ICMs can serve as a barrier to effective immune responses. The role of ICMs in the setting of systemic low-grade inflammation as in aging and antiretroviral therapy (ART)-suppressed HIV infection is not known. In this study, we made use of stored samples from the FLORAH cohort of HIV-infected ART-suppressed adults (age range 19–77 years.) and age-matched HIV-uninfected controls. We measured the expression levels of ICMs: PD-1, LAG-3, TIGIT, TIM-3, and 2B4 on resting CD4 and CD8 T cells and maturation subsets. To determine how expression of these molecules can affect T cell function, we stimulated peripheral blood mononuclear cell with HIV Gag or p09/H1N1 antigen and performed intracellular cytokine staining by multiparameter flow cytometry. ICMs were expressed at higher levels in CD8 compared with CD4. PD-1 was the only molecule that remained significantly higher in HIV-infected individuals compared with controls. LAG-3 expression increased with age in CD4 and CD8 T cells. 2B4 expression on CD8 T cells was negatively associated with IL-2 production but showed no effect on CD4 T cell function. TIM-3 expression was negatively associated with IL-21 production in CD4 and CD8 T cells and also negatively correlated with flu vaccine responses in HIV-negative individuals. Taken altogether, this study demonstrates the marked variation in ICM expression in T cells among adults and sheds light on the biology of these molecules and their effects on antigen-specific T cell functions. Overall, our results point to TIM-3 as a potential biomarker for immune function in HIV + individuals on ART

Inhibition of Intracellular Bacterial Replication in Fibroblasts Is Dependent on the Perforin-Like Protein (Perforin-2) Encoded by Macrophage-Expressed Gene 1

Fibroblasts are known to eliminate intracellular bacteria, but the lethal hit of the bactericidal mechanism has not been defined. We show that primary embryonic and established fibroblasts can be induced by interferons or by intracellular bacterial infection to express a perforin-like mRNA previously described as macrophage expressed gene 1 (mpeg1). The presence and level of the perforin-like mRNA correlate with the ability of primary mouse embryonic fibroblasts (MEF) to eliminate intracellular bacteria. In addition, siRNA knock-down of the perforin-like molecule abolishes bactericidal activity and allows intracellular bacterial replication. Complementation of MEF in which the endogenous perforin-like molecule has been knocked down with an RFP-tagged version restores bactericidal activity. The perforin-like molecule has broad bactericidal specificity for pathogenic and non-pathogenic bacteria including Gram positive, Gram negative and acid fast bacteria. The perforin-like molecule renders previously lysozyme-resistant bacteria sensitive to lysis by lysozyme suggesting physical damage of the outer cell wall by the perforin-like protein. MEFs damage cell walls of intracellular bacteria by insertion, polymerization and pore-formation of the perforin-like protein, analogous to pore-formers of complement and Perforin-1 of cytolytic lymphocytes. We propose the name Perforin-2

An improved method for specific-target preamplification PCR analysis of single blastocysts useful for embryo sexing and high-throughput gene expression analysis

Gene expression analysis in preimplantation embryos has been used for answering fundamental questions related to development, prediction of pregnancy outcome, and other topics. Limited amounts of mRNA in preimplantation embryos hinders progress in studying the preimplantation embryo. Here, a method was developed involving direct synthesis and specific-target preamplification (STA) of cDNA for gene expression analysis in single blastocysts. Effective cell lysis and genomic DNA removal steps were incorporated into the method. In addition, conditions for real-time PCR of cDNA generated from these processes were improved. By using this system, reliable embryo sexing results based on expression of sex-chromosome linked genes was demonstrated. Calibration curve analysis of PCR results using the Fluidigm Biomark microfluidic platform (Fluidigm, South San Francisco, CA) was performed to evaluate 96 STA cDNA from single blastocysts. In total, 93.75% of the genes were validated. Robust amplification was detected even when STA cDNA from a single blastocyst was diluted 1,024-fold. Further analysis showed that within-assay variation increased when cycle threshold values exceeded 18. Overall, STA quantitative real-time PCR analysis was shown to be useful for analysis of gene expression of multiple specific targets in single blastocysts

Circulating inflammatory monocytes contribute to impaired influenza vaccine responses in HIV-infected participants

Antibody responses are often impaired in old age and in HIV-positive (HIV+) infection despite virologic control with antiretroviral therapy but innate immunologic determinants are not well understood. Monocytes and natural killer cells were examined for relationships to age, HIV infection and influenza vaccine responses. Virologically suppressed HIV+ (n = 139) and HIV-negative (HIV-) (n = 137) participants classified by age as young (18-39 years), middle-aged (40-59 years) and old (≥60 years) were evaluated preinfluenza and postinfluenza vaccination. Prevaccination frequencies of inflammatory monocytes were highest in old HIV+ and HIV-, with old HIV+ exhibiting higher frequency of integrin CD11b on inflammatory monocytes that was correlated with age, expression of C-C chemokine receptor-2 (CCR2) and plasma soluble tumor necrosis factor receptor-1 (sTNFR1), with inverse correlation with postvaccination influenza H1N1 antibody titers. Higher frequencies of CD11b+ inflammatory monocytes (CD11b(hi), >48.4%) compared with low frequencies of CD11b+ inflammatory monocytes (<15.8%) was associated with higher prevaccination frequencies of total and inflammatory monocytes and higher CCR2 MFI, higher plasma sTNFR1 and CXCL-10 with higher lipopolysaccharide stimulated expression of TNFα and IL-6, concomitant with lower postvaccination influenza antibody titers. In HIV+ CD11b(hi) expressers, the depletion of inflammatory monocytes from peripheral blood mononuclear cells resulted in enhanced antigen-specific CD4+ T-cell proliferation. Immature CD56(hi) natural killer cells were lower in young HIV+ compared with young HIV- participants. Perturbations of innate immunity and inflammation signified by high CD11b on inflammatory monocytes are exacerbated with aging in HIV+ and negatively impact immune function involved in Ab response to influenza vaccination