Type I interferon restricts type 2 immunopathology through the regulation of group 2 innate lymphoid cells.

Viral respiratory tract infections are the main causative agents of the onset of infection-induced asthma and asthma exacerbations that remain mechanistically unexplained. Here we found that deficiency in signaling via type I interferon receptor led to deregulated activation of group 2 innate lymphoid cells (ILC2 cells) and infection-associated type 2 immunopathology. Type I interferons directly and negatively regulated mouse and human ILC2 cells in a manner dependent on the transcriptional activator ISGF3 that led to altered cytokine production, cell proliferation and increased cell death. In addition, interferon-γ (IFN-γ) and interleukin 27 (IL-27) altered ILC2 function dependent on the transcription factor STAT1. These results demonstrate that type I and type II interferons, together with IL-27, regulate ILC2 cells to restrict type 2 immunopathology.

An Assessment of Blood Vessel Remodeling of Nanofibrous Poly(ε-Caprolactone) Vascular Grafts in a Rat Animal Model.

The development of an ideal vascular prosthesis represents an important challenge in terms of the treatment of cardiovascular diseases with respect to which new materials are being considered that have produced promising results following testing in animal models. This study focuses on nanofibrous polycaprolactone-based grafts assessed by means of histological techniques 10 days and 6 months following suturing as a replacement for the rat aorta. A novel stereological approach for the assessment of cellular distribution within the graft thickness was developed. The cellularization of the thickness of the graft was found to be homogeneous after 10 days and to have changed after 6 months, at which time the majority of cells was discovered in the inner layer where the regeneration of the vessel wall was found to have occurred. Six months following implantation, the endothelialization of the graft lumen was complete, and no vasa vasorum were found to be present. Newly formed tissue resembling native elastic arteries with concentric layers composed of smooth muscle cells, collagen, and elastin was found in the implanted polycaprolactone-based grafts. Moreover, the inner layer of the graft was seen to have developed structural similarities to the regular aortic wall. The grafts appeared to be well tolerated, and no severe adverse reaction was recorded with the exception of one case of cartilaginous metaplasia close to the junctional suture.

Physiological evidence of integrin-antibody reactive proteins influencing the innate cellular immune responses of larval Galleria mellonella hemocytes.

Larval Galleria mellonella (L.) hemocytes form microaggregates in response to stimulation by Gram-positive bacteria. Hemocyte adhesion to foreign materials is mediated by the cAMP/ protein kinase A pathway and the ß-subunit of cholera toxin using a cAMP-independent mechanism. Cholera toxin-induced microaggregation was inhibited by the integrin inhibitory RGDS peptide, implying integrins may be part of the mechanism. Based on the types of mammalian integrin-antibody reactive proteins affecting hemocyte adhesion and bacterial-induced responses α5 , αv , ß1 , and ß3 subunits occurred on both granular cell and plasmatocyte hemocyte subtypes. A fluorescent band representing the binding of rabbit α5 -integrin subunit antibodies occurred between adhering heterotypic hemocytes. The frequency of the bands was increased by cholera toxin. The α5 and ß1 rabbit integrin subunit antibodies inhibited removal of Bacillus subtilis (Cohn) from the hemolymph in vivo. A α5 ß1 -specific synthetic peptide blocker similarly diminished hemocyte function whereas the αv ß3 -specific inhibitory peptide and the corresponding integrin subunit antibodies did not influence nonself hemocyte activities. Western blots revealed several proteins reacting with a given integrin-antibody subtype. Thus integrin-antibody reactive proteins (which may include integrins) with possible α5 and ß1 epitopes modulate immediate hemocyte function. Confocal microscopy established plasmatocyte adhesion to and rosetting over substrata followed by granular cell microaggregate adhesion to plasmatocytes during early stage nodulation.

Best Tourniquet Holding and Strap Pulling Technique.

BACKGROUND: Appropriate strap pressure before tightening-system use is an important aspect of nonelastic, limb tourniquet application. METHODS: Using different two-handed techniques, the strap of the Generation 7 Combat Application Tourniquet (C-A-T7), Tactical Ratcheting Medical Tourniquet (Tac RMT), Tactical Mechanical Tourniquet (TMT), Parabelt, and Generation 3 SOF® Tactical Tourniquet-Wide (SOFTTW) was secured mid-thigh by 20 appliers blinded to pressure data and around a thigh-sized ballistic gel cylinder by gravity and 23.06kg. RESULTS: Pulling only outward (90° to strap entering buckle) achieved the lowest secured pressures on thighs and gel. For appliers, the best holding location was above the buckle, and the best strap-pulling direction was tangential to the thigh or gel (0° to strap entering buckle). Preceding tangential pulling with outward pulling resulted in higher secured pressures on the gel but did not aid appliers. Appliers generally did not reach secured pressures achievable for their strength. Of 80 thigh applications per tourniquet, 77 C-A-T7, 41 Tac RMT, 35 TMT, 16 Parabelt, and 10 SOFTTW applications had secured pressures greater than 100mmHg. CONCLUSIONS: The default for best tourniquet strap-application technique is to hold above the buckle and pull the strap tangential to the limb at the buckle. Additionally, neither strength nor experience guarantees desirable strap pressures in the absence of pressure knowledge.

Development of an Injectable Nitric Oxide Releasing Poly(ethylene) Glycol-Fibrin Adhesive Hydrogel.

Fibrin microparticles were incorporated into poly(ethylene) glycol (PEG)-fibrinogen hydrogels to create an injectable, composite that could serve as a wound healing support and vehicle to deliver therapeutic factors for tissue engineering. Nitric oxide (NO), a therapeutic agent in wound healing, was loaded into fibrin microparticles by blending S-Nitroso-N-acetyl penicillamine (SNAP) with a fibrinogen solution. The incorporation of microparticles affected swelling behavior and improved tissue adhesivity of composite hydrogels. Controlled NO release was induced via photolytic and thermal activation, and modulated by weight percent of particles incorporated. These NO-releasing composites were non-cytotoxic in culture. Cells maintained morphology, viability, and proliferative character. Fibrin microparticles loaded with SNAP and incorporated into a PEG-fibrinogen matrix, creates a novel injectable composite hydrogel that offers improved tissue adhesivity and inducible NO-release for use as a regenerative support for wound healing and tissue engineering applications.

Synthesis and Characterization of the Novel Nitric Oxide (NO) Donating Compound, S-nitroso-N-acetyl-D-penicillamine Derivatized Cyclam (SNAP-Cyclam).

Nitric oxide (NO) has been heavily studied over the past two decades due to its multitude of physiological functions and its potential therapeutic promise. Of major interest is the desire to fabricate or coat implanted devices with an NO releasing material that will impart the appropriate dose and duration of NO release to positively mediate the biological response to the medical device, thereby improving its safety and efficacy. To date, this goal has not yet been achieved, despite very promising early research. Herein, we describe the synthesis and NO release properties of a novel NO donor which covalently links the S-nitrosothiol, S-nitroso-N-acetyl-D-penicillamine (SNAP), to the macrocycle, cyclam (SNAP-cyclam). This compound can then be blended into a wide variety of polymer matrices, imparting NO release to the polymer system. This release can be initiated and controlled by transition metal catalysis, thermal degradation or photolytic release of NO from the composite NO-releasing material. SNAP-cyclam is capable of releasing physiologically relevant levels of NO for up to 3 months in vitro when blended into poly(l-lactic acid) thin films.

Transition-Metal-Mediated Release of Nitric Oxide (NO) from S-Nitroso-N-acetyl-d-penicillamine (SNAP): Potential Applications for Endogenous Release of NO at the Surface of Stents Via Corrosion Products.

Nitric oxide (NO), identified over the last several decades in many physiological processes and pathways as both a beneficial and detrimental signaling molecule, has been the subject of extensive research. Physiologically, NO is transported by a class of donors known as S-nitrosothiols. Both endogenous and synthetic S-nitrosothiols have been reported to release NO during interactions with certain transition metals, primarily Cu(2+) and Fe(2+). Ag(+) and Hg(2+) have also been identified, although these metals are not abundantly present in physiological systems. Here, we evaluate Pt(2+), Fe(2+), Fe(3+), Mg(2+), Zn(2+), Mn(2+), Co(2+), Ni(2+), and Cu(2+) for their ability to generate NO from S-nitroso-N-acetyl-d-penicillamine (SNAP) under physiological pH conditions. Specifically, we report NO generation from RSNOs initiated by three transition metal ions; Co(2+), Ni(2+), and Zn(2+), which have not been previously reported to generate NO. Additionally, preliminary in vivo evidence of zinc wires implanted in the rat arterial wall and circulating blood is presented which demonstrated inhibited thrombus formation after 6 months. One potentially useful application of these metal ions capable of generating NO from RSNOs is their use in the fabrication of biodegradable metallic stents capable of generating NO at the stent-blood interface, thereby reducing stent-related thrombosis and restenosis.

Bioactive vapor deposited calcium-phosphate silica sol-gel particles for directing osteoblast behavior.

Silica-based materials are being developed and used for a variety of applications in orthopedic tissue engineering. In this work, we characterize the ability of a novel silica sol vapor deposition system to quickly modify biomaterial substrates and modulate surface hydrophobicity, surface topography, and composition. We were able to show that surface hydrophobicity, surface roughness, and composition could be rapidly modified. The compositional modification was directed towards generating apatitic-like surface mineral compositions (Ca/P ratios ∼1.30). Modified substrates were also capable of altering cell proliferation and differentiation behavior of preosteoblasts (MC3T3) and showed potential once optimized to provide a simple means to generate osteo-conductive substrates for tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2135-2148, 2016.

Fabrication and Short-Term in Vivo Performance of a Natural Elastic Lamina-Polymeric Hybrid Vascular Graft.

Although significant advances have been made in the development of artificial vascular grafts, small-diameter grafts still suffer from excessive platelet activation, thrombus formation, smooth muscle cell intimal hyperplasia, and high occurrences of restenosis. Recent discoveries demonstrating the excellent blood-contacting properties of the natural elastic lamina have raised the possibility that an acellular elastic lamina could effectively serve as a patent blood-contacting surface in engineered vascular grafts. However, the elastic lamina alone lacks the requisite mechanical properties to function as a viable vascular graft. Here, we have screened a wide range of biodegradable and biostable medical-grade polymers for their ability to adhere to the outer surface of the elastic lamina and allow cellular repopulation following engraftment in the rat abdominal aorta. We demonstrate a novel method for the fabrication of elastic lamina-polymeric hybrid small-diameter vascular grafts and identify poly(ether urethane) (PEU 1074A) as ideal for this purpose. In vivo results demonstrate graft patency over 21 days, with low thrombus formation, mild inflammation, and the general absence of smooth muscle cell hyperplasia on the graft's luminal surface. The results provide a new direction for developing small-diameter vascular grafts that are mass-producible, shelf-stable, and universally compatible due to a lack of immune response and inhibit the in-graft restenosis response that is common to nonautologous materials.

Biomaterial guides for lymphatic endothelial cell alignment and migration.

Axillary dissection during breast cancer surgery produces extensive lymphatic vessel damage that often leads to lifelong secondary lymphedema of the arm. We have developed a biodegradable material conduit for lymphatic vessel reconstruction where fibers electrospun along the conduit lumen promote endothelial cell alignment and migration in vitro. The diameter and density of the electrospun fibers were optimized for cell migration and direction on two-dimensional substrates by seeding human lymphatic endothelial cells (LECs) onto aligned fibers of varying diameters and densities, randomly oriented fibers, and film substrates with no fibers. We found that LECs became aligned in the fiber direction, with cells seeded on the randomly oriented fibers becoming oriented in random directions, whereas cells seeded on the highly aligned fibers became highly aligned. Cell migration was dependent upon fiber alignment and density, with optimal migration found on 1300 nm diameter aligned fibers of low density. Blood endothelial cells seeded on the fibers exhibited similar behavior as the LECs. Fiber alignment was preserved upon rolling the two-dimensional substrate into the tubular geometry of a lymphatic vessel. The data suggest that aligned electrospun fibers may promote endothelial migration across the conduit in a manner that is independent of lymphatic growth factors.

Varying the diameter of aligned electrospun fibers alters neurite outgrowth and Schwann cell migration.

Aligned, electrospun fibers have shown great promise in facilitating directed neurite outgrowth within cell and animal models. While electrospun fiber diameter does influence cellular behavior, it is not known how aligned, electrospun fiber scaffolds of differing diameter influence neurite outgrowth and Schwann cell (SC) migration. Thus, the goal of this study was to first create highly aligned, electrospun fiber scaffolds of varying diameter and then assess neurite and SC behavior from dorsal root ganglia (DRG) explants. Three groups of highly aligned, electrospun poly-l-lactic acid (PLLA) fibers were created (1325+383 nm, large diameter fibers; 759+179 nm, intermediate diameter fibers; and 293+65 nm, small diameter fibers). Embryonic stage nine (E9) chick DRG were cultured on fiber substrates for 5 days and then the explants were stained against neurofilament and S100. DAPI stain was used to assess SC migration. Neurite length and SC migration distance were determined. In general, the direction of neurite extension and SC migration were guided along the aligned fibers. On the small diameter fiber substrate, the neurite length was 42% and 36% shorter than those on the intermediate and large fiber substrates, respectively. Interestingly, SC migration did not correlate with that of neurite extension in all situations. SCs migrated equivalently with extending neurites in both the small and large diameter scaffolds, but lagged behind neurites on the intermediate diameter scaffolds. Thus, in some situations, topography alone is sufficient to guide neurites without the leading support of SCs. Scanning electron microscopy images show that neurites cover the fibers and do not reside exclusively between fibers. Further, at the interface between fibers and neurites, filopodial extensions grab and attach to nearby fibers as they extend down the fiber substrate. Overall, the results and observations suggest that fiber diameter is an important parameter to consider when constructing aligned, electrospun fibers for nerve regeneration applications.