Background: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to target and deliver novel therapeutics to treat a host of human diseases. Methods: We engineered the surfaces of cell-derived nanovesicles to act as decoys in the treatment of inflammation by antagonizing the major proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Results: Decoy exosomes were generated by displaying the TNFα binding domain of human TNF receptor-1 (hTNFR1) on the outer surface of exosomes using stably transfected HEK293 cells. We developed an efficient method to purify the engineered exosomes from conditioned medium based on sequential centrifugation, ultrafiltration, and precipitation. We characterized decoy exosomes using immune-quantification, nanoparticle tracking analysis, and confocal microscopy to confirm that they retain the correct orientation, size, and shape of naturally produced exosomes. We demonstrated the engineered decoy exosomes specifically antagonize activities of TNFα using an inflammatory reporter cell line. Conclusions: Decoy exosomes produced in human cells serve as a novel biologic reagent for antagonizing inflammatory signaling mediated by TNFα.

Targeted delivery of lysosomal enzymes to the endocytic compartment of human cells represents a transformative technology for treating a large family of lysosomal storage diseases (LSDs). Gaucher disease is one of the most common types of LSDs caused by mutations to the lysosomal β-glucocerebrosidase (GBA). Here, we describe a genetic strategy to produce engineered exosomes loaded with GBA in two different spatial configurations for targeted delivery to the endocytic compartment of recipient cells. By fusing human GBA to an exosome-anchoring protein: vesicular stomatitis virus glycoprotein (VSVG), we demonstrate that the chimeric proteins were successfully integrated into exosomes which were secreted as extracellular vesicles (EVs) by producer cells. Isolation and molecular characterization of EVs confirmed that the fusion proteins were loaded onto exosomes without altering their surface markers, particle size or distribution. Further, enzyme-loaded exosomes/EVs added to cultured medium were taken up by recipient cells. Further, the endocytosed exosomes/EVs targeted to endocytic compartments exhibited a significant increase in GBA activity. Together, we have developed a novel method for targeting and delivery of lysosomal enzymes to their natural location: the endocytic compartment of recipient cells. Since exosomes/EVs have an intrinsic ability to cross the blood-brain-barrier, our technology may provide a new approach to treat severe types of LSDs, including Gaucher disease with neurological complications.

The native exosome transmembrane protein, CD63 can be modified for surface engineering applications.

What is already known about this subject • Written consumer medicine information has been associated with increased medicine knowledge, satisfaction and adherence to therapy, especially in combination with verbal counselling.• Increasing internationally standardized and compulsory consumer medicine information leaflets are being prepared, primarily by pharmaceutical manufacturers. • However, no studies have investigated the impact of Vietnamese standardized written information leaflets on consumers' medicine knowledge and satisfaction with information received.What this study adds • Vietnamese written medicine information was developed for three drug classes.• There was an increase in consumers' medicine knowledge and satisfaction with the information received, after receiving written and verbal medicine information in Vietnamese (the consumers' native language).• The increases in satisfaction and knowledge were maintained 2 months after receiving the information. AimsTo develop three Vietnamese medicine information leaflets and to evaluate their impact in combination with verbal counselling (in Vietnamese) on consumers' medicine knowledge and satisfaction with the written information received. MethodsLeaflets in Vietnamese were developed for three drug classes: biguanides, calcium channel blockers and HMG-CoA reductase inhibitors. A two-group (comparison and intervention), randomized, repeated measures design was used. The comparison group (n = 51) received routine verbal counselling in English. The intervention group (n = 44) received verbal and written information in Vietnamese. Australian Vietnamese-speaking consumers were recruited. Data on consumer knowledge and satisfaction were collected from both groups at three time points: baseline (prior to receiving leaflets and verbal counselling), at 1-2 weeks (first postintervention) and 8 weeks after receiving the information. ResultsAll intervention group respondents were satisfied with the information. Mean medicine knowledge and satisfaction scores increased with time in the intervention group, and were significantly higher than in the comparison group (knowledge, F = 18.5, d.f. = 2, P < 0.0001; satisfaction, F = 19.8, d.f. = 2, P < 0.0001). The increases observed in the intervention group's mean knowledge and satisfaction scores at first postintervention were significantly higher compared with baseline for all drug classes. ConclusionsDelivery of written and verbal medicine information in Vietnamese resulted in an increase in consumers' medicine knowledge and satisfaction with written information. This study highlights the importance of providing information in languages other than English and in consumers' native tongue to aid in the quality use of medicines.

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In the transtibial osteomyoplastic amputation (TOA) technique, the distal ends of the tibia and fibula are surgically joined to form a ''bone bridge'' to stabilize the bony anatomy of the distal residuum. The distal-most muscles also are secured to reestablish a length-tension relationship. Unlike conventional amputation techniques in which the muscles are not secured and do not retain length-tension relationship, the TOA procedure is anticipated to allow muscles to actively contract and retain normal physiological function. In this case series, outcomes of the TOA procedure were investigated by measuring electromyography signals from the tibialis anterior and gastrocnemius muscles in the residuum and forces at the residuum socket interface (RSI) in unilateral transtibial amputees with TOA during three types of gait activities (self-paced walking, brisk 2-minute walking, and walking over a distance of 25 ft while carrying various loads). Results confirmed the presence of loadings at the distal residuum and the activity in the residuum muscles during these gaits. Furthermore, statistical analysis showed that when the distal RSI force variation was higher, the residual tibialis anterior muscle was more active compared with its activity at lower distal RSI force variation. (J Prosthet Orthot. 2013;25:151Y158.)

Background: Recent technological advancements have enabled live-cell imaging of intracellular organelles to monitor their biogenesis in mammalian cells. However, applying this method to gain insight into extracellular organelles, such as extracellular vesicles (EVs), presents unique challenges that require special considerations in design and engineering.Results: We have developed a dual-reporter system that combines genetic fusion, fluorescence microcopy and magnetic beads capture of EVs to study the biogenesis of EVs in mammalian cell cultures. First, we genetically produced a series of reporters by fusing a green fluorescent protein (GFP) and an affinity peptide (6xHis), with either the endogenous transmembrane protein, CD63, or EVs targeting vesicular stomatitis viral glycoprotein (VSVG). Transfection of these reporters into human 293T cells resulted in expression and integration of these reporters into pre-exosome compartments, which were subsequently released into the culture medium. Confocal imaging and nano-particle tracking analysis demonstrated that EVs were appropriately labeled and exhibited a single dominant peak in the 80–110 nm size range, indicating that isolated EVs were comprised of micro-vesicles and/or exosome subpopulations. Incubation of isolated EVs with nickel-coated magnetic beads resulted in successful capture of GFP-positive EVs. Finally, addition of EVs into culture medium was able to reveal the cellular uptake of GFP-labeled EVs by recipient cells. Taken together, our dual-reporter system provides a powerful method for both monitoring and capturing of EVs in mammalian cell culture systems.Conclusion: A dual-reporter system provides a robust tool to study the life cycle of EVs in mammalian cells from biogenesis and excretion to cellular uptake.

This article elucidates the effect of prosthetic foot on the residuum-socket interface (RSI) pressure and gait characteristics in a man with transtibial osteomyoplastic amputation (TOA). The study evaluates the effect of three prosthetic feet, including 1) Renegade Foot\ from Freedom Innovations (Irvine, CA), 2) Venture Footi from College Park (Fraser, MI), and 3) Proprio Foot\ from Ö ssur (Reykjavik, Iceland) in six gait activities: walking forward at ''normal'' pace, walking forward at fast pace, ascending and descending a staircase, and ascending and descending a ramp. Force resistive sensors were placed at six locations, including distal anterior end-bearing, middle posterior, and four proximal points inside the prosthetic socket, to capture real-time RSI pressures. Whereas nominal values of pressure were observed in the proximal region, greater pressure was observed at the distal anterior end-bearing region of the socket, which confirmed one of the intended outcomes of the TOA procedure. Of 36 statistical tests (t-test, p G 0.05), 35 tests (97.2%) confirmed the hypothesis that when the same prosthetic foot was used in the same gait activity, peak and mean pressures are greater at the distal anterior end-bearing location than at other locations. Furthermore, 182 of 216 (84.2%) statistical tests (t-test, significance level of 0.05) supported the hypothesis that at the same measured location during the same gait activity, different prosthetic feet result in different peak (or mean) RSI pressures. Coefficients of variation of the mean sustained pressures showed that when the gait activity was changed, each prosthetic foot affected the sustained pressure differently, even at the same measured location. Each prosthetic foot also had a direct effect on temporal gait parameters such as stance phase and gait cycle durations. These results elucidate the importance for clinicians to understand the characteristics of different prosthetic foot designs to match with the specific needs of the client with amputation. (J Prosthet Orthot. 2012;24:211Y220.) KEY INDEXING TERMS: prosthetic socket pressure, transtibial osteomyoplastic amputation, gait activity, residuum-socket interface T he prosthetic socket plays a critical role in providing comfort, appropriate load transmission, and efficient movement control in people with transtibial amputation (TTA). 1 The adverse consequences of ill-fitting socket or improper loading include soft tissue injury, bleeding, bruising, pressure sores, and pain, which in the short-term reduce the functional ability of people with amputation. If left unaddressed, these can diminish the individual's health and quality of life. 2,3 The interface pressure distribution between the prosthetic socket and the residual limb (residuum-socket interface [RSI]) is a critical consideration in the design and fit of a socket to a patient. 4,5 Proper distribution of an individual's weight across the surface of the residuum plays a critical role in the comfort and function of people with TTA. 6,7 Furthermore, th...