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Background Respiratory tract infections (RTIs) are a leading cause of hospitalization and death in people age ≥65 years. RTIs are caused by multiple viruses, most of which lack effective treatments. An immunotherapy that enhances pan-antiviral innate immunity may reduce RTI incidence in older adults. Inhibition of targets downstream of target of rapamycin complex 1 (TORC1) was reported to upregulate pan-antiviral gene expression and protect mice from a viral RTI (York AG et al. Cell 2015). We evaluated whether TORC1 inhibition increased antiviral gene expression and decreased RTI incidence in older adults. Methods A randomized, double-blind, placebo, controlled study was conducted to determine whether the TORC1 inhibitor RTB101 alone or in combination with the TORC1 inhibitor everolimus reduced the incidence of laboratory-confirmed RTIs. The study enrolled 652 older adults at increased risk of RTI-related morbidity and mortality (defined as age ≥85 years, or age ≥65 years with asthma, COPD, type 2 diabetes mellitus, or current smokers). Subjects were treated for 16 weeks during winter cold and flu season with oral RTB101 5 mg or 10 mg once daily (QD), RTB101 10 mg twice daily, RTB101 10 mg + everolimus 0.1 mg QD, or matched placebo. The primary endpoint was the percentage of subjects with ≥1 laboratory-confirmed RTI through Week 16. Results RTB101 was well tolerated. In the intent-to-treat analysis, RTB101 10 mg QD was observed to: reduce the percentage of subjects with laboratory-confirmed RTIs by 30.6% compared with placebo (P = 0.025); reduce the incidence of RTIs caused by multiple different viruses; and upregulate interferon-stimulated pan-antiviral gene expression in whole blood (P = 0.00001 vs. placebo, Figure 1). Furthermore, RTB101 10 mg QD was observed to reduce the time to alleviation of moderate to severe RTI symptoms by 5 days, and to reduce the rate of all-cause hospitalization (rate ratio 0.439, 90% CI 0.196–0.983, P = 0.047). Conclusion RTB101 10 mg QD was associated with a significant reduction in laboratory-confirmed RTIs due to multiple viral pathogens that lack effective medicines for treatment or prevention. RTB101 was observed to upregulate interferon-stimulated pan-antiviral gene expression, which may underlie the reduction in RTI incidence. Disclosures Joan Mannick, MD, resTORbio (Employee, Shareholder), Amelia Tomlinson, PhD, resTORbio (Employee), Sarb Shergill, PhD, resTORbio (Employee), Grace Teo, PhD, resTORbio (Employee), Lloyd Klickstein, MD, PhD, resTORbio (Employee).

Early events of mesenchymal stem/stromal cell (MSC) adhesion to and transmigration through the vascular wall following systemic infusion are important for MSC trafficking to inflamed sites, yet are poorly characterized in vivo. Here, we used intravital confocal imaging to determine the acute extravasation kinetics and distribution of culture-expanded MSC (2–6 hours postinfusion) in a murine model of dermal inflammation. By 2 hours postinfusion, among the MSC that arrested within the inflamed ear dermis, 47.8% ± 8.2% of MSC had either initiated or completed transmigration into the extravascular space. Arrested and transmigrating MSCs were equally distributed within both small capillaries and larger venules. This suggested existence of an active adhesion mechanism, since venule diameters were greater than those of the MSC. Heterotypic intravascular interactions between distinct blood cell types have been reported to facilitate the arrest and extravasation of leukocytes and circulating tumor cells. We found that 42.8% ± 24.8% of intravascular MSC were in contact with neutrophil-platelet clusters. A role for platelets in MSC trafficking was confirmed by platelet depletion, which significantly reduced the preferential homing of MSC to the inflamed ear, although the total percentage of MSC in contact with neutrophils was maintained. Interestingly, although platelet depletion increased vascular permeability in the inflamed ear, there was decreased MSC accumulation. This suggests that increased vascular permeability is unnecessary for MSC trafficking to inflamed sites. These findings represent the first glimpse into MSC extravasation kinetics and microvascular distribution in vivo, and further clarify the roles of active adhesion, the intravascular cellular environment, and vascular permeability in MSC trafficking. Stem Cells 2015;33:265–277

Capture and isolation of flowing cells and particulates from body fluids has enormous implications in diagnosis, monitoring, and drug testing, yet monovalent adhesion molecules used for this purpose result in inefficient cell capture and difficulty in retrieving the captured cells. Inspired by marine creatures that present long tentacles containing multiple adhesive domains to effectively capture flowing food particulates, we developed a platform approach to capture and isolate cells using a 3D DNA network comprising repeating adhesive aptamer domains that extend over tens of micrometers into the solution. The DNA network was synthesized from a microfluidic surface by rolling circle amplification where critical parameters, including DNA graft density, length, and sequence, could readily be tailored. Using an aptamer that binds to protein tyrosine kinase-7 (PTK7) that is overexpressed on many human cancer cells, we demonstrate that the 3D DNA network significantly enhances the capture efficiency of lymphoblast CCRF-CEM cells over monovalent aptamers and antibodies, yet maintains a high purity of the captured cells. When incorporated in a herringbone microfluidic device, the 3D DNA network not only possessed significantly higher capture efficiency than monovalent aptamers and antibodies, but also outperformed previously reported cell-capture microfluidic devices at high flow rates. This work suggests that 3D DNA networks may have broad implications for detection and isolation of cells and other bioparticles.

We examine the effect of bone marrow transplantation (BMT) on retinal cell turnover by performing simultaneous cell tracking of native microglia and engrafting donor bone marrow-derived cell (BMDC) populations in the retinae of live mice using a custom-built multi-color confocal scanning laser ophthalmoscope (SLO) specifically developed for murine retinal imaging. CX3CR1GFP/+ mice whose retinal microglia express the green fluorescent protein (GFP) were exposed to a lethal dose of gamma radiation and subsequently rescued with bone marrow cells from universal DsRed donor mice. Over a time course of four months after the irradiation and BMT, progressive loss of GFP+ microglia was accompanied by delayed engraftment of DsRed+ BMDC. Morphologic examination revealed that the remaining GFP+ microglia were ramified, while engrafting DsRed+ cells exhibited both ramification and dendriform shape. Leukocyte endothelial interaction, normally absent in healthy retinal vasculature, was observed even after three months, ...

Cell surfaces are fertile ground for chemists and material scientists to manipulate or augment cell functions and phenotypes. This not only helps to answer basic biology questions but also has diagnostic and therapeutic applications. In this review, we summarize the most recent advances in the engineering of the cell surface. In particular, we focus on the potential applications of surface engineered cells for 1) targeting cells to desirable sites in cell therapy, 2) programming assembly of cells for tissue engineering, 3) bioimaging and sensing, and ultimately 4) manipulating cell biology., National Institutes of Health (U.S.) (Grabt R03DE019191), American Heart Association (Grant 0970178N)

Tissue engineering strategies date back to the seventies and eighties for developing skin substitutes. Despite early approaches for replacement, repair, and regeneration of failing organs, the true emergence of tissue engineering as a medical field started in the early nineties when tissue engineering was defined as an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain or improve tissue function. Multiple challenges remain for translation of tissue-engineered products to the clinic. Cell type, source, and manipulation are critical parameters that need to be further studied and defined, in order to achieve the best clinical outcomes. Many approaches are too complex for scale-up to industrial level manufacture.

Systemically administered adult mesenchymal stem cells (MSCs), which are being explored in clinical trials to treat inflammatory disease, exhibit the critical ability to extravasate at sites of inflammation. We aimed to characterize the basic cellular processes mediating this extravasation and compare them to those involved in leukocyte transmigration. Using high-resolution confocal and dynamic microscopy, we show that, like leukocytes, human bone marrow-derived MSC preferentially adhere to and migrate across tumor necrosis factor-α-activated endothelium in a vascular cell adhesion molecule-1 (VCAM-1) and G-protein-coupled receptor signaling-dependent manner. As several studies have suggested, we observed that a fraction of MSC was integrated into endothelium. In addition, we observed two modes of transmigration not previously observed for MSC: Paracellular (between endothelial cells) and transcellular (directly through individual endothelial cells) diapedesis through discrete gaps and pores in the endothelial monolayer, in association with VCAM-1-enriched “transmigratory cups”. Contrasting leukocytes, MSC transmigration was not preceded by significant lateral migration and occurred on the time scale of hours rather than minutes. Interestingly, rather than lamellipodia and invadosomes, MSC exhibited nonapoptotic membrane blebbing activity that was similar to activities previously described for metastatic tumor and embryonic germ cells. Our studies suggest that low avidity binding between endothelium and MSC may grant a permissive environment for MSC blebbing. MSC blebbing was associated with early stages of transmigration, in which blebs could exert forces on underlying endothelial cells indicating potential functioning in breaching the endothelium. Collectively, our data suggest that MSC transmigrate actively into inflamed tissues via both leukocyte-like and novel mechanisms. STEM CELLS2012;30:2472–2486

This paper describes a magnetic, nonoperative device and control system designed to treat long-gap esophageal atresia LEA. This congenital disorder occurs in approximately 100 newborn infants every year and is characterized by a discontinuity in the esophagus between the mouth and stomach. Our device builds upon previous work investigating the use of internal permanent magnets to stretch the proximal and distal esophageal pouches together until anastomosis occurs. We implement a hydraulic standoff device for the proximal magnet assembly to control the distance between the two magnets independent of the esophageal gap size. The standoff allows for controllable, intermittent force between the two pouches and provides a layer of safety from runaway magnetic forces that could potentially damage delicate esophageal tissue. The proximal device comes in two variations: a convex tip for stretching the esophagus and a concave mating tip for meeting the distal end during anastomosis. A light emitting diode LED and phototransistor pair estimates the esophageal gap size for the duration of the procedure, and a fluid pressure sensor enables the force on the esophageal tissue to be calculated. The external control circuitry, physician interface, and pump are described that demonstrate the core functionality of the system.

A cell’s fate is tightly controlled by its microenvironment. Key factors contributing to this microenvironment include physical contacts with the extracellular matrix and neighboring cells, in addition to soluble factors produced locally or distally. Alterations to these cues can drive homeostatic processes, such as tissue regeneration/wound healing, or may lead to pathologic tissue dysfunction. In vitro models of cell and tissue microenvironments are desirable for enhanced understanding of the biology and ultimately for improved treatment. However, mechanisms to exert specific control over cellular microenvironments remains a significant challenge. Genetic modification has been used but is limited to products that can be manufactured by cells and release kinetics of therapeutics cannot easily be controlled. Herein we describe a non-genetic approach to engineer cells with an intracellular depot of phenotype altering agent/s that can be used for altering cell fate via intracrine-, paracrine-, and endocrine-like mechanisms. Specifically, we show that human mesenchymal stem cells (MSCs) can be engineered with poly lactide-co-glycolic acid (PLGA) particles containing dexamethasone, which acts on cytoplasmic receptors. The controlled release properties of these particles allowed for sustained intracellular and extracellular delivery of agent to promote differentiation of particle-carrying cells, as well as neighboring cells and distant cells that do not contain particles.

Covalently conjugated sialyl Lewis X (SLeX) on the mesenchymal stem cell (MSC) surface through a biotin-streptavidin bridge imparts leukocyte-like rolling characteristics without altering the cell phenotype and the multilineage differentiation potential. We demonstrate that the conjugation of SLeX on the MSC surface is stable, versatile, and induces a robust rolling response on P-selectin coated substrates. These results indicate the potential to increase the targeting efficiency of any cell type to specific tissue.

The study of MSC trafficking is clinically relevant for minimally invasive cell therapy to promote regeneration of damaged tissue, to treat inflammation, and to promote angiogenesis. However, these studies are complicated by the diverse methods used to culture, characterize, and deliver MSCs and by the variety of methods used to assess homing events. This review provides a critical analysis of the methods used to track homing of exogenously infused MSCs and discusses strategies for enhancing their trafficking to particular tissues.