Triclosan (TCS) is a high-volume chemical used as an antimicrobial ingredient in more than 2000 consumer products, such as toothpaste, cosmetics, kitchenware, and toys. We report that brief exposure to TCS, at relatively low doses, causes low-grade colonic inflammation, increases colitis, and exacerbates colitis-associated colon cancer in mice. Exposure to TCS alters gut microbiota in mice, and its proinflammatory effect is attenuated in germ-free mice. In addition, TCS treatment increases activation of Toll-like receptor 4 (TLR4) signaling in vivo and fails to promote colitis in mice. Together, our results demonstrate that this widely used antimicrobial ingredient could have adverse effects on colonic inflammation and associated colon tumorigenesis through modulation of the gut microbiota and TLR4 signaling. Together, these results highlight the need to reassess the effects of TCS on human health and potentially update policies regulating the use of this widely used antimicrobial.

The impermeability of the plasma membrane towards large, hydrophilic biomolecules is a major obstacle in their use and development against intracellular targets. To overcome such limitations, protein transduction domains (PTDs) have been used as protein carriers, however they often require covalent fusion to the protein for efficient delivery. In an effort to develop more efficient and versatile biological vehicles, a series of PTD-inspired polyoxanorbornene-based synthetic mimics with identical chemical compositions but different hydrophobic/hydrophilic segregation were used to investigate the role of sequence segregation on protein binding and uptake into Jurkat T cells and HEK293Ts. This series was composed of a strongly segregated block copolymer, an intermediately segregated gradient copolymer, and a non-segregated homopolymer. To assess how protein isoelectric point, the study was extended to other proteins (bovine serum albumin, avidin, and streptavidin). Among the series, the block copolymer maximized both protein binding and translocation efficiencies, closely followed by the gradient copolymer, resulting in two protein transporter molecules more efficacious than currently commercially available agents. These two polymers were also used to deliver the biologically active Cre recombinase into a loxP-reporter T cell line. Since exogenous Cre must reach the nucleus and retain its activity to induce gene recombination, this in vitro experiment better exemplifies the broad applicability of this synthetic system. This study shows that increasing segregation between hydrophobic and cationic moieties in these polymeric mimics improves non-covalent protein delivery, providing crucial design parameters for the creation of more potent biological delivery agents for research and biomedical applications.

Targeting cellular proteins with antibodies, to better understand cellular signaling pathways in the context of disease modulation, is a fast-growing area of investigation. Humanized antibodies are increasingly gaining attention for their therapeutic potential, but the collection of cellular targets is limited to those secreted from cells or expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as putative targets for antibody binding. Protein kinase Cθ (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic capable of efficiently delivering an antibody against phosphorylated PKCθ (Thr538) into human peripheral mononuclear blood cells and altering expression of downstream indicators of T cell activation and differentiation. We used a humanized, lymphocyte transfer model of graft-versus-host disease, to evaluate the durability of protein transduction domain mimic:Anti-pPKCθ modulation, when delivered into human peripheral mononuclear blood cells ex vivo. We demonstrate that protein transduction domain mimic:Antibody complexes can be readily introduced with high efficacy into hard-to-transfect human peripheral mononuclear blood cells, eliciting a biological response sufficient to alter disease progression. Thus, protein transduction domain mimic:Antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.

The immune-mediated tissue destruction of graft- vs -host disease (GvHD) remains a major barrier to greater use of hematopoietic stem cell transplantation (HSCT). Mesenchymal stem cells (MSCs) have intrinsic immunosuppressive qualities and are being actively investigated as a therapeutic strategy for treating GvHD. We characterized Cymerus™ MSCs, which are derived from adult, induced pluripotent stem cells (iPSCs), and show they display surface markers and tri-lineage differentiation consistent with MSCs isolated from bone marrow (BM). Administering iPSC-MSCs altered phosphorylation and cellular localization of the T cell-specific kinase, Protein Kinase C theta (PKCθ), attenuated disease severity, and prolonged survival in a humanized mouse model of GvHD. Finally, we sevaluated a constellation of pro-inflammatory molecules on circulating PBMCs that correlated closely with disease progression and which may serve as biomarkers to monitor therapeutic response. Altogether, our data suggest Cymerus iPSC-MSCs offer the potential for an off-the-shelf, cell-based therapy to treat GvHD.

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