Matthew Mold

Copper prevents amyloid-β 1–42 from forming amyloid fibrils under near-physiological conditions in vitro

The aggregation and deposition of amyloid-β(1–42) (Aβ42) in the brain is implicated in the aetiology of Alzheimers disease (AD). While the mechanism underlying its deposition in vivo is unknown its precipitation in vitro is influenced by metal ions. For example, Aβ42 is known to bind copper, Cu(II), in vitro and binding results in aggregation of the peptide. The biophysical properties of Cu(II)-Aβ42 aggregates are of significant importance to their putative involvement in the amyloid cascade hypothesis of AD and are currently the subject of strong debate. In particular the question has been raised if sub- and super-stoichiometric concentrations of Cu(II) act in opposing ways in respectively accelerating and preventing amyloid fibril formation by Aβ42. Herein we have used fluorimetry and transmission electron microscopy to provide unequivocal evidence that under near-physiological conditions both sub- and super-stoichiometric concentrations of Cu(II) prevented the assembly of Aβ42 into ThT-positive β-sheet rich amyloid fibrils.

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.

Copper Abolishes the β-Sheet Secondary Structure of Preformed Amyloid Fibrils of Amyloid-β42

The observation of the co-deposition of metals and amyloid-beta(42) (Abeta(42)) in brain tissue in Alzheimers disease prompted myriad investigations into the role played by metals in the precipitation of this peptide. Copper is bound by monomeric Abeta(12) and upon precipitation of the copper-peptide complex thereby prevents Abeta(42) from adopting a beta-sheet secondary structure. Copper is also bound by beta-sheet conformers of Abeta(42), and herein we have investigated how this interaction affects the conformation of the precipitated peptide. Copper significantly reduced the thioflavin T fluorescence of aged, fibrillar Abeta(42) with, for example, a 20-fold excess of the metal resulting in a ca 90% reduction in thioflavin T fluorescence. Transmission electron microscopy showed that copper significantly reduced the quantities of amyloid fibrils while Congo red staining and polarized light demonstrated a copper-induced abolition of apple-green birefringence. Microscopy under cross-polarized light also revealed the first observation of spherulites of Abeta(42). The size and appearance of these amyloid structures were found to be very similar to spherulites identified in Alzheimers disease tissue. The combined results of these complementary methods strongly suggested that copper abolished the beta-sheet secondary structure of pre-formed, aged amyloid fibrils of Abeta(42). Copper may protect against the presence of beta-sheets of Abeta(42) in vivo, and its binding by fibrillar Abeta(42) could have implications for Alzheimers disease therapy.

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 Al3+ 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.

Aluminum content of human semen: implications for semen quality.

A deterioration of human semen quality has been observed over recent decades. A possible explanation could be an increased exposure to environmental pollutants, including aluminum. Our aim was to measure the aluminum concentration in the semen of 62 patients and to carry out a preliminary evaluation on its impact on specific semen parameters. For each patient, semen analyses were performed according to WHO guidelines. A graphite furnace atomic absorption spectrometry method was used to determine semen aluminum concentration. A cytological analysis using an aluminum-specific fluor, lumogallion, was also performed. The mean aluminum concentration in human semen was 339 μg/L. Patients with oligozoospermia had a statistically higher aluminum concentration than others. No significant difference was observed for other semen parameters. Cytological analysis showed the presence of aluminum in spermatozoa. This study provided unequivocal evidence of high concentrations of aluminum in human semen and suggested possible implications for spermatogenesis and sperm count.

Serum Amyloid P Component Accelerates the Formation and Enhances the Stability of Amyloid Fibrils in a Physiologically Significant Under-Saturated Solution of Amyloid-β42

The mechanism whereby an under-saturated solution of amyloid-β (Aβ)42 precipitates as β sheets in vivo in Alzheimers disease remains to be elucidated. Herein we present in vitro evidence that serum amyloid P component may mediate this process through its acceleration of amyloid formation from an under-saturated solution of Aβ42 and subsequently its stabilization of the amyloid fibrils formed over physiologically significant timeframes. Our observations support serum amyloid P component as a therapeutic target in Alzheimers disease.

Aluminium in brain tissue in autism

Autism spectrum disorder is a neurodevelopmental disorder of unknown aetiology. It is suggested to involve both genetic susceptibility and environmental factors including in the latter environmental toxins. Human exposure to the environmental toxin aluminium has been linked, if tentatively, to autism spectrum disorder. Herein we have used transversely heated graphite furnace atomic absorption spectrometry to measure, for the first time, the aluminium content of brain tissue from donors with a diagnosis of autism. We have also used an aluminium-selective fluor to identify aluminium in brain tissue using fluorescence microscopy. The aluminium content of brain tissue in autism was consistently high. The mean (standard deviation) aluminium content across all 5 individuals for each lobe were 3.82(5.42), 2.30(2.00), 2.79(4.05) and 3.82(5.17) μg/g dry wt. for the occipital, frontal, temporal and parietal lobes respectively. These are some of the highest values for aluminium in human brain tissue yet recorded and one has to question why, for example, the aluminium content of the occipital lobe of a 15year old boy would be 8.74 (11.59) μg/g dry wt.? Aluminium-selective fluorescence microscopy was used to identify aluminium in brain tissue in 10 donors. While aluminium was imaged associated with neurones it appeared to be present intracellularly in microglia-like cells and other inflammatory non-neuronal cells in the meninges, vasculature, grey and white matter. The pre-eminence of intracellular aluminium associated with non-neuronal cells was a standout observation in autism brain tissue and may offer clues as to both the origin of the brain aluminium as well as a putative role in autism spectrum disorder.

Copper is a potent inhibitor of the propensity for human ProIAPP1-48 to form amyloid fibrils in vitro

Abstract Background: The amyloidogenic peptides IAPP and ProIAPP1-48 are implicated in β cell death in type 2 diabetes mellitus. While the mechanism of their deposition in vivo is unknown we have shown

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.

Further insight into the role of metals in amyloid formation by IAPP1-37 and ProIAPP1-48

Abstract Background: IAPP1-37 and ProIAPP1-48 are amyloidogenic peptides implicated in β-cell death in diabetes. Interactions with metals may be involved in both the cytotoxicity of these