Suppression of EAE and Prevention of Blood-Brain Barrier Breakdown after Vaccination with Novel Bifunctional Peptide Inhibitor

The efficacy of bifunctional peptide inhibitor (BPI) in preventing blood-brain barrier (BBB) breakdown during onset of experimental autoimmune encephalomyelitis (EAE) and suppression of the disease was evaluated in mice. The mechanism that defines how BPI prevents the disease was investigated by measuring the in vitro cytokine production of splenocytes. Peptides were injected 5 to 11 days prior to induction of EAE, and the severity of the disease was monitored by a standard clinical scoring protocol and change in body weight. The BBB breakdown in diseased and treated mice was compared to that in normal control mice by determining deposition of gadolinium diethylenetriaminepentaacetate (Gd-DTPA) in the brain using magnetic resonance imaging (MRI). Mice treated with PLP-BPI showed no or low indication of EAE as well as normal increase in body weight. In contrast, mice treated with the control peptide or PBS showed a decrease in body weight and a high disease score. The diseased mice had high deposition of Gd-DTPA in the brain, indicating breakdown in the BBB. However, the deposition of Gd-DTPA in PLP-BPI-treated mice was similar to that in normal control mice. Thus, PLP-BPI can suppress EAE when administered as a peptide vaccine and maintain the integrity of the BBB

FRET Detection of Lymphocyte Function-Associated Antigen-1 Conformational Extension

Lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18, αLβ2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1-specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation

A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4 + T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention