Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles.

Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell-related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV-based therapeutics at the preclinical and clinical levels. This review outlines topic-specific action items that, if addressed, will enhance the development of best-practice models for EV therapies. Stem Cells Translational Medicine 2017;6:1730-1739.

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

Challenges with advanced therapy medicinal products and how to meet them.

Advanced therapy medicinal products (ATMPs), which include gene therapy medicinal products, somatic cell therapy medicinal products and tissue-engineered products, are at the cutting edge of innovation and offer a major hope for various diseases for which there are limited or no therapeutic options. They have therefore been subject to considerable interest and debate. Following the European regulation on ATMPs, a consolidated regulatory framework for these innovative medicines has recently been established. Central to this framework is the Committee for Advanced Therapies (CAT) at the European Medicines Agency (EMA), comprising a multidisciplinary scientific expert committee, representing all EU member states and European Free Trade Association countries, as well as patient and medical associations. In this article, the CAT discusses some of the typical issues raised by developers of ATMPs, and highlights the opportunities for such companies and research groups to approach the EMA and the CAT as a regulatory advisor during development.

Dual role of SLP-76 in mediating T cell receptor-induced activation of phospholipase C-gamma1.

Phospholipase C-gamma1 (PLC-gamma1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-gamma1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-gamma1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-gamma1 GEM recruitment, but were required for PLC-gamma1 phosphorylation at Tyr(783). Thus, GEM recruitment can be insufficient for full activation of PLC-gamma1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-gamma1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-gamma1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-gamma1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.

A new tyrosine phosphorylation site in PLC gamma 1: the role of tyrosine 775 in immune receptor signaling.

Phospholipase Cgamma (PLCgamma) is a ubiquitous gatekeeper of calcium mobilization and diacylglycerol-mediated events induced by the activation of Ag and growth factor receptors. The activity of PLCgamma is regulated through its controlled membrane translocation and tyrosine (Y) phosphorylation. Four activation-induced tyrosine phosphorylation sites have been previously described (Y472, Y771, Y783, and Y1254), but their specific roles in Ag receptor-induced PLCgamma1 activation are not fully elucidated. Unexpectedly, we found that the phosphorylation of a PLCgamma1 construct with all four sites mutated to phenylalanine was comparable with that observed with wild-type PLCgamma1, suggesting the existence of an unidentified site(s). Sequence alignment with known phosphorylation sites in PLCgamma2 indicated homology of PLCgamma1 tyrosine residue 775 (Y775) with PLCgamma2 Y753, a characterized phosphorylation site. Tyrosine 775 was characterized as a phosphorylation site using phospho-specific anti-Y775 antiserum, and by mutational analysis. Phosphorylation of Y775 did not depend on the other tyrosines, and point mutation of PLCgamma1 Y775, or the previously described Y783, substantially reduced AgR-induced calcium, NF-AT, and AP-1 activation. Mutation of Y472, Y771, and Y1254 had no effect on overall PLCgamma1 phosphorylation or activation. Although the concomitant mutation of Y775 and Y783 abolished downstream PLCgamma1 signaling, these two tyrosines were sufficient to reconstitute the wild-type response in the absence of functional Y472, Y771, and Y1254. These data establish Y775 as a critical phosphorylation site for PLCgamma1 activation and confirm the functional importance of Y783.

Surface expression of Fc epsilon RI on Langerhans' cells of clinically uninvolved skin is associated with disease activity in atopic dermatitis, allergic asthma, and rhinitis.

BACKGROUND: Fc epsilon RI expressed on the surface of human epidermal Langerhans' cells facilitates uptake of IgE-associated allergens and plays a pivotal role in the pathogenesis of atopic dermatitis. Seminal results from studies investigating Langerhans' cell Fc epsilon RI in skin biopsy sections or epidermal cell suspensions demonstrate the highest receptor expression in lesional skin of patients with active atopic dermatitis. OBJECTIVE: We sought to investigate and localize Fc epsilon RI expression on Langerhans' cells within a minimally disturbed tissue environment in clinically uninvolved skin and to compare receptor expression between healthy donors and patients with atopic dermatitis or other allergic diseases. METHODS: Intact epidermal sheets from skin suction blisters, immunofluorescently stained with Langerhans' cell markers and anti-Fc epsilon RI alpha (mAbs 15E5 and 22E7) or anti-IgE, were examined by means of confocal microscopy. Samples incubated with anti-Fc epsilon RI alpha before or after cell fixation-permeabilization were compared to discriminate between cytoplasmic and membrane localization. RESULTS: Cytoplasmic Fc epsilon RI alpha chain was found in Langerhans' cells from all donors, irrespective of atopic status. Surface Fc epsilon RI-bound IgE was detected in the skin of individuals with active atopic dermatitis and in the skin of those with active asthma or rhinitis. No surface Fc epsilon RI was expressed in the skin of patients with a clinical history of atopic dermatitis, asthma, or rhinitis whose disease was in remission or in the skin of nonatopic individuals. CONCLUSION: In clinically uninvolved skin, Langerhans' cell-surface Fc epsilon RI expression is not only linked to atopic dermatitis but is also generally associated with allergic disease. This supports the concept of a systemic regulatory mechanism associated with active allergic disease, which is further aggravated by local inflammation in atopic skin lesions.