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Respiratory sensitization is a complex immunological process eventually leading to hypersensitivity following re-exposure to the chemical. A frequent consequence is occupational asthma, which may occur after long latency periods. Although chemical-induced respiratory hypersensitivity has been known for decades, there are currently no comprehensive and validated approaches available for the prospective identification of chemicals that induce respiratory sensitization, while the expectations of new approach methodologies (NAMs) are high. A great hope is that due to a better understanding of the molecular key events, new methods can be developed now. However, this is a big challenge due to the different chemical classes to which respiratory sensitizers belong, as well as because of the complexity of the response and the late manifestation of symptoms. In this review article, the current information on respiratory sensitization related processes is summarized by introducing it in the available adverse outcome pathway (AOP) concept. Potentially useful models for prediction are discussed. Knowledge gaps and gaps of regulatory concern are identified.
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The European Union (EU) Chemicals Strategy for Sustainability regards chemicals that affect the immune system among the most harmful ones. The Extended One-Generation Reproductive Toxicity study (EOGRTS; Organisation for Economic Co-Operation and Development (OECD) Test Guideline (TG) 443), addresses, among others, potential effects of chemicals on development. In specific cases, the EOGRTS is performed with addition of a so-called cohort 3, that addresses potential effects on the developing immune system, by means of a central assay measuring the T-cell dependent antibody response (TDAR). This assay is based on an interplay of antigen presentation, T-cell help and antibody production by B-cells, and together comprises a functional immune response. In the context of the EOGRTS review project of the European Chemicals Agency (ECHA), we evaluated 15 available TDARs for compliance with conduct and reporting requirements. Collectively, the majority of the TDAR studies were considered to be adequately conducted. We however observed: (i) the protocols differed by the antigen used (sheep red blood cells (SRBC) or KLH), the route of administration (intravenous, intraperitoneal, or subcutaneous), prime or prime/boost immunizations, and whether IgG was measured. (ii) There was major variation in the effects of the positive control for immunosuppression, cyclophosphamide. (iii) Proficiency was not always shown. (iv) Statistical analysis was not always done or reported. (v) Results of effects on lymphocyte populations or other immunotoxicity observations obtained in cohort 1 (or 2) of the EOGRTS were not always discussed together with results of the TDAR. Taken together, next to an improved quality of reporting, this may suggest a need to better define the conduct of the TDAR in OECD TG 443 and OECD Guidance Document (GD) 151, at least for certain aspects.
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União Europeia , Reprodução , Linfócitos T , Testes de Toxicidade , Animais , Testes de Toxicidade/métodos , Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Formação de Anticorpos/efeitos dos fármacos , HumanosRESUMO
Macrophages are well known for their involvement in the biocompatibility, as well as biodistribution, of nano(bio)materials. Although there are a number of rodent cell lines, they may not fully recapitulate primary cell responses, particularly those of human cells. Isolation of tissue-resident macrophages from humans is difficult and may result in insufficient cells with which to determine the possible interaction with nano(bio)materials. Isolation of primary human monocytes and differentiation to monocyte-derived macrophages may provide a useful tool with which to further study these interactions. To that end, we developed a standard operating procedure for this differentiation, as part of the Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices (REFINE) project, and used it to measure the secretion of bioactive molecules from M1 and M2 differentiated monocytes in response to model nano(bio)materials, following an initial assessment of pyrogenic contamination, which may confound potential observations. The SOP was deployed in two partner institutions with broadly similar results. The work presented here shows the utility of this assay but highlights the relevance of donor variability in responses to nano(bio)materials. Whilst donor variability can provide some logistical challenges to the application of such assays, this variability is much closer to the heterogeneous cells that are present in vivo, compared to homogeneous non-human cell lines.
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Materiais Biocompatíveis , Diferenciação Celular , Macrófagos , Monócitos , Fenótipo , Humanos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/citologia , Células CultivadasRESUMO
As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).
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For the batch release of vaccines, potency release assays are required. Non-animal in vitro tests have numerous advantages and are preferred; however, several vaccines are still released using in vivo assays. Their major drawback is the inherent variability with its practical implications. We quantified the variability of in vivo potency release assays for whole-cell pertussis, inactivated polio and meningococcal B (MenB) vaccines which showed large CV (Coefficient of Variation) ranging from 34% to 125%. As inherent variability might potentially be attributed to the highly variable immune system between individual animals, we evaluated the antibody titres to four MenB antigens in 344 individual outbred mice. These varied strongly, with more than 100-fold differences in antibody titres in responsive mice. Furthermore, within individual mice there was generally no correlation between the strengths of the responses to the four antigens. A mouse with a very low or no response to one antigen in many cases exhibited a strong response to another antigen. The large differences between individual animals is likely a considerable contributor to the inherent variability of in vivo potency assays. Our data again support the notion that it is preferred to move away from in vivo potency assays for monitoring batch to batch consistency as part of vaccine batch release testing.
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Vacinas Meningocócicas , Coqueluche , Camundongos , Animais , Vacinas de Produtos InativadosRESUMO
BACKGROUND: The establishment of reliable and robust in vitro models for hazard assessment, a prerequisite for moving away from animal testing, requires the evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface (ALI) are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as a monoculture and, to increase the physiologic relevance of the model, also as a co-culture with macrophages (either derived from the THP-1 monocyte cell line or from human blood monocytes). The lung model was exposed to NMs using the VITROCELL® Cloud12 system at physiologically relevant dose levels. RESULTS: Overall, the results of the 7 participating laboratories are quite similar. After exposing Calu-3 alone and Calu-3 co-cultures with macrophages, no effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 particles on the cell viability and barrier integrity were detected. LPS exposure induced moderate cytokine release in the Calu-3 monoculture, albeit not statistically significant in most labs. In the co-culture models, most laboratories showed that LPS can significantly induce cytokine release (IL-6, IL-8 and TNF-α). The exposure to quartz and TiO2 particles did not induce a statistically significant increase in cytokine release in both cell models probably due to our relatively low deposited doses, which were inspired by in vivo dose levels. The intra- and inter-laboratory comparison study indicated acceptable interlaboratory variation for cell viability/toxicity (WST-1, LDH) and transepithelial electrical resistance, and relatively high inter-laboratory variation for cytokine production. CONCLUSION: The transferability and reproducibility of a lung co-culture model and its exposure to aerosolized particles at the ALI were evaluated and recommendations were provided for performing inter-laboratory comparison studies. Although the results are promising, optimizations of the lung model (including more sensitive read-outs) and/or selection of higher deposited doses are needed to enhance its predictive value before it may be taken further towards a possible OECD guideline.
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Lipopolissacarídeos , Quartzo , Animais , Humanos , Técnicas de Cocultura , Reprodutibilidade dos Testes , Pulmão , CitocinasRESUMO
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.
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Hipersensibilidade a Drogas , Hipersensibilidade , Humanos , Reprodutibilidade dos Testes , Ativação do Complemento , Lipossomos , Proteínas do Sistema ComplementoRESUMO
Physiologically-based kinetic (PBK) modeling is a valuable tool to understand the kinetics of nanoparticles (NPs) in vivo. However, estimating PBK parameters remains challenging and commonly requires animal studies. To develop predictive models to estimate PBK parameter values based on NP characteristics, a database containing PBK parameter values and corresponding NP characteristics is needed. As a first step toward this objective, this study estimates PBK parameters for gold NPs (AuNPs) and provides a comparison of two different NPs. Two animal experiments are conducted in which varying doses of AuNPs attached with polyethylene glycol (PEG) are administered intravenously to rats. The resulting Au concentrations are used to estimate PBK model parameters. The parameters are compared with PBK parameters previously estimated for poly(alkyl cyanoacrylate) NPs loaded with cabazitaxel and for LipImage 815. This study shows that a small initial database of PBK parameters collected for three NPs is already sufficient to formulate new hypotheses on NP characteristics that may be predictive of PBK parameter values. Further research should focus on developing a larger database and on developing quantitative models to predict PBK parameter values.
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Ouro , Nanopartículas Metálicas , Ratos , Animais , Cinética , Polietilenoglicóis , CianoacrilatosRESUMO
Air-liquid interface (ALI) lung cell models cultured on permeable transwell inserts are increasingly used for respiratory hazard assessment requiring controlled aerosolization and deposition of any material on ALI cells. The approach presented herein aimed to assess the transwell insert-delivered dose of aerosolized materials using the VITROCELL® Cloud12 system, a commercially available aerosol-cell exposure system. An inter-laboratory comparison study was conducted with seven European partners having different levels of experience with the VITROCELL® Cloud12. A standard operating procedure (SOP) was developed and applied by all partners for aerosolized delivery of materials, i.e., a water-soluble molecular substance (fluorescence-spiked salt) and two poorly soluble particles, crystalline silica quartz (DQ12) and titanium dioxide nanoparticles (TiO2 NM-105). The material dose delivered to transwell inserts was quantified with spectrofluorometry (fluorescein) and with the quartz crystal microbalance (QCM) integrated in the VITROCELL® Cloud12 system. The shape and agglomeration state of the deposited particles were confirmed with transmission electron microscopy (TEM). Inter-laboratory comparison of the device-specific performance was conducted in two steps, first for molecular substances (fluorescein-spiked salt), and then for particles. Device- and/or handling-specific differences in aerosol deposition of VITROCELL® Cloud12 systems were characterized in terms of the so-called deposition factor (DF), which allows for prediction of the transwell insert-deposited particle dose from the particle concentration in the aerosolized suspension. Albeit DF varied between the different labs from 0.39 to 0.87 (mean (coefficient of variation (CV)): 0.64 (28%)), the QCM of each VITROCELL® Cloud 12 system accurately measured the respective transwell insert-deposited dose. Aerosolized delivery of DQ12 and TiO2 NM-105 particles showed good linearity (R2 > 0.95) between particle concentration of the aerosolized suspension and QCM-determined insert-delivered particle dose. The VITROCELL® Cloud 12 performance for DQ12 particles was identical to that for fluorescein-spiked salt, i.e., the ratio of measured and salt-predicted dose was 1.0 (29%). On the other hand, a ca. 2-fold reduced dose was observed for TiO2 NM-105 (0.54 (41%)), which was likely due to partial retention of TiO2 NM-105 agglomerates in the vibrating mesh nebulizer of the VITROCELL® Cloud12. This inter-laboratory comparison demonstrates that the QCM integrated in the VITROCELL® Cloud 12 is a reliable tool for dosimetry, which accounts for potential variations of the transwell insert-delivered dose due to device-, handling- and/or material-specific effects. With the detailed protocol presented herein, all seven partner laboratories were able to demonstrate dose-controlled aerosolization of material suspensions using the VITROCELL® Cloud12 exposure system at dose levels relevant for observing in vitro hazard responses. This is an important step towards regulatory approved implementation of ALI lung cell cultures for in vitro hazard assessment of aerosolized materials.
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Extremidade Superior , Fluoresceína , Correlação de DadosRESUMO
Usage of injectable dermal fillers applied for aesthetic purposes has extensively increased over the years. As such, the number of related adverse reactions has increased, including patients showing severe complications such as product migration, topical swelling and inflammatory reactions of the skin. In order to understand the underlying molecular events of these adverse reactions we performed a genome-wide gene expression study on the multi-cell type human Phenion® Full-Thickness Skin Model exposed to five experimental hyaluronic acid (HA) preparations with increasing cross-linking degree, four commercial fillers from Perfectha®, and non-resorbable filler Bio-Alcamid®. In addition, we evaluated whether cross-linking degree or particle size of the HA-based fillers could be associated with the occurrence of adverse effects. In all cases, exposure to different HA fillers resulted in a clearly elevated gene expression of cytokines and chemokines related to acute inflammation as part of the foreign body response. Furthermore, for one experimental filler genes of OXPHOS complexes I-V were significantly down-regulated (adjusted p-value < 0.05), resulting in mitochondrial dysfunction which can be linked to over-expression of pro-inflammatory cytokines TNFα and IL-1ß and chemokine CCL2. Our hypothesis that cross-linking degree or particle size of the HA-based fillers is related to the biological responses induced by these fillers could only partially be confirmed for particle size. In conclusion, our innovative approach resulted in gene expression changes from a human 3D skin model exposed to dermal fillers that mechanistically substantiate aforementioned adverse reactions, and thereby adds to the weight of evidence that these fillers may induce inflammatory and fibrotic responses.
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Preenchedores Dérmicos , Corpos Estranhos , Envelhecimento da Pele , Humanos , Ácido Hialurônico/farmacologia , Preenchedores Dérmicos/efeitos adversos , Transcriptoma , Materiais Biocompatíveis/efeitos adversos , Citocinas/genéticaRESUMO
Safety and potency assessment for batch release testing of established vaccines still relies partly on animal tests. An important avenue to move to batch release without animal testing is the consistency approach. This approach is based on thorough characterization of the vaccine to identify critical quality attributes that inform the use of a comprehensive set of non-animal tests to release the vaccine, together with the principle that the quality of subsequent batches follows from their consistent production. Many vaccine antigens are by themselves not able to induce a protective immune response. The antigens are therefore administered together with adjuvant, most often by adsorption to aluminium salts. Adjuvant function is an important component of vaccine potency, and an important quality attribute of the final product. Aluminium adjuvants are capable of inducing NLRP3 inflammasome activation. The aim of this study was to develop and evaluate an in vitro assay for NLRP3 inflammasome activation by aluminium-adjuvanted vaccines. We evaluated the effects of Diphtheria-Tetanus-acellular Pertussis combination vaccines from two manufacturers and their respective adjuvants, aluminium phosphate (AP) and aluminium hydroxide (AH), in an in vitro assay for NLRP3 inflammasome activation. All vaccines and adjuvants tested showed a dose-dependent increase in IL-1ß production and a concomitant decrease in cell viability, suggesting NLRP3 inflammasome activation. The results were analysed by benchmark dose modelling, showing a similar 50% effective dose (ED50) for the two vaccine batches and corresponding adjuvant of manufacturer A (AP), and a similar ED50 for the two vaccine batches and corresponding adjuvant of manufacturer B (AH). This suggests that NLRP3 inflammasome activation is determined by the adjuvant only. Repeated freeze-thaw cycles reduced the adjuvant biological activity of AH, but not AP. Inflammasome activation may be used to measure adjuvant biological activity as an important quality attribute for control or characterization of the adjuvant.
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Vacinas contra Difteria, Tétano e Coqueluche Acelular , Difteria , Tétano , Coqueluche , Adjuvantes Imunológicos/farmacologia , Alumínio , Hidróxido de Alumínio/farmacologia , Anticorpos Antibacterianos , Linhagem Celular , Difteria/prevenção & controle , Vacina contra Difteria, Tétano e Coqueluche , Humanos , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Vacina contra Coqueluche , Tétano/prevenção & controle , Coqueluche/prevenção & controleRESUMO
Nanoparticles including nanomedicines are known to be recognised by and interact with the immune system. As these interactions may result in adverse effects, for safety evaluation, the presence of such interactions needs to be investigated. Nanomedicines in particular should not unintendedly interact with the immune system, since patient's exposure is not minimised as in the case of 'environmental' nanoparticles, and repeated exposure may be required. NLRP3 inflammasome activation and dendritic cell (DC) maturation are two types of immune mechanisms known to be affected by nanoparticles including nanomedicines. NLRP3 inflammasome activation results in production of the pro-inflammatory cytokines IL-1ß and IL-18, as well as a specific type of cell death, pyroptosis. Moreover, chronic NLRP3 inflammasome activation has been related to several chronic diseases. Upon maturation, DC activate primary T cells; interference with this process may result in inappropriate activation and skewing of the adaptive immune response. Here, we evaluated the effect of two nanomedicines, representing nanostructured lipid carriers and polymers, on these two assays. Moreover, with a view to possible future standardisation and regulatory application, these assays were subject to an inter-laboratory comparison study using common SOPs. One laboratory performed three independent NLRP3 inflammasome activation experiments, while the other performed a single experiment. Two laboratories each performed three independent DC maturation experiments. While the nanostructured lipid carrier only showed marginal effects, the polymers showed major cytotoxicity. No evidence for inflammasome activation or DC maturation was demonstrated. Intra- and inter-laboratory comparison showed clearly reproducible results.
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Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Células Dendríticas , Humanos , Inflamassomos/metabolismo , Lipídeos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nanomedicina , PolímerosRESUMO
Nanotechnologies such as nanoparticles are established components of new medical devices and pharmaceuticals. The use and distribution of these materials increases the requirement for standardized evaluation of possible adverse effects, starting with a general cytotoxicity screening. The Horizon 2020 project "Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices (REFINE)" identified in vitro cytotoxicity quantification as a central task and first step for risk assessment and development for medical nanocarriers. We have performed an interlaboratory comparison on a cell-assay matrix including a kinetic lactate dehydrogenase (LDH) release cell death and WST-8 cell viability assay adapted for testing organic nanocarriers in four well-characterized cell lines of different organ origins. Identical experiments were performed by three laboratories, namely the Biomedical Technology Center (BMTZ) of the University of Münster, SINTEF Materials and Chemistry (SINTEF), and the National Institute for Public Health and the Environment (RIVM) of the Netherlands according to new standard operating procedures (SOPs). The experiments confirmed that LipImage™ 815 lipidots® are non-cytotoxic up to a concentration of 128 µg/mL and poly(alkyl cyanoacrylate) (PACA) nanoparticles for drug delivery of cytostatic agents caused dose-dependent cytotoxic effects on the cell lines starting from 8 µg/mL. PACA nanoparticles loaded with the active pharmaceutical ingredient (API) cabazitaxel showed a less pronounced dose-dependent effect with the lowest concentration of 2 µg/mL causing cytotoxic effects. The mean within laboratory standard deviation was 4.9% for the WST-8 cell viability assay and 4.0% for the LDH release cell death assay, while the between laboratory standard deviation was 7.3% and 7.8% for the two assays, respectively. Here, we demonstrated the suitability and reproducibility of a cytotoxicity matrix consisting of two endpoints performed with four cell lines across three partner laboratories. The experimental procedures described here can facilitate a robust cytotoxicity screening for the development of organic nanomaterials used in medicine.
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Nanopartículas , Linhagem Celular , Sobrevivência Celular , L-Lactato Desidrogenase/metabolismo , Nanopartículas/toxicidade , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Resorbable tissue fillers for aesthetic purposes can induce severe complications including product migration, late swelling, and inflammatory reactions. The relation between product characteristics and adverse effects is not well understood. We hypothesized that the degree of cross-linking hyaluronic acid (HA) fillers was associated with the occurrence of adverse effects. Five experimental HA preparations similar to HA fillers were synthesized with an increasing degree of cross-linking. Furthermore, a series of commercial fillers (Perfectha®) was obtained that differ in degradation time based on the size of their particulate HA components. Cytotoxic responses and cytokine production by human THP-1-derived macrophages exposed to extracts of the evaluated resorbable HA fillers were absent to minimal. Gene expression analysis of the HA-exposed macrophages revealed the responses related to cell cycle control and immune reactivity. Our results could not confirm the hypothesis that the level of cross-linking in our experimental HA fillers or the particulate size of commercial HA fillers is related to the induced biological responses. However, the evaluation of cytokine induction and gene expression in macrophages after biomaterial exposure presents promising opportunities for the development of methods to identify cellular processes that may be predictive for biomaterial-induced responses in patients.
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Preenchedores Dérmicos , Ácido Hialurônico , Materiais Biocompatíveis/efeitos adversos , Citocinas , Preenchedores Dérmicos/farmacologia , Humanos , Ácido Hialurônico/efeitos adversos , MacrófagosRESUMO
The application of nanomaterials in medicine has led to novel pharmaceuticals and medical devices that have demonstrated a strong potential for increasing the efficacy/performance and safety of therapeutic and diagnostic procedures to address a wide range of diseases. However, the successful translation of these technologies from their inception (proof-of-concept) to clinical practice has been challenged by substantial gaps in the scientific and technical capacity of R&D companies, especially SMEs, to keep up with the ever-evolving regulatory expectations in the emerging area of nanomedicine. To address these challenges, the EU Horizon 2020 project REFINE has developed a Decision Support System (DSS) to support developers of nanotechnology-enabled health products in bringing their products to the clinic. The REFINE DSS has been developed to support experts, innovators, and regulators in the implementation of intelligent testing strategies (ITS) for efficient preclinical assessment of nanotechnology-enabled health products. The DSS applies logical rules provided by REFINE experts which generate prioritized lists of assays to be performed (i.e. ITSs) for physicochemical characterisation and for immunotoxicological endpoints. The DSS has been tested against several case studies and was validated by internal project experts as well as external ones.
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Nanomedicina , Nanoestruturas , Nanomedicina/métodos , Nanotecnologia/métodosRESUMO
The widespread and increasing use of engineered nanomaterials (ENM) increases the risk of human exposure, generating concern that ENM may provoke adverse health effects. In this respect, their physicochemical characteristics are critical. The immune system may respond to ENM through inflammatory reactions. The NLRP3 inflammasome responds to a wide range of ENM, and its activation is associated with various inflammatory diseases. Recently, anisotropic ENM have become of increasing interest, but knowledge of their effects on the immune system is still limited. The objective of the study was to compare the effects of gold ENM of different shapes on NLRP3 inflammasome activation and related signalling pathways. Differentiated THP-1 cells (wildtype, ASC- or NLRP3-deficient), were exposed to PEGylated gold nanorods, nanostars, and nanospheres, and, thus, also different surface chemistries, to assess NLRP3 inflammasome activation. Next, the exposed cells were subjected to gene expression analysis. Nanorods, but not nanostars or nanospheres, showed NLRP3 inflammasome activation. ASC- or NLRP3-deficient cells did not show this effect. Gene Set Enrichment Analysis revealed that gold nanorod-induced NLRP3 inflammasome activation was accompanied by downregulated sterol/cholesterol biosynthesis, oxidative phosphorylation, and purinergic receptor signalling. At the level of individual genes, downregulation of Paraoxonase-2, a protein that controls oxidative stress, was most notable. In conclusion, the shape and surface chemistry of gold nanoparticles determine NLRP3 inflammasome activation. Future studies should include particle uptake and intracellular localization.
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Ouro , Nanopartículas Metálicas , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nanotubos , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
Biodistribution of nanoencapsulated bioactive compounds is primarily determined by the size, shape, chemical composition and surface properties of the encapsulating nanoparticle, and, thus, less dependent on the physicochemical properties of the active pharmaceutical ingredient encapsulated. In the current work, we aimed to investigate the impact of formulation type on biodistribution profile for two clinically relevant nanoformulations. We performed a comparative study of biodistribution in healthy rats at several dose levels and durations up to 14-day post-injection. The studied nanoformulations were nanostructured lipid carriers incorporating the fluorescent dye IR780-oleyl, and polymeric nanoparticles containing the anticancer agent cabazitaxel. The biodistribution was approximated by quantification of the cargo in blood and relevant organs. Several clear and systematic differences in biodistribution were observed, with the most pronounced being a much higher (more than 50-fold) measured concentration ratio between cabazitaxel in all organs vs. blood, as compared to IR780-oleyl. Normalized dose linearity largely showed opposite trends between the two compounds after injection. Cabazitaxel showed a higher brain accumulation than IR780-oleyl with increasing dose injected. Interestingly, cabazitaxel showed a notable and prolonged accumulation in lung tissue compared to other organs. The latter observations could warrant further studies towards a possible therapeutic indication within lung and conceivably brain cancer for nanoformulations of this highly antineoplastic compound, for which off-target toxicity is currently dose-limiting in the clinic.
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Antineoplásicos , Nanopartículas , Nanoestruturas , Animais , Portadores de Fármacos/química , Lipídeos/química , Nanopartículas/química , Polímeros , Ratos , Distribuição TecidualRESUMO
BACKGROUND: Ensuring consistency of tetanus neurotoxin (TeNT) production by Clostridium tetani could help to ensure consistent product quality in tetanus vaccine manufacturing, ultimately contributing to reduced animal testing. The aim of this study was to identify RNA signatures related to consistent TeNT production using standard and non-standard culture conditions. METHODS: We applied RNA sequencing (RNA-Seq) to study C. tetani gene expression in small-scale batches under several culture conditions. RESULTS: We identified 1381 time-dependent differentially expressed genes (DEGs) reflecting, among others, changes in growth rate and metabolism. Comparing non-standard versus standard culture conditions identified 82 condition-dependent DEGs, most of which were specific for one condition. The tetanus neurotoxin gene (tetX) was highly expressed but showed expression changes over time and between culture conditions. The tetX gene showed significant down-regulation at higher pH levels (pH 7.8), which was confirmed by the quantification data obtained with the recently validated targeted LC-MS/MS approach. CONCLUSIONS: Non-standard culture conditions lead to different gene expression responses. The tetX gene appears to be the best transcriptional biomarker for monitoring TeNT production as part of batch-to-batch consistency testing during tetanus vaccine manufacturing.
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Clostridium tetani/genética , Clostridium tetani/metabolismo , Neurotoxinas/biossíntese , Neurotoxinas/genética , Toxoide Tetânico/biossíntese , Toxoide Tetânico/normas , Sequência de Bases , Células Cultivadas , Regulação Bacteriana da Expressão GênicaRESUMO
INTRODUCTION: Before release, vaccine batches are assessed for quality to evaluate whether they meet the product specifications. Vaccine batch tests, in particular of inactivated and toxoid vaccines, still largely rely on in vivo methods. Improved vaccine production processes, ethical concerns, and suboptimal performance of some in vivo tests have led to the development of in vitro alternatives. AREAS COVERED: This review describes the scientific constraints that need to be overcome for replacement of in vivo batch tests, as well as potential solutions. Topics include the critical quality attributes of vaccines that require testing, the use of cell-based assays to mimic aspects of in vivo vaccine-induced immune responses, how difficulties with testing adjuvanted vaccines in vitro can be overcome, the use of altered batches to validate new in vitro test methods, and how cooperation between different stakeholders is key to moving the transition forward. EXPERT OPINION: For safety testing, many in vitro alternatives are already available or at an advanced level of development. For potency testing, in vitro alternatives largely comprise immunochemical methods that assess several, but not all critical vaccine properties. One-to-one replacement by in vitro alternatives is not always possible and a combination of methods may be required.