Exposure of the basophilic cell line KU812 to liposomes reveals activation profiles associated with potential anaphylactic responses linked to physico-chemical characteristics

Lipidic nanoparticles (LNP), particularly liposomes, have been proven to be a successful and versatile platform for intracellular drug delivery for decades. Whilst primarily developed for small molecule delivery, liposomes have recently undergone a renaissance due to their success in vaccination strategies, delivering nucleic acids, in the COVID-19 pandemic. As such, liposomes are increasingly being investigated for the delivery of nucleic acids, beyond mRNA, as non-viral gene delivery vectors. Although not generally considered toxic, liposomes are increasingly shown to not be immunologically inert, which may have advantages in vaccine applications but may limit their use in other conditions where immunological responses may lead to adverse events, particularly those associated with complement activation. We sought to assess a small panel of liposomes varying in a number of physico-chemical characteristics associated with complement activation and inflammatory responses, and examine how basophil-like cells may respond to them. Basophils, as well as other cell types, are involved in the anaphylactic responses to liposomes but are difficult to isolate in sufficient numbers to conduct large scale analysis. Here, we report the use of the human KU812 cell line as a surrogate for primary basophils. Multiple phenotypic markers of activation were assessed, as well as the release of histamine and inflammasome activity within the cells. We found that larger liposomes were more likely to result in KU812 activation, and that non-PEGylated liposomes were potent stimulators of inflammasome activity (four-fold greater IL-1β secretion than untreated controls), and a lower ratio of cholesterol to lipid was also associated with greater IL-1β secretion ([Cholesterol:DSPC ratio] 1:10; 0.35 pg/mL IL-1β vs. 5:10; 0.1 pg/mL). Additionally, PEGylation appeared to be associated with direct KU812 activation. These results suggest possible mechanisms related to the consequences of complement activation that may be underpinned by basophilic cells, in addition to other immune cell types. Investigation of the mechanisms behind these responses, and their impact on use in vivo, are now warranted

Childhood bereavement services : a survey of UK provision.

The purpose of the study was to identify the location, range and type of childhood bereavement service provision in the UK. A questionnaire was mailed to 127 services who were either solely dedicated to childhood bereavement or who offered a service within the range of work of a host organization and for which there was a supporting organizational structure. Responses were received from 108 services (a response rate of 85%). The findings identified that 85% of childhood bereavement services are located in the voluntary sector; 14% are dedicated childhood bereavement services, while 86% are offered as part of a host organization. Forty-four per cent of host organizations are hospices. The majority of services (73%) relied on both paid and unpaid staff, with 11% relying entirely on paid staff and 14% of services relying entirely on unpaid staff. The interventions offered ranged from individual family work (86%), individual child work (62%), groupwork with families (53%) and groupwork with children (45%). In addition, services offered prebereavement support (64%), a ’drop-inflservice (17%), information and advice (95%), training (32%) and the provision of resources (88%). As well as offering a service to children and their families, 74% of childhood bereavement services provided a service to ’secondary users‘, such as schools (66%), the emergency services (28%) and other professionals (63%). In terms of funding, 12% of services relied solely on external sources of funding, including donations, legacies, revenue from the host organization or grants, while 12% of services relied solely on internal sources of funding, including fundraising and training. The majority of services (73%), however, gained income from a range of sources. The study identifies the diversity of provision that has implications for the evaluation of childhood bereavement services

Application of KU812 cells for assessing complement activation related effects by nano(bio)materials

Immunocompatibility issues related to nano(bio)materials, particularly liposomal formulations, involving activation of the complement system have been relatively well described however, they highlight the importance of preclinical evaluation of such interactions. These complement-mediated hypersensitivity reactions, in which basophils are implicated, are associated with complement activation-related pseudoallergy (CARPA). Ex vivo investigation of such events using primary basophils is technically challenging due to the relatively limited number of circulating basophils in peripheral blood. In the current work, the KU812 cell line has been applied as an in vitro model for basophil activation to investigate CARPA-related responses following exposure to test materials obtained from the REFINE consortium. To that end, we developed a standard operating procedure measuring a panel of cell-surface markers indicative of basophilic activation. Two laboratories performed the assays, demonstrating a clear difference in responses between liposomal and polymeric nano(bio)materials, while interlaboratory comparison of the standard operating procedure demonstrated reproducibility in results, between the two facilities. These results suggest the potential to use this protocol as a screening method for such responses however, validation using primary basophils is now warranted

Exposure of the basophilic cell line KU812 to liposomes reveals activation profiles associated with, potential, anaphylactic responses linked to physico-chemical characteristics

Lipidic nanoparticles (LNP), particularly liposomes, have been proven to be a successful and versatile platform for intracellular drug delivery for decades. Whilst primarily developed for small molecule delivery, liposomes have recently undergone a renaissance due to their success in vaccination strategies, delivering nucleic acids, in the COVID-19 pandemic. As such, liposomes are increasingly being investigated for the delivery of nucleic acids, beyond mRNA, as non-viral gene delivery vectors. Although not, generally, considered toxic, liposomes are increasingly shown to not be immunologically inert; which may have advantages in vaccine applications but, may limit their use in other conditions were immunological responses may lead to advers events, particularly those associated with complement activation. We sought to assess a small panel of liposomes varying in a number of physico-chemical characteristics, associated with complement activation and inflammatory responses, and examine how basophil-like cells may respond to them. Basophils, as well as other cell types, are involved in the anaphylactic responses to liposomes but, are difficult to isolate in sufficient numbers to conduct large scale analysis. Here, we report the use of the, human, KU812 cell line as a surrogate for primary basophils, in this investigation. Mulitple phenotypic markers of activation were assessed, as well as the release of histamine, and inflammasome activity within the cells. We found that larger liposomes were more likely to result in KU812 activation, and that non-PEGylated liposomes were potent stimulators of inflammasome activity (4-fold greater IL-1β secretion than untreated controls), and a lower ratio of cholesterol to lipid was also associated with greater IL-1β secretion ([Cholesterol:DSPC ratio] 1:10; 0.35pg/mL IL-1β vs 5:10; 0.1pg/mL). Additionally, PEGylation appeared to be associated with direct KU812 actvation. These results suggest possible mechanisms, related to the consequences of complement activation, that may be underpinned by basophilic cells, in addition to other immune cell types. Investigation of the mechanisms behind these responses, and their impact on use in vivo, are now warranted