Gender comparison in a murine model of allergen-driven airway inflammation and the response to budesonide treatment

BACKGROUND: Evidence suggests that gender differences exist in the severity of many immunological diseases and their response to glucocorticosteroid treatment. In this report, we have used a murine model of ovalbumin-induced lung inflammation to address whether gender could affect the systemic response, airway inflammation and hyperreactivity and their responses to budesonide. RESULTS: Following an acute ovalbumin challenge, actively sensitised BALB/c mice developed a time-dependent increase in interleukin-4 and interleukin-5 production and inflammatory cell influx into bronchoalveolar lavage fluid. Apart from an increased number of lymphocytes in female mice at day 3 post-challenge, none of the above parameters were affected by gender. Blood leukocyte numbers were also unaffected, whereas a two-fold increase in total serum immunoglobulin E was observed in female mice. Budesonide, given intranasally, did not affect the blood parameters, but dose-dependently inhibited the pulmonary inflammation and airway hyperreactivity in both male and female mice. Female mice were slightly less sensitive to budesonide's inhibitory action on interleukin-5 production and the development of airway hyperreactivity. CONCLUSIONS: Our results suggest that, apart from a 2-fold increase in serum immunoglobulin E levels observed in female mice, gender is not a major factor in the present murine model of ovalbumin-induced lung inflammation. In contrast, gender might slightly influence the potency of test compounds such as steroids

Development of downstream processing options for the commercial scale purification of stem cell derived exosomes

Exosomes (or extracellular vesicles) are rapidly gaining momentum as a novel type of regenerative medicine. Exosomes are nano-vesicles in the size range of 20-150nm that are secreted by mammalian cell types (including stem cells). They can induce potent biological functions in surrounding target cells to induce effects that mimic those of the producing cell. These functions can be regenerative, immunomodulatory, anti-scarring and even anti-cancer depending on the state of the producer cell. Consequently, there is enormous potential to develop a range of function-specific products across a wide variety of indications similar to macromolecular blockbusters, but which extend beyond symptom management to produce curative outcomes. However, current methods for purifying exosomes have been based on technologies grandfathered in from the viral industry which are not suitable for large scale, high purity production but are robust enough to perform preliminary characterisation of the exosome population composition. Please click Additional Files below to see the full abstract

Manufacturing Exosomes:A Promising Therapeutic Platform

Extracellular vesicles, in particular the subclass exosomes, are rapidly emerging as a novel therapeutic platform. However, currently very few clinical validation studies and no clearly defined manufacturing process exist. As exosomes progress towards the clinic for treatment of a vast array of diseases, it is important to define the engineering basis for their manufacture early in the development cycle to ensure they can be produced cost-effectively at the appropriate scale. We hypothesize that transitioning to defined manufacturing platforms will increase consistency of the exosome product and improve their clinical advancement as a new therapeutic tool. We present manufacturing technologies and strategies that are being implemented and consider their application for the transition from bench-scale to clinical production of exosomes

A step closer to industrial scale manufacture of exosomes - Adaptation of clinical grade neural stem cells from 2D to 3D culture

Exosomes derived from the clinical grade neural stem cell line CTX (ReNeuron) are the basis of a new class of therapy for the treatment of degenerative disorders. Thus far we have generated CTX-derived exosomes at research scale in 2D planar cultures. Now the cell culture process needs to be scaled up in order to deliver commercially relevant quantities of exosomes that have the correct quality attributes. To meet these demands, CTX cells, which are adherent and habitually grown in a 2D static environment, must be adapted for growth in 3D agitated bioreactor systems. Please click Additional Files below to see the full abstract

The Development of Stem Cell-derived Exosomes as a Cell-free Regenerative Medicine

A successful strategy in regenerative medicine over the last decade has been the translation of stem cell therapy to repair diseased or damaged tissue in a wide range of indications, despite limited evidence attributing any therapeutic benefit to cell survival or differentiation. Recent findings, however, have demonstrated that the conditioned media from stem cell cultures can produce similar efficacious effects compared to those observed for cells. This has led to the stem cell paracrine hypothesis, proposing that secreted factors released from the stem cells contribute significantly to their beneficial effects. It has been well documented that stem cells have the ability to release a range of growth factors, cytokines and chemokines relevant to their function; however, these factors are released at levels too low to account for the reported therapeutic effects. Further purification of the conditioned media has since identified that not only are small molecules released by the stem cells, but so too are a large quantity of membrane-bound vesicles, including exosomes, in a functionally relevant manner. In this review, we present our current understanding and explore the evidence supporting the development of stem cell-derived exosomes as a cell-free regenerative medicine

Investigating heparin affinity chromatography for extracellular vesicle purification and fractionation

The purification of extracellular vesicles (EVs) remains a major hurdle in the progression of fundamental research and the commercial application of EV-based products. In this study, we evaluated the potential of heparin affinity chromatography (HAC) to purify neural stem cell-derived EVs as part of a multistep process. Bind-elute chromatography, such as HAC, is an attractive method of purification because it is highly scalable, robust and can be automated. Our findings support an interaction between EVs and heparin. The recovery of EVs using HAC based on particle counts was a minimum of 68.7%. We found HAC could remove on average 98.8% and 99.0% of residual protein and DNA respectively. In addition to EV purification, HAC was used to separate EVs into three populations based on their affinity to the heparin column. Within these populations, we detected differences in the expression of the exosome-associated protein TSG101 and the tetraspanin immunophenotype. However, the significance of these observations is not clear. Overall HAC shows promise as a potential purification method to capture EVs and this study proposes a novel application of HAC for EV fractionation. Moving forward, a better understanding of the heparin-EV interaction would be required before HAC can be more widely adopted for these applications

The role of TRPC channels in human airway smooth muscle cell homeostasis

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