In-depth mechanistic analysis including high-throughput RNA sequencing in the prediction of functional and structural cardiotoxicants using hiPSC cardiomyocytes.

BACKGROUND: Cardiotoxicity remains one of the most reported adverse drug reactions that lead to drug attrition during pre-clinical and clinical drug development. Drug-induced cardiotoxicity may develop as a functional change in cardiac electrophysiology (acute alteration of the mechanical function of the myocardium) and/or as a structural change, resulting in loss of viability and morphological damage to cardiac tissue. RESEARCH DESIGN AND METHODS: Non-clinical models with better predictive value need to be established to improve cardiac safety pharmacology. To this end, high-throughput RNA sequencing (ScreenSeq) was combined with high-content imaging (HCI) and Ca2+ transience (CaT) to analyze compound-treated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). RESULTS: Analysis of hiPSC-CMs treated with 33 cardiotoxicants and 9 non-cardiotoxicants of mixed therapeutic indications facilitated compound clustering by mechanism of action, scoring of pathway activities related to cardiomyocyte contractility, mitochondrial integrity, metabolic state, diverse stress responses and the prediction of cardiotoxicity risk. The combination of ScreenSeq, HCI and CaT provided a high cardiotoxicity prediction performance with 89% specificity, 91% sensitivity and 90% accuracy. CONCLUSIONS: Overall, this study introduces mechanism-driven risk assessment approach combining structural, functional and molecular high-throughput methods for pre-clinical risk assessment of novel compounds.

The influence of aluminium and copper upon the early aggregatory behaviour and size of Islet amyloid polypeptide under simulated physiological conditions.

BACKGROUND AND AIM: Islet amyloid polypeptide/amylin deposition in the form of amyloid plaques is a common pathological feature observed in the pancreatic tissue of those with Type II Diabetes Mellitus. Its propensity to form amyloid fibrils and the resultant toxicity of this peptide in vivo is influenced by both the concentration and species of metal present in situ. Herein, we examine the influence of Al (III) and Cu (II), applied at equimolar and supra-stoichiometric concentrations on the initial aggregatory behaviour of amylin under near physiological conditions. METHODS: Dynamic light scattering measurements, which monitored the aggregation status and size of the peptide in real time, were performed during the early lag-phase of fibrillogenesis (T ≤ 30 min) in the absence or presence of metal ions. RESULTS: Islet amyloid polypeptide (10 µM) rapidly aggregated when introduced into a physiological medium favouring the formation of large, agglomerated structures (> 1000 nm) after 30 min incubation. Neither the addition of equimolar or excess metals significantly influenced the size of the peptide when intensity distributions were consulted; however, number distributions indicated that both Al (III) and Cu (II) may have had, an albeit temporary, stabilising influence upon the conformations present within solution. CONCLUSION: These results infer that small oligomeric species are likely transient entities that are rapidly incorporated into large agglomerates during the very initial stages of fibrillogenesis. While both Al (III) and Cu (II) both inhibited agglomeration to some degree, their stabilising affect upon peptide aggregation was limited over the juncture of the experiments performed herein; hence, it is difficult to say whether these metal ions play a role in enhancing the toxicity of these peptides through influencing their aggregation in the short-term.

Monitoring the early aggregatory behaviour and size of Aß1-42 in the absence & presence of metal ions using dynamic light scattering.

BACKGROUND AND AIM: Aß1-42 is an amyloidogenic peptide found within senile plaques extracted from those who died with a diagnosis of Alzheimer's disease. The potent neurotoxicity of this peptide is related to its propensity to form aggregated conformations in vivo, a process that is influenced by the species and concentration of metal ions present within the local environment. This study examines the impact of different metals upon the early aggregatory behaviour and size of Aß1-42 under simulated physiological conditions. METHODS: The size and aggregatory behaviour of Aß1-42 in the presence and absence of metal ions was monitored during the initial 30 min of fibril formation in real-time using dynamic light scattering. RESULTS: Intensity scattering measurements showed a clear tendency towards aggregation with regards to Aß1-42 only solutions (10 µM). Both equimolar Al3+ & Cu2+ lowered and stabilised the dimensions of Aß1-42 aggregates; however, a diminutive but significant increase in size was still observed over a 30-min period. While excess Al3+ continued to supress the size of Aß1-42, a 10-fold increase in the concentration of Cu2+ accelerated peptide aggregation relative to that observed for equimolar metal but not compared to Aß1-42 alone. CONCLUSION: These results infer that Al3+ ions stabilise and aid in the maintenance of smaller, toxic intermediates while excess Cu2+ facilitates the formation of larger, more inert, amorphous species exceeding 1 µm in size. Furthermore, we propose that metal-induced toxicity of Aß1-42 is reflective of their ability to preserve smaller oligomeric species in vitro.

The measurement and full statistical analysis including Bayesian methods of the aluminium content of infant vaccines.

BACKGROUND: Aluminium salts are the most common adjuvants in infant vaccines. The aluminium content of a vaccine is provided by the manufacturer and is indicated on the patient information leaflet. There is no independent verification, for example by the European Medicines Agency, of the aluminium content of infant vaccines. METHODS: We have measured the aluminium content of thirteen infant vaccines using microwave-assisted acid and peroxide digestion followed by transversely heated graphite furnace atomic absorption spectrometry. Our data are compared with manufacturer's data using full statistical analyses including Bayesian methods. RESULTS: We found that only three vaccines contained the amount of aluminium indicated by the manufacturer. Six vaccines contained a statistically significant (P < 0.05) greater quantity while four vaccines contained a statistically significant (P < 0.05) lower quantity. The range of content for any single vaccine varied considerably, for example, from 0.172 to 0.602 mg/vaccine for Havrix. CONCLUSIONS: The data have raised specific questions about the significance of the aluminium content of vaccines and identified areas of extremely limited information. Since aluminium is a known toxin in humans and specifically a neurotoxin, its content in vaccines should be accurate and independently monitored to ensure both efficacy and safety.

The interaction of aluminium-based adjuvants with THP-1 macrophages in vitro: Implications for cellular survival and systemic translocation.

Within clinical vaccinations, recombinant antigens are routinely entrapped inside or adsorbed onto the surface of aluminium salts in order to increase their immunological potency in vivo. The efficacy of these immunisations is highly dependent upon the recognition and uptake of these complexes by professional phagocytes and their subsequent delivery to the draining lymph nodes for further immunological processing. While monocytes have been shown to internalise aluminium adjuvants and their adsorbates, the role of macrophages in this respect has not been fully established. Furthermore, this study explored the interaction of THP-1 macrophages with aluminium-based adjuvants (ABAs) and how this relationship influenced the survival of such cells in vitro. THP-1 macrophages were exposed to low concentrations of ABAs (1.7 µg/mL Al) for a maximum of seven days. ABA uptake was determined using lumogallion staining and cell viability by both DAPI (4',6-diamidino-2-phenylindole) staining and LDH (lactate dehydrogenase) assay. Evidence of ABA particle loading was identified within cells at early junctures following treatment and appeared to be quite prolific (>90% cells positive for Al signal after 24 h). Total sample viability (% LDH release) in treated samples was predominantly similar to untreated cells and low levels of cellular death were consistently observed in populations positive for Al uptake. It can thus be concluded that aluminium salts can persist for some time within the intracellular environment of these cells without adversely affecting their viability. These results imply that macrophages may play a role in the systemic translocation of ABAs once administered in the form of an inoculation.

The size of micro-crystalline tyrosine (MCT®) influences its recognition and uptake by THP-1 macrophages in vitro.

The physicochemical hallmarks of particulate immunopotentiators play a pivotal role with regards to their adjuvanticity in vivo. These properties have not been fully characterised in the case of MCT®, an amino acid-based adjuvant used as an alternative to aluminium salts in subcutaneous allergy immunotherapy (SCIT). This study presents a full characterisation of MCT® and in a preliminary capacity reveals how parameters, specifically particle size, might influence the recognition of MCT® by antigen presenting cells (APCs) in vitro. Light microscopic analysis demonstrated that MCT® was composed of highly crystalline needles, the majority of which exceeded 10 µm in length under physiological conditions (median size - 20.8 µm). While the substantial length of crystals presented a significant barrier to cellular recognition and uptake, isolated incidences of perpendicular recognition were observed owing to the smaller comparative width of crystallites (median size - 2.8 µm). This appeared to allow a small proportion of material to be ingested both fully and partially by THP-1 macrophages, although further studies are required to unequivocally confirm this observation. Preferential recognition of needle tips also favoured the direct presentation of antigen to immune cells as proteinaceous adsorption appeared to be isolated to these regions. Furthermore, the data herein provide valuable insights into the mechanisms surrounding how this adjuvant potentiates an immunological response following administration.

Aggregation of the diabetes-related peptide ProIAPP1-48 measured by dynamic light scattering.

Islet amyloid polypeptide (IAPP1-37) or amylin is implicated in the aetiology of diabetes. It is found as amyloid along with its precursor ProIAPP1-48 in the islets of Langerhans in the pancreas. Metals have been implicated in amyloidogenesis of both IAPP and ProIAPP. Herein we have used dynamic light scattering (DLS) to investigate how Al(III) and Cu(II) influence aggregation of ProIAPP1-48 under near-physiological conditions and in a biologically-relevant timeframe. ProIAPP1-48 formed primarily sub-micron particles within 5 min (e.g. 470 nm at 15µM peptide) that grew to micron-sized particles (1310 nm) within a 30 min timeframe. Equimolar Al(III) had little influence upon particle size at either 5 (656 nm) or 30 min (1250 nm) while Cu(II) tended to increase particle size over the same time period (731-1300 nm). It is suggested that any effects of Al(III) and Cu(II) reflected their well known tendencies to support ß-sheet or amorphous aggregates of ProIAPP1-48 respectively.

Unraveling the enigma: elucidating the relationship between the physicochemical properties of aluminium-based adjuvants and their immunological mechanisms of action.

Aluminium salts are by far the most commonly used adjuvants in vaccines. There are only two aluminium salts which are used in clinically-approved vaccines, Alhydrogel® and AdjuPhos®, while the novel aluminium adjuvant used in Gardasil® is a sulphated version of the latter. We have investigated the physicochemical properties of these two aluminium adjuvants and specifically in milieus approximating to both vaccine vehicles and the composition of injection sites. Additionally we have used a monocytic cell line to establish the relationship between their physicochemical properties and their internalisation and cytotoxicity. We emphasise that aluminium adjuvants used in clinically approved vaccines are chemically and biologically dissimilar with concomitantly potentially distinct roles in vaccine-related adverse events.

Intracellular tracing of amyloid vaccines through direct fluorescent labelling.

Alzheimer's disease is a debilitating neurodegenerative condition that progressively causes synaptic loss and major neuronal damage. Immunotherapy utilising Aß as an active immunogen or via passive treatment utilising antibodies raised to amyloid have shown therapeutic promise. The migratory properties of peripheral blood-borne monocytes and their ability to enter the central nervous system, suggests a beneficial role in mediating tissue damage and neuroinflammation. However, the intrinsic phagocytic properties of such cells have pre-disposed them to internalise misfolded amyloidogenic peptides that could act as seeds capable of nucleating amyloid formation in the brain. Mechanisms governing the cellular fate of amyloid therefore, may prove to be key in the development of future vaccination regimes. Herein, we have developed unequivocal and direct conformation-sensitive fluorescent molecular probes that reveal the intracytoplasmic and intranuclear persistence of amyloid in a monocytic T helper 1 (THP-1) cell line. Use of the pathogenic Aß42 species as a model antigen in simulated vaccine formulations suggested differing mechanisms of cellular internalisation, in which fibrillar amyloid evaded lysosomal capture, even when co-deposited on particulate adjuvant materials. Taken collectively, direct fluorescent labelling of antigen-adjuvant complexes may serve as critical tools in understanding subsequent immunopotentiation in vaccines directed against amyloidosis and wider dementia.

Insight into the cellular fate and toxicity of aluminium adjuvants used in clinically approved human vaccinations.

Aluminium adjuvants remain the most widely used and effective adjuvants in vaccination and immunotherapy. Herein, the particle size distribution (PSD) of aluminium oxyhydroxide and aluminium hydroxyphosphate adjuvants was elucidated in attempt to correlate these properties with the biological responses observed post vaccination. Heightened solubility and potentially the generation of Al(3+) in the lysosomal environment were positively correlated with an increase in cell mortality in vitro, potentially generating a greater inflammatory response at the site of simulated injection. The cellular uptake of aluminium based adjuvants (ABAs) used in clinically approved vaccinations are compared to a commonly used experimental ABA, in an in vitro THP-1 cell model. Using lumogallion as a direct-fluorescent molecular probe for aluminium, complemented with transmission electron microscopy provides further insight into the morphology of internalised particulates, driven by the physicochemical variations of the ABAs investigated. We demonstrate that not all aluminium adjuvants are equal neither in terms of their physical properties nor their biological reactivity and potential toxicities both at the injection site and beyond. High loading of aluminium oxyhydroxide in the cytoplasm of THP-1 cells without immediate cytotoxicity might predispose this form of aluminium adjuvant to its subsequent transport throughout the body including access to the brain.

From Stock Bottle to Vaccine: Elucidating the Particle Size Distributions of Aluminum Adjuvants Using Dynamic Light Scattering.

The physicochemical properties of aluminum salts are key determinants of their resultant adjuvanticity in vivo when administered as part of a vaccine. While there are links between particle size and the efficacy of the immune response, the limited literature directly characterizing the PSD of aluminum adjuvants has stymied the elucidation of such a relationship for these materials. Hence, this comparative study was undertaken to monitor the PSD of aluminum adjuvants throughout the process of vaccine formulation using DLS. A significant proportion of the stock suspensions was highly agglomerated (>9 µm) and Alhydrogel® exhibited the smallest median size (2677 ± 120 nm) in comparison to Adju-Phos® or Imject alum® (7152 ± 308 and 7294 ± 146 nm respectively) despite its large polydispersity index (PDI). Dilution of these materials induced some degree of disaggregation within all samples with Adju-Phos® being the most significantly affected. The presence of BSA caused the median size of Alhydrogel® to increase but these trends were not evident when model vaccines were formulated with either Adju-Phos® or Imject alum®. Nevertheless, Alhydrogel® and Adju-Phos® exhibited comparable median sizes in the presence of this protein (4194 ± 466 and 4850 ± 501 nm respectively) with Imject alum® being considerably smaller (2155 ± 485 nm). These results suggest that the PSD of aluminum adjuvants is greatly influenced by dilution and the degree of protein adsorption experienced within the vaccine itself. The size of the resultant antigen-adjuvant complex may be important for its immunological recognition and subsequent clearance from the injection site.

Unequivocal identification of intracellular aluminium adjuvant in a monocytic THP-1 cell line.

Aluminium-based adjuvants (ABA) are the predominant adjuvants used in human vaccinations. While a consensus is yet to be reached on the aetiology of the biological activities of ABA several studies have identified shape, crystallinity and size as critical factors affecting their adjuvanticity. In spite of recent advances, the fate of ABA following their administration remains unclear. Few if any studies have demonstrated the unequivocal presence of intracellular ABA. Herein we demonstrate for the first time the unequivocal identification of ABA within a monocytic T helper 1 (THP-1) cell line, using lumogallion as a fluorescent molecular probe for aluminium. Use of these new methods revealed that particulate ABA was only found in the cell cytoplasm. Transmission electron microscopy revealed that ABA were contained within vesicle-like structures of approximately 0.5-1 µm in diameter.