Effects of HLA single chain trimer design on peptide presentation and stability.

MHC class I "" molecules, coupling MHC heavy chain, β-microglobulin, and a specific peptide into a single polypeptide chain, are widely used in research. To more fully understand caveats associated with this design that may affect its use for basic and translational studies, we evaluated a set of engineered single-chain trimers with combinations of stabilizing mutations across eight different classical and non-classical human class I alleles with 44 different peptides, including a novel human/murine chimeric design. While, overall, single-chain trimers accurately recapitulate native molecules, care was needed in selecting designs for studying peptides longer or shorter than 9-mers, as single-chain trimer design could affect peptide conformation.

Silymarin suppresses basal and stimulus-induced activation, exhaustion, differentiation, and inflammatory markers in primary human immune cells.

Silymarin (SM), and its flavonolignan components, alter cellular metabolism and inhibit inflammatory status in human liver and T cell lines. In this study, we hypothesized that SM suppresses both acute and chronic immune activation (CIA), including in the context of HIV infection. SM treatment suppressed the expression of T cell activation and exhaustion markers on CD4+ and CD8+ T cells from chronically-infected, HIV-positive subjects.

Uncovering biologically significant lipid isomers with liquid chromatography, ion mobility spectrometry and mass spectrometry.

Understanding how biological molecules are generated, metabolized and eliminated in living systems is important for interpreting processes such as immune response and disease pathology. While genomic and proteomic studies have provided vast amounts of information over the last several decades, interest in lipidomics has also grown due to improved analytical technologies revealing altered lipid metabolism in type 2 diabetes, cancer, and lipid storage disease. Mass spectrometry (MS) measurements are currently the dominant approach for characterizing the lipidome by providing detailed information on the spatial and temporal composition of lipids.

Natural Products as Tools for Defining How Cellular Metabolism Influences Cellular Immune and Inflammatory Function during Chronic Infection.

Chronic viral infections like those caused by hepatitis C virus (HCV) and human immunodeficiency virus (HIV) cause disease that establishes an ongoing state of chronic inflammation. While there have been tremendous improvements towards curing HCV with directly acting antiviral agents (DAA) and keeping HIV viral loads below detection with antiretroviral therapy (ART), there is still a need to control inflammation in these diseases. Recent studies indicate that many natural products like curcumin, resveratrol and silymarin alter cellular metabolism and signal transduction pathways via enzymes such as adenosine monophosphate kinase (AMPK) and mechanistic target of rapamycin (mTOR), and these pathways directly influence cellular inflammatory status (such as NF-κB) and immune function.

Silymarin Suppresses Cellular Inflammation By Inducing Reparative Stress Signaling.

Silymarin, a characterized extract of the seeds of milk thistle (Silybum marianum), suppresses cellular inflammation. To define how this occurs, transcriptional profiling, metabolomics, and signaling studies were performed in human liver and T cell lines. Cellular stress and metabolic pathways were modulated within 4 h of silymarin treatment: activation of Activating Transcription Factor 4 (ATF-4) and adenosine monophosphate protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) signaling, the latter being associated with induction of DNA-damage-inducible transcript 4 (DDIT4).

Independent, parallel pathways to CXCL10 induction in HCV-infected hepatocytes.

Background & Aims: The pro-inflammatory chemokine CXCL10 is induced by HCV infection in vitro and in vivo, and is associated with outcome of IFN (interferon)-based therapy. We studied how hepatocyte sensing of early HCV infection via TLR3 (Toll-like receptor 3) and RIG-I (retinoic acid inducible gene I) led to expression of CXCL10.

Methods: CXCL10, type I IFN, and type III IFN mRNAs and proteins were measured in PHH (primary human hepatocytes) and hepatocyte lines harboring functional or non-functional TLR3 and RIG-I pathways following HCV infection or exposure to receptor-specific stimuli.

The role of amino acid changes in the human immunodeficiency virus type 1 transmembrane domain in antibody binding and neutralization.

The detailed interactions between antibodies and the HIV-1 envelope protein that lead to neutralization are not well defined. Here, we show that several conservative substitutions in the envelope gp41 led to a ~100 fold increase in neutralization sensitivity to monoclonal antibodies (MAbs) that target gp41: 4E10 and 2F5. Substitution at position 675 alone did not impact neutralization susceptibility to MAbs that recognize more distal sites in gp120 (b12, VRC01, PG9).

Stabilization of α-conotoxin AuIB: influences of disulfide connectivity and backbone cyclization.

α-Conotoxins are peptides isolated from the venom ducts of cone snails that target nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential applications for treating a range of conditions in humans, including pain. However, like all peptides, conotoxins are susceptible to degradation, and to enhance their therapeutic potential it is important to elucidate the factors contributing to instability and to develop approaches for improving stability.

Differential effects of selective HDAC inhibitors on macrophage inflammatory responses to the Toll-like receptor 4 agonist LPS.

Broad-spectrum inhibitors of HDACs are therapeutic in many inflammatory disease models but exacerbated disease in a mouse model of atherosclerosis. HDAC inhibitors have anti- and proinflammatory effects on macrophages in vitro. We report here that several broad-spectrum HDAC inhibitors, including TSA and SAHA, suppressed the LPS-induced mRNA expression of the proinflammatory mediators Edn-1, Ccl-7/MCP-3, and Il-12p40 but amplified the expression of the proatherogenic factors Cox-2 and Pai-1/serpine1 in primary mouse BMM.

Selective induction of the Notch ligand Jagged-1 in macrophages by soluble egg antigen from Schistosoma mansoni involves ERK signalling.

Soluble egg antigen (SEA) from the helminth Schistosoma mansoni promotes T helper type 2 (Th2) responses by modulating antigen-presenting cell function. The Jagged/Notch pathway has recently been implicated in driving Th2 development. We show here that SEA rapidly up-regulated mRNA and protein expression of the Notch ligand Jagged-1 in both murine bone marrow-derived macrophages (BMMs) and human monocyte-derived macrophages (HMDMs).

Cyclic MrIA: a stable and potent cyclic conotoxin with a novel topological fold that targets the norepinephrine transporter.

Conotoxins, disulfide-rich peptides from the venom of cone snails, have created much excitement over recent years due to their potency and specificity for ion channels and their therapeutic potential. One recently identified conotoxin, MrIA, a 13-residue member of the chi-conotoxin family, inhibits the human norepinephrine transporter (NET) and has potential applications in the treatment of pain. In the current study, we show that the beta-hairpin structure of native MrIA is retained in a synthetic cyclic version, as is biological activity at the NET.

Solution structure of chi-conopeptide MrIA, a modulator of the human norepinephrine transporter.

The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a beta-hairpin with the two strands connected by an inverse gamma-turn.