The Antiviral Efficacy of HIV-Specific CD8+ T-Cells to a Conserved Epitope Is Heavily Dependent on the Infecting HIV-1 Isolate

A major challenge to developing a successful HIV vaccine is the vast diversity of viral sequences, yet it is generally assumed that an epitope conserved between different strains will be recognised by responding T-cells. We examined whether an invariant HLA-B8 restricted Nef90–97 epitope FL8 shared between five high titre viruses and eight recombinant vaccinia viruses expressing Nef from different viral isolates (clades A–H) could activate antiviral activity in FL8-specific cytotoxic T-lymphocytes (CTL). Surprisingly, despite epitope conservation, we found that CTL antiviral efficacy is dependent on the infecting viral isolate. Only 23% of Nef proteins, expressed by HIV-1 isolates or as recombinant vaccinia-Nef, were optimally recognised by CTL. Recognition of the HIV-1 isolates by CTL was independent of clade-grouping but correlated with virus-specific polymorphisms in the epitope flanking region, which altered immunoproteasomal cleavage resulting in enhanced or impaired epitope generation. The finding that the majority of virus isolates failed to present this conserved epitope highlights the importance of viral variance in CTL epitope flanking regions on the efficiency of antigen processing, which has been considerably underestimated previously. This has important implications for future vaccine design strategies since efficient presentation of conserved viral epitopes is necessary to promote enhanced anti-viral immune responses

Novel ketone diet enhances physical and cognitive performance.

Ketone bodies are the most energy-efficient fuel and yield more ATP per mole of substrate than pyruvate and increase the free energy released from ATP hydrolysis. Elevation of circulating ketones via high-fat, low-carbohydrate diets has been used for the treatment of drug-refractory epilepsy and for neurodegenerative diseases, such as Parkinson's disease. Ketones may also be beneficial for muscle and brain in times of stress, such as endurance exercise. The challenge has been to raise circulating ketone levels by using a palatable diet without altering lipid levels. We found that blood ketone levels can be increased and cholesterol and triglycerides decreased by feeding rats a novel ketone ester diet: chow that is supplemented with (R)-3-hydroxybutyl (R)-3-hydroxybutyrate as 30% of calories. For 5 d, rats on the ketone diet ran 32% further on a treadmill than did control rats that ate an isocaloric diet that was supplemented with either corn starch or palm oil (P < 0.05). Ketone-fed rats completed an 8-arm radial maze test 38% faster than did those on the other diets, making more correct decisions before making a mistake (P < 0.05). Isolated, perfused hearts from rats that were fed the ketone diet had greater free energy available from ATP hydrolysis during increased work than did hearts from rats on the other diets as shown by using [31P]-NMR spectroscopy. The novel ketone diet, therefore, improved physical performance and cognitive function in rats, and its energy-sparing properties suggest that it may help to treat a range of human conditions with metabolic abnormalities.-Murray, A. J., Knight, N. S., Cole, M. A., Cochlin, L. E., Carter, E., Tchabanenko, K., Pichulik, T., Gulston, M. K., Atherton, H. J., Schroeder, M. A., Deacon, R. M. J., Kashiwaya, Y., King, M. T., Pawlosky, R., Rawlins, J. N. P., Tyler, D. J., Griffin, J. L., Robertson, J., Veech, R. L., Clarke, K. Novel ketone diet enhances physical and cognitive performance.A.J.M. thanks the Research Councils UK for supporting his Academic Fellowship. This work was supported by the Defense Advanced Research Projects Agency.This is the final version of the article. It first appeared from FASEB at https://doi.org/10.1096/fj.201600773R

Coding variants in NOD-like receptors: An association study on risk and survival of colorectal cancer

Nod-like receptors (NLRs) are important innate pattern recognition receptors and regulators of inflammation or play a role during development. We systematically analysed 41 non-synonymous single nucleotide polymorphisms (SNPs) in 21 NLR genes in a Czech discovery cohort of sporadic colorectal cancer (CRC) (1237 cases, 787 controls) for their association with CRC risk and survival. Five SNPs were found to be associated with CRC risk and eight with survival at 5% significance level. In a replication analysis using data of two large genome-wide association studies (GWASs) from Germany (DACHS: 1798 cases and 1810 controls) and Scotland (2210 cases and 9350 controls) the associations found in the Czech discovery set were not confirmed. However, expression analysis in human gut-related tissues and immune cells revealed that the NLRs associated with CRC risk or survival in the discovery set were expressed in primary human colon or rectum cells, CRC tissue and/or cell lines, providing preliminary evidence for a potential involvement of NLRs in general in CRC development and/or progression. Most interesting was the finding that the enigmatic development-related NLRP5 (also known as MATER) was not expressed in normal colon tissue but in colon cancer tissue and cell lines. Future studies may show whether regulatory variants instead of coding variants might affect the expression of NLRs and contribute to CRC risk and survival

Identification of novel interactions between microRNAs and pattern-recognition receptor signalling in dendretic cells

Dendritic cells are equipped with a range of different pattern-recognition receptors (PRR) aimed at recognizing foreign pathogens. Recent evidence has suggested that PRR signalling regulates the expression of microRNAs (miRNAs), important post-transcriptional regulators of gene expression, which have been shown to fine-tune innate immune responses. This thesis describes the discovery of miR-650, a novel PRR-responsive miRNA that is down regulated in monocyte-derived dendritic cells (DCs) on PRR stimulation. Chapter 4 describes the characterisation of miR-650 expression in DCs matured by exposure to a variety of different pathogen-derived ligands, or during Influenza A virus infection. When correlating the level of miR-650 to the induction of DC activation markers on the cell surface, an inverse correlation was observed, suggesting a relationship between miR-650 down regulation and the effective dose of the ligand. Work presented in this thesis further explores the potential function of miR-650 by us- ing a multi pronged approach encompassing computational biology, genome-wide expression profiling and individual reporter assays, to gain insight into the gene networks regulated by miR-650. While Chapter 5 focuses on the identification and confirmation of individual miR-650:target interactions, Chapter 6 investigates both direct as well as secondary effects exerted by miR-650 on a global level. The work in these two chapters identifies a number of novelmiR-650 targets and suggests a dual role for miR-650 in the innate immune response. Firstly, it is shown that miR-650 directly regulates a group of interferon-stimulated genes with known antiviral activity. Supporting its role in antiviral host defence, miR-650 is also shown to directly target components of the autophagic machinery, and even more importantly, down regulation of miR-650 induces autophagosome formation. Secondly, identified targets also include negative regulators of in- nate signalling suggesting that, in addition to its antiviral function, PRR-mediated down regulation of miR-650 expression may also provide a negative feedback loop controlling in- flammatory responses.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Identification of novel interactions between MicroRNAs and pattern-recognition receptor signalling in dentritic cells

Dendritic cells are equipped with a range of different pattern-recognition receptors (PRR) aimed at recognizing foreign pathogens. Recent evidence has suggested that PRR signalling regulates the expression of microRNAs (miRNAs), important post-transcriptional regulators of gene expression, which have been shown to fine-tune innate immune responses. This thesis describes the discovery of miR-650, a novel PRR-responsive miRNA that is down regulated in monocyte-derived dendritic cells (DCs) on PRR stimulation. Chapter 4 describes the characterisation of miR-650 expression in DCs matured by exposure to a variety of different pathogen-derived ligands, or during Influenza A virus infection. When correlating the level of miR-650 to the induction of DC activation markers on the cell surface, an inverse correlation was observed, suggesting a relationship between miR-650 down regulation and the effective dose of the ligand. Work presented in this thesis further explores the potential function of miR-650 by using a multi-pronged approach encompassing computational biology, genome-wide expression profiling and individual reporter assays, to gain insight into the gene networks regulated by miR-650. While Chapter 5 focuses on the identification and confirmation of individual miR-650:target interactions, Chapter 6 investigates both direct as well as secondary effects exerted by miR-650 on a global level. The work in these two chapters identifies a number of novel miR-650 targets and suggests a dual role for miR-650 in the innate immune response. Firstly, it is shown that miR-650 directly regulates a group of interferon-stimulated genes with known antiviral activity. Supporting its role in antiviral host defence, miR-650 is also shown to directly target components of the autophagic machinery, and even more importantly, down regulation of miR-650 induces autophagosome formation. Secondly, identified targets also include negative regulators of innate signalling suggesting that, in addition to its antiviral function, PRR-mediated down regulation of miR-650 expression may also provide a negative feedback loop controlling inflammatory responses. Notably, miR-650 displays reciprocal target regulation with miR-155, a well-studied miRNA with established functions in the innate immune system, thus suggesting cooperativity between the two miRNAs. The original aim of this thesis was to examine the effect of HIV-1 infection on the global miRNAome of DCs using a genome-wide profiling method. However, as outlined in Chapter 3, the data generated suggest that HIV-1 infection has little or no impact on miRNA expression. Further work is needed to establish if this represents deliberate immune evasion by HIV-1, or just indicates the limits of the methodology employed.</p

Identification of novel interactions between MicroRNAs and pattern-recognition receptor signalling in dentritic cells

Dendritic cells are equipped with a range of different pattern-recognition receptors (PRR) aimed at recognizing foreign pathogens. Recent evidence has suggested that PRR signalling regulates the expression of microRNAs (miRNAs), important post-transcriptional regulators of gene expression, which have been shown to fine-tune innate immune responses. This thesis describes the discovery of miR-650, a novel PRR-responsive miRNA that is down regulated in monocyte-derived dendritic cells (DCs) on PRR stimulation. Chapter 4 describes the characterisation of miR-650 expression in DCs matured by exposure to a variety of different pathogen-derived ligands, or during Influenza A virus infection. When correlating the level of miR-650 to the induction of DC activation markers on the cell surface, an inverse correlation was observed, suggesting a relationship between miR-650 down regulation and the effective dose of the ligand. Work presented in this thesis further explores the potential function of miR-650 by using a multi-pronged approach encompassing computational biology, genome-wide expression profiling and individual reporter assays, to gain insight into the gene networks regulated by miR-650. While Chapter 5 focuses on the identification and confirmation of individual miR-650:target interactions, Chapter 6 investigates both direct as well as secondary effects exerted by miR-650 on a global level. The work in these two chapters identifies a number of novel miR-650 targets and suggests a dual role for miR-650 in the innate immune response. Firstly, it is shown that miR-650 directly regulates a group of interferon-stimulated genes with known antiviral activity. Supporting its role in antiviral host defence, miR-650 is also shown to directly target components of the autophagic machinery, and even more importantly, down regulation of miR-650 induces autophagosome formation. Secondly, identified targets also include negative regulators of innate signalling suggesting that, in addition to its antiviral function, PRR-mediated down regulation of miR-650 expression may also provide a negative feedback loop controlling inflammatory responses. Notably, miR-650 displays reciprocal target regulation with miR-155, a well-studied miRNA with established functions in the innate immune system, thus suggesting cooperativity between the two miRNAs. The original aim of this thesis was to examine the effect of HIV-1 infection on the global miRNAome of DCs using a genome-wide profiling method. However, as outlined in Chapter 3, the data generated suggest that HIV-1 infection has little or no impact on miRNA expression. Further work is needed to establish if this represents deliberate immune evasion by HIV-1, or just indicates the limits of the methodology employed.This thesis is not currently available via ORA