Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory agent produced primarily by activated monocytes and macrophages. TNF-alpha is synthesized as a precursor protein of M(r) 26,000 (26K) which is processed to a secreted 17K mature form by cleavage of an Ala-Val bond between residues 76-77. The enzyme(s) responsible for processing pro-TNF-alpha has yet to be identified. Here, we describe the capacity of a metalloproteinase inhibitor, GI 129471, to block TNF-alpha secretion both in vitro and in vivo. The inhibition is specific to TNF-alpha; the production of other secreted cytokines, such as the interleukins IL-1 beta, IL-2, or IL-6, is not inhibited. The mechanism of inhibition occurs at a post-translational step in TNF-alpha production. Our data suggest that TNF-alpha processing is mediated by a unique Zn2+ endopeptidase which is inhibited by GI 129471 and would represent a novel target for therapeutic intervention in TNF-alpha associated pathologies.

Any polar-ordered material with a spatially uniform polarization field is internally frustrated: The symmetry-required local preference for polarization is to be nonuniform, i.e., to be locally bouquet-like or "splayed." However, it is impossible to achieve splay of a preferred sign everywhere in space unless appropriate defects are introduced into the field. Typically, in materials like ferroelectric crystals or liquid crystals, such defects are not thermally stable, so that the local preference is globally frustrated and the polarization field remains uniform. Here, we report a class of fluid polar smectic liquid crystals in which local splay prevails in the form of periodic supermolecular-scale polarization modulation stripes coupled to layer undulation waves. The polar domains are locally chiral, and organized into patterns of alternating handedness and polarity. The fluid-layer undulations enable an extraordinary menagerie of filament and planar structures that identify such phases.

Assembling structures to divide space controllably and spontaneously into subunits at the nanometer scale is a significant challenge, although one that biology has solved in two distinct ways: prokaryotes and eukaryotes. Prokaryotes have a single compartment delimited by one or more lipid-protein membranes. Eukaryotes have nested-membrane structures that provide internal compartments--such as the cell nucleus and cell organelles in which specialized functions are carried out. We have developed a simple method of creating nested bilayer compartments in vitro via the "interdigitated" bilayer phase formed by adding ethanol to a variety of saturated phospholipids. At temperatures below the gel-liquid crystalline transition, T(m), the interdigitated lipid-ethanol sheets are rigid and flat; when the temperature is raised above T(m), the sheets become flexible and close on themselves and the surrounding solution to form closed compartments. During this closure, the sheets can entrap other vesicles, biological macromolecules, or colloidal particles. The result is efficient and spontaneous encapsulation without disruption of even fragile materials to form biomimetic nano-environments for possible use in drug delivery, colloidal stabilization, or as microreactors. The vesosome structure can take full advantage of the 40 years of progress in liposome development including steric stabilization, pH loading of drugs, and intrinsic biocompatibility. However, the multiple compartments of the vesosome give better protection to the interior contents in serum, leading to extended release of model compounds in comparison to unilamellar liposomes.

Unilamellar vesicles or "liposomes" are commonly used as simple cell models and as drug delivery vehicles. A major limitation of unilamellar liposomes in these applications has been premature contents release in physiological environments. This premature release is likely due to enzyme degradation or protein insertion into the liposome membrane, which significantly increases the bilayer permeability. Encapsulating unilamellar liposomes within a second bilayer to form multicompartment "vesosomes" extends contents retention by 2 orders of magnitude by preventing enzymes and/or proteins from reaching the interior bilayers. The multicompartment structure of the vesosome can also allow for independent optimization of the interior compartments and exterior bilayer; however, just the bilayer-within-a-bilayer structure of the vesosome is sufficient to increase drug retention from minutes to hours. The vesosome is a better mimic of eukaryotic cell structure and demonstrates the benefits of multiple internal bilayer-enclosed compartments.

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Degenerative disc disease (DDD) primarily affects the central part of the intervertebral disc namely the nucleus pulposus (NP). DDD explains about 40% of low back pain and is characterized by massive cellular alterations that ultimately result in the disappearance of resident NP cells. Thus, repopulating the NP with regenerative cells is a promising therapeutic approach and remains a great challenge. The objectives of this study were to evaluate the potential of growth factor-driven protocols to commit human adipose stromal cells (hASCs) toward NP-like cell phenotype and the involvement of Smad proteins in this differentiation process. Here, we demonstrate that the transforming growth factor-b1 and the growth differentiation factor 5 synergistically drive the nucleopulpogenic differentiation process. The commitment of the hASCs was robust and highly specific as attested by the expression of NP-related genes characteristic of young healthy human NP cells. In addition, the engineered NP-like cells secreted an abundant aggrecan and type II collagen rich extracellular matrix comparable with that of native NP. Furthermore, we demonstrate that these in vitro engineered cells survived, maintained their specialized phenotype and secretory activity after in vivo transplantation in nude mice subcutis. Finally, we provide evidence suggesting that the Smad 2/3 pathway mainly governed the acquisition of the NP cell molecular identity while the Smad1/5/8 pathway controlled the NP cell morphology. This study offers valuable insights for the development of biologically-inspired treatments for DDD by generating adapted and exhaustively characterized autologous regenerative cells. STEM CELLS 2016;34:653-667 SIGNIFICANCE STATEMENTIn the present manuscript, we investigated whether human adipose stromal cells (hASCs) can be a clinically relevant source of stem cells for the generation of phenotypically stable and biologically active NPCyte-like cells. We successfully generated NPCyte-like cells from hASCs using a reproducible, robust and accurate growth factor-based induction protocol. We also demonstrated by experimental organogenesis in nude mice subcutis, that NPCyte-like cells seeded in an instructive hydrogel survived, maintained their specialized phenotype and presented secretory activities in vivo. In addition to highlighting the robustness and reproducibility of our protocol, we also document the temporal role of Smad pathways towards NP commitment by using specific chemical inhibitors. These data strengthen the specificity of the NP cell commitment by demonstrating new insights into the role of Smad proteins.

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Vesosome with distinct interior (green, orange) and exterior (blue) bilayer membranes. Drugs are encapsulated within the interior compartments and are protected from blood serum components by the exterior membrane. The outer membrane can be decorated with PEG lipids (red) to prevent aggregation in the blood as well as specific labels or targeting ligands.