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.

Multiple sclerosis (MS) is a disabling demyelinating autoimmune disorder of the central nervous system (CNS) which is driven by IL-23-and IL-1β-induced autoreactive Th17 cells that traffic to the CNS and secrete proinflammatory cytokines. Th17 pathogenicity in MS has been correlated with the dysregulation of microRNA (miRNA) expression, and specific miRNAs have been shown to promote the pathogenic Th17 phenotype. In the present study, we demonstrate, using the animal model of MS, experimental autoimmune encephalomyelitis (EAE), that let-7 miRNAs confer protection against EAE by negatively regulating the proliferation, differentiation and chemokine-mediated migration of pathogenic Th17 cells to the CNS. Specifically, we found that let-7 miRNAs may directly target the cytokine receptors Il1r1 and Il23r, as well as the chemokine receptors Ccr2 and Ccr5. Therefore, our results identify a novel regulatory role for let-7 miRNAs in pathogenic Th17 differentiation during EAE development, suggesting a promising therapeutic application for disease treatment.

Vijayaraghavan J, Maggi EC, Crabtree JS. miR-24 regulates menin in the endocrine pancreas. Am J Physiol Endocrinol Metab 307: E84 -E92, 2014. First published May 13, 2014; doi:10.1152/ajpendo.00542.2013.-Menin, the product of the MEN1 gene, functions as a tumor suppressor and was first identified in 1997 due to its causative role in the endocrine tumor disorder multiple endocrine neoplasia, type 1 (MEN1). More recently, menin has been identified as a key player in pancreatic islet biology with the observation of an inverse relationship between menin levels and pancreatic islet proliferation. However, the factors regulating menin and the MEN1 gene in the pancreas are poorly understood. Here, we describe the regulation of menin by miR-24 and demonstrate that miR-24 directly decreases menin levels and impacts downstream cell cycle inhibitors in MIN6 insulinoma cells and in ␤lox5 immortalized ␤-cells. This regulation of menin impacts cell viability and proliferation in ␤lox5 cells. Furthermore, our data show a feedback regulation between miR-24 and menin that is present in the pancreas, suggesting that miR-24 regulates menin levels in the pancreatic islet. menin; MEN1; pancreatic islet; miR-24; miRNA THE ENDOCRINE PANCREAS is responsible for maintaining normal glucose homeostasis through the tightly controlled, regulated release of insulin from the pancreatic islet. Pancreatic islets have the unique ability to dynamically and reversibly expand to adapt to changes in insulin demand through careful balance of cell growth and renewal (including ␤-cell replication, neogenesis, transdifferentiation, and hypertrophy), and cell death (apoptosis, atrophy, and autophagy). When this process becomes compromised, it leads to the clinical manifestation of gestational diabetes or type 2 diabetes (T2D). However, detailed knowledge of the mechanisms underlying this process is lacking.MicroRNAs (miRNAs), an evolutionarily conserved class of posttranscriptional regulators, are short ϳ22-nucleotide noncoding RNA sequences that regulate the expression of mRNA targets by binding to the 3=-UTR and inhibiting translation and/or targeting the mRNA for degradation. There is growing evidence that miRNAs regulate key biological processes in the pancreatic islet (13,14,17,27,33,49). For example, miR-375, the first miRNA identified in ␤-cell function, regulates insulin secretion and is essential for normal glucose homeostasis and turnover of ␣-and ␤-cells (40, 41). Other recent studies indicate a role for miR-338-3p and miR-181a in ␤-cell differentiation and insulin sensitivity, respectively (7, 23). p38 (MAPK) is downregulated by miR-24 through the insulinresponsive glucose transporter 4 (GLUT4) to affect peripheral insulin resistance (21). miR-24 also regulates insulin production by targeting the insulin transcriptional repressors Sox6 and Bhlhe22 (35).

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.

Neuroendocrine tumors (NETs), which can have survival rates as low as 4%, currently have limited therapeutic interventions available highlighting the dire need for the identification of novel biological targets for use as new potential drug targets. One such potential target is retinoblastoma-binding protein 2 (RBP2), an H3K4 demethylase whose overexpression has been linked to cancer formation and metastasis in non-endocrine tumor types. We measured RBP2 mRNA and protein levels in enteropancreatic NETs by measuring RBP2 in matched human normal and NET tissue samples. Further, proliferation, migration, invasion and colony formation assays were performed in the physiologically relevant NET cell lines βlox5, H727 and QGP-1 to understand the role of RBP2 and its demethylase activity on end points of tumorigenesis. Our data indicate a strong correlation between RBP2 mRNA and protein expression in NET specimens. RBP2 was overexpressed relative to tissue-matched normal controls in 80% of the human tumors measured. In vitro studies showed RBP2 overexpression significantly increased proliferation, migration, invasion and colony formation, whereas knockdown significantly decreases the same parameters in a demethylase-independent manner. The cell cycle inhibitors p21 and p57 decreased with RBP2 overexpression and increased upon its depletion, suggesting a regulatory role for RBP2 in cellular proliferation. Taken together, our results support the hypothesis that the aberrant overexpression of RBP2 is a frequent contributing factor to tumor formation and metastasis in enteropancreatic NETs.