Evaluation of the Toxic Effects of Aluminum Hydroxide Nanoparticles as Adjuvants in Vaccinated Neonatal Mice.

Aluminum hydroxide nanoparticles have been employed in many industries, which are widely abundant in many aspects of human life. The role of the aluminum hydroxide nanoparticles adjuvant is to enhance the immune response. However, the impact of nanoparticles exposure has not been perfectly investigated yet. Accordingly, some questions have been raised about their potentially harmful effects, based on which the current research aims to answer them. This study aimed to investigate the histological effects of aluminum hydroxide nanoparticles and bulk-aluminum hydroxide (bulk Al[OH]3) on the liver, lung, heart, and kidney tissues. For this reason, an experiment was implemented on the aluminum hydroxide nanoparticles adjuvant in five neonatal mice. Intramuscularly, the mice were injected with 0.125 mL of adjuvanted vaccine, while five neonatal mice were injected with bulk and nanoparticles of Al (OH)3 and then sacrificed after one and two months, respectively. Vaccines were controlled by evaluating the histopathological response in neonatal mice. Subsequently, the pathological effect of both adjuvants was surveyed using the histological study of the lung, liver, heart, and kidney of the animals. The obtained recorded data indicated that both types of vaccine adjuvants caused pathological lesions on the histology sections of the liver, lung, heart, and kidney tissues. Moreover, bulk Al (OH)3 adjuvant vaccine was more effective and had a higher pathological response than aluminum hydroxide nanoparticles adjuvant vaccine. In addition, the total DNA content in both groups was estimated using Fluorometer from Promega. Compared to aluminum hydroxide nanoparticles groups, the tissues indicated a decrease in total DNA content obtained in bulk Al (OH)3 groups. Therefore, it can be concluded that the exposure to aluminum hydroxide nanoparticles would result in less pronounced toxicity, as well as systemic inflammation, compared to the bulk Al (OH)3 aluminum hydroxide.

The REACH registration process: A case study of metallic aluminium, aluminium oxide and aluminium hydroxide.

The European Union's REACH Regulation requires determination of potential health and environmental effects of chemicals in commerce. The present case study examines the application of REACH guidance for health hazard assessments of three high production volume (HPV) aluminium (Al) substances: metallic aluminium, aluminium oxide, and aluminium hydroxide. Among the potential adverse health consequences of aluminium exposure, neurotoxicity is one of the most sensitive targets of Al toxicity and the most critical endpoint. This case study illustrates integration of data from multiple lines of evidence into REACH weight of evidence evaluations. This case study then explains how those results support regulatory decisions on classification and labelling. Challenges in the REACH appraisal of Al compounds include speciation, solubility and bioavailability, application of assessment factors, read-across rationale and differences with existing regulatory standards. Lessons learned from the present case study relate to identification and evaluation of toxicologic and epidemiologic data; assessing data relevance and reliability; development of derived no-effect levels (DNELs); addressing data gaps and preparation of chemical safety reports.

Aluminum hydroxide nebulization-induced redox imbalance and acute lung inflammation in mice.

Purpose: Aluminum is the third most abundant metal in the earth's crust and is widely used in industry. Chronic contact with aluminum results in a reduction in the activity of electron transport chain complexes, leading to excessive production of reactive oxygen species (ROS) and oxidative stress. This study aimed to evaluate the effects of short-term exposure of aluminum hydroxide on oxidative stress and pulmonary inflammatory response.Materials and methods: Male BALB/c mice were divided into three groups: control group (CG); phosphate buffered saline group (PBSG) and aluminum hydroxide group (AHG). CG was exposed to ambient air, while PBSG and AHG were exposed to PBS or aluminum hydroxide solutions via nebulization, three times per day for five consecutive days. Twenty-four hours after the last exposure, all animals were euthanized for subsequent analysis.Results: Exposure to aluminum hydroxide in the blood resulted in lower platelet levels, higher neutrophils, and lower monocytes compared to CG and PBSG. Aluminum hydroxide promoted the recruitment of inflammatory cells to the lung. Macrophage, neutrophil and lymphocyte counts were higher in AHG compared to CG and PBSG. Protein oxidation and superoxide dismutase activity were higher, while catalase activity and reduced and oxidizes glutathione ratio in AHG were lower compared to CG and PBSG. Furthermore, there was an increase in the inflammatory markers CCL2 and IFN-γ in AHG compared to CG and PBSG.Conclusion: In conclusion, short-term nebulization with aluminum hydroxide induces the influx of inflammatory cells and oxidative stress in adult BALB/c mice.

Physical, morphological and chemical modification of Al-based nanofillers in by-products of incinerated nanocomposites and related biological outcome.

The number of products containing nanomaterials is increasing this last ten years. Information and literature about the end-of-life of nanocomposites often remains partial and does not address the overall fate and transformations of nanoparticles that may affect biological responses. This paper underlines that the physico-chemical features of nanoparticles can be modified by the incineration process and the available toxicological data on pristine nanofillers might not be relevant to assess the modified nanoparticles included in soot. Combustion tests have been performed at lab-scale using a cone calorimeter modified to collect fumes (particulate matter and gas phase) and have been characterized using various techniques. Nanocomposites selected were poly(ethylene vinyl acetate) containing Al-based nanoparticles, i.e. boehmites or alumina. Evaluations of in vitro cytotoxicity responses on pristine nanofillers, soot and residual ash, show that safe boehmite nanoparticles, become toxic due to a chemical modification after incineration process.

Self-stacking of exfoliated charged nanosheets of LDHs and graphene as biosensor with real-time tracking of dopamine from live cells.

This study introduces a new strategy for periodic stacking of positively charged NiAl layered double hydroxides (LDHs) nanosheets with negatively charged monolayers of graphene (G) by systematically optimizing several parameters in a controlled co-feeding fashion and resultant heterostacked NiAl LDH/G LBL nanocomposites have been practically applied in sensitive detection of dopamine released from live cells as early Parkinson's disease (PD) diagnostic tool. PD is the second most chronic neurodegenerative disorder with gradual progressive loss of movement and muscle control causing substantial disability and threatening the life seriously. Unfortunately majority of dopaminergic neurons present in substantia nigra of PD patients are destroyed before it is being clinically diagnosed, so early stages PD diagnosis is essential. Because of direct neighboring of extremely conductive graphene to semiconductive LDHs layers, enhanced intercalation capability of LDHs, and huge surface area with numerous active sites, good synergy effect is harvested in heteroassembled NiAl LDH/G LBL material, which in turn shows admirable electrocatalytic ability in DA detection. The interference induced by UA and AA is effectively eliminated especially after the modifying the electrode with Nafion. The outstanding electrochemical sensing performance of NiAl LDH/G LBL modified electrode has been achieved in terms of broad linear range and lowest real detection limit of 2 nM (S/N = 3) towards DA oxidation. Benefitting from superior efficiency, biosensor has been successfully used for real-time in-vitro tracking of DA efflux from live human nerve cell after being stimulated. We believe that our biosensing platform of structurally integrated well-ordered LBL heteroassembly by inserting graphene directly to the interlayer galleries of LDHs material will open up new avenue in diseases determination window.

Comparative efficacy and toxicity of two vaccine candidates against Sporothrix schenckii using either Montanide™ Pet Gel A or aluminum hydroxide adjuvants in mice.

Sporotrichosis is an important zoonosis in Brazil and the most frequent subcutaneous mycosis in Latin America, caused by different Sporothrix species. Currently, there is no effective vaccine available to prevent this disease. In this study, the efficacy and toxicity of the adjuvant Montanide™ Pet Gel A (PGA) formulated with S. schenckii cell wall proteins (ssCWP) was evaluated and compared with that of aluminum hydroxide (AH). Balb/c mice received two subcutaneous doses (1st and 14th days) of either the unadjuvanted or adjuvanted vaccine candidates. On the 21st day, anti-ssCWP antibody levels (ELISA), the phagocytic index, as well as the ex vivo release of IFN-γ, IL-4, and IL-17 by splenocytes and IL-12 by peritoneal macrophages were assessed. Cytotoxicity of the vaccine formulations was evaluated in vitro and by histopathological analysis of the inoculation site. Both adjuvanted vaccine formulations increased anti-ssCWP IgG, IgG1, IgG2a, and IgG3 levels, although IgG2a levels were higher in response to PGA+CWP100, probably contributing to the increase in S. schenckii yeast phagocytosis by macrophages in the opsonophagocytosis assay when using serum from PGA+CWP100-immunized mice. Immunization with AH+CWP100 led to a mixed Th1/Th2/Th17 ex vivo cytokine release profile, while PGA+CWP100 stimulated a preferential Th1/Th2 profile. Moreover, PGA+CWP100 was less cytotoxic in vitro, caused less local toxicity and led to a similar reduction in fungal load in the liver and spleen of S. schenckii- or S. brasiliensis-challenged mice as compared with AH+CWP100. These results suggest that PGA may be an effective and safe adjuvant for a future sporotrichosis vaccine.

Non-linear dose-response of aluminium hydroxide adjuvant particles: Selective low dose neurotoxicity.

Aluminium (Al) oxyhydroxide (Alhydrogel®), the main adjuvant licensed for human and animal vaccines, consists of primary nanoparticles that spontaneously agglomerate. Concerns about its safety emerged following recognition of its unexpectedly long-lasting biopersistence within immune cells in some individuals, and reports of chronic fatigue syndrome, cognitive dysfunction, myalgia, dysautonomia and autoimmune/inflammatory features temporally linked to multiple Al-containing vaccine administrations. Mouse experiments have documented its capture and slow transportation by monocyte-lineage cells from the injected muscle to lymphoid organs and eventually the brain. The present study aimed at evaluating mouse brain function and Al concentration 180days after injection of various doses of Alhydrogel® (200, 400 and 800µg Al/kg of body weight) in the tibialis anterior muscle in adult female CD1 mice. Cognitive and motor performances were assessed by 8 validated tests, microglial activation by Iba-1 immunohistochemistry, and Al level by graphite furnace atomic absorption spectroscopy. An unusual neuro-toxicological pattern limited to a low dose of Alhydrogel® was observed. Neurobehavioural changes, including decreased activity levels and altered anxiety-like behaviour, were observed compared to controls in animals exposed to 200µg Al/kg but not at 400 and 800µg Al/kg. Consistently, microglial number appeared increased in the ventral forebrain of the 200µg Al/kg group. Cerebral Al levels were selectively increased in animals exposed to the lowest dose, while muscle granulomas had almost completely disappeared at 6 months in these animals. We conclude that Alhydrogel® injected at low dose in mouse muscle may selectively induce long-term Al cerebral accumulation and neurotoxic effects. To explain this unexpected result, an avenue that could be explored in the future relates to the adjuvant size since the injected suspensions corresponding to the lowest dose, but not to the highest doses, exclusively contained small agglomerates in the bacteria-size range known to favour capture and, presumably, transportation by monocyte-lineage cells. In any event, the view that Alhydrogel® neurotoxicity obeys "the dose makes the poison" rule of classical chemical toxicity appears overly simplistic.

Safety Assessment of Alumina and Aluminum Hydroxide as Used in Cosmetics.

This is a safety assessment of alumina and aluminum hydroxide as used in cosmetics. Alumina functions as an abrasive, absorbent, anticaking agent, bulking agent, and opacifying agent. Aluminum hydroxide functions as a buffering agent, corrosion inhibitor, and pH adjuster. The Food and Drug Administration (FDA) evaluated the safe use of alumina in several medical devices and aluminum hydroxide in over-the-counter drugs, which included a review of human and animal safety data. The Cosmetic Ingredient Review (CIR) Expert Panel considered the FDA evaluations as part of the basis for determining the safety of these ingredients as used in cosmetics. Alumina used in cosmetics is essentially the same as that used in medical devices. This safety assessment does not include metallic or elemental aluminum as a cosmetic ingredient. The CIR Expert Panel concluded that alumina and aluminum hydroxide are safe in the present practices of use and concentration described in this safety assessment.

Biodistribution and toxicity of spherical aluminum oxide nanoparticles.

With the rapid development of the nano-industry, concerns about their potential adverse health effects have been raised. Thus, ranking accurately their toxicity and prioritizing for in vivo testing through in vitro toxicity test is needed. In this study, we used three types of synthesized aluminum oxide nanoparticles (AlONPs): γ-aluminum oxide hydroxide nanoparticles (γ-AlOHNPs), γ- and α-AlONPs. All three AlONPs were spherical, and the surface area was the greatest for γ-AlONPs, followed by the α-AlONPs and γ-AlOHNPs. In mice, γ-AlOHNPs accumulated the most 24 h after a single oral dose. Additionally, the decreased number of white blood cells (WBC), the increased ratio of neutrophils and the enhanced secretion of interleukin (IL)-8 were observed in the blood of mice dosed with γ-AlOHNPs (10 mg kg(-1)). We also compared their toxicity using four different in vitro test methods using six cell lines, which were derived from their potential target organs, BEAS-2B (lung), Chang (liver), HACAT (skin), H9C2 (heart), T98G (brain) and HEK-293 (kidney). The results showed γ-AlOHNPs induced the greatest toxicity. Moreover, separation of particles was observed in a transmission electron microscope (TEM) image of cells treated with γ-AlOHNPs, but not γ-AlONPs or α-AlONPs. In conclusion, our results suggest that the accumulation and toxicity of AlONPs are stronger in γ-AlOHNPs compared with γ-AlONPs and α-AlONPs owing their low stability within biological system, and the presence of hydroxyl group may be an important factor in determining the distribution and toxicity of spherical AlONPs.

Non-clinical safety assessment of single and repeated intramuscular administration of a human papillomavirus-16/18 vaccine in rabbits and rats.

The human papillomavirus (HPV)-16/18 vaccine (Cervarix®) is a prophylactic vaccine for the prevention of cervical cancer. The vaccine contains recombinant virus-like particles assembled from the L1 major capsid proteins of the cervical cancer-causing viral types HPV-16 and HPV-18, and Adjuvant System 04 (AS04), which contains the immunostimulant MPL and aluminium salt. To evaluate potential local and systemic toxic effects of the HPV-16/18 vaccine or AS04 alone, three repeated-dose studies were performed in rabbits and rats. One rabbit study also included a single-dose evaluation. In rabbits (~2.5 kg), the full human dose (HD) of the vaccine was evaluated (0.5 ml per injection site), and in rats (~250 g), 1/5 HD of vaccine was evaluated, corresponding to ≥ 12 times the dosage in humans relative to body weight. In both animal models, the treatment-related changes included a slight transient increase in the number of circulating neutrophils as well as a local inflammatory reaction at the injection site. These treatment-related changes were less pronounced after four doses of AS04 alone than after four doses of the HPV-16/18 vaccine. Additional treatment-related changes in the rat included lower albumin/globulin ratios and microscopic signs of inflammation in the popliteal lymph nodes. In both animal models, 13 weeks after the fourth dose, recovery was nearly complete, although at the injection site in some animals there were signs of discoloration, muscle-fibre regeneration and focal points of macrophage infiltration. Therefore, in these non-clinical models, the single and repeated dose administrations of the HPV-16/18 vaccine or AS04 alone were safe and well tolerated.

Systematic review of potential health risks posed by pharmaceutical, occupational and consumer exposures to metallic and nanoscale aluminum, aluminum oxides, aluminum hydroxide and its soluble salts.

Abstract Aluminum (Al) is a ubiquitous substance encountered both naturally (as the third most abundant element) and intentionally (used in water, foods, pharmaceuticals, and vaccines); it is also present in ambient and occupational airborne particulates. Existing data underscore the importance of Al physical and chemical forms in relation to its uptake, accumulation, and systemic bioavailability. The present review represents a systematic examination of the peer-reviewed literature on the adverse health effects of Al materials published since a previous critical evaluation compiled by Krewski et al. (2007) . Challenges encountered in carrying out the present review reflected the experimental use of different physical and chemical Al forms, different routes of administration, and different target organs in relation to the magnitude, frequency, and duration of exposure. Wide variations in diet can result in Al intakes that are often higher than the World Health Organization provisional tolerable weekly intake (PTWI), which is based on studies with Al citrate. Comparing daily dietary Al exposures on the basis of "total Al"assumes that gastrointestinal bioavailability for all dietary Al forms is equivalent to that for Al citrate, an approach that requires validation. Current occupational exposure limits (OELs) for identical Al substances vary as much as 15-fold. The toxicity of different Al forms depends in large measure on their physical behavior and relative solubility in water. The toxicity of soluble Al forms depends upon the delivered dose of Al(+3) to target tissues. Trivalent Al reacts with water to produce bidentate superoxide coordination spheres [Al(O2)(H2O4)(+2) and Al(H2O)6 (+3)] that after complexation with O2(•-), generate Al superoxides [Al(O2(•))](H2O5)](+2). Semireduced AlO2(•) radicals deplete mitochondrial Fe and promote generation of H2O2, O2 (•-) and OH(•). Thus, it is the Al(+3)-induced formation of oxygen radicals that accounts for the oxidative damage that leads to intrinsic apoptosis. In contrast, the toxicity of the insoluble Al oxides depends primarily on their behavior as particulates. Aluminum has been held responsible for human morbidity and mortality, but there is no consistent and convincing evidence to associate the Al found in food and drinking water at the doses and chemical forms presently consumed by people living in North America and Western Europe with increased risk for Alzheimer's disease (AD). Neither is there clear evidence to show use of Al-containing underarm antiperspirants or cosmetics increases the risk of AD or breast cancer. Metallic Al, its oxides, and common Al salts have not been shown to be either genotoxic or carcinogenic. Aluminum exposures during neonatal and pediatric parenteral nutrition (PN) can impair bone mineralization and delay neurological development. Adverse effects to vaccines with Al adjuvants have occurred; however, recent controlled trials found that the immunologic response to certain vaccines with Al adjuvants was no greater, and in some cases less than, that after identical vaccination without Al adjuvants. The scientific literature on the adverse health effects of Al is extensive. Health risk assessments for Al must take into account individual co-factors (e.g., age, renal function, diet, gastric pH). Conclusions from the current review point to the need for refinement of the PTWI, reduction of Al contamination in PN solutions, justification for routine addition of Al to vaccines, and harmonization of OELs for Al substances.

Toxicity of boehmite nanoparticles: impact of the ultrafine fraction and of the agglomerates size on cytotoxicity and pro-inflammatory response.

Boehmite (γ-AlOOH) nanoparticles (NPs) are used in a wide range of industrial applications. However, little is known about their potential toxicity. This study aimed at a better understanding of the relationship between the physico-chemical properties of these NPs and their in vitro biological activity. After an extensive physico-chemical characterization, the cytotoxicity, pro-inflammatory response and oxidative stress induced by a bulk industrial powder and its ultrafine fraction were assessed using RAW264.7 macrophages. Although the bulk powder did not trigger a significant biological activity, pro-inflammatory response was highly enhanced with the ultrafine fraction. This observation was confirmed with boehmite NPs synthesized at the laboratory scale, with well-defined and tightly controlled physico-chemical features: toxicity was increased when NPs were dispersed. In conclusion, the agglomerates size of boehmite NPs has a major impact on their toxicity, highlighting the need to study not only raw industrial powders containing NPs but also the ultrafine fractions representative of respirable particles.

Stabilising metal(loid)s in soil with iron and aluminium-based products: microbial, biochemical and plant growth impact.

Four iron and aluminium-based products, including red mud (RM), hematite (Fe2O3), an iron-rich water treatment residual (Fe-WTR) and amorphous Al hydroxide (Al-OH), were evaluated for their effectiveness at stabilising As and heavy metals (i.e. Cd, Cu, Pb, Zn) in a circumneutral contaminated soil [As (2105 mg kg(-1)), Cd (18 mg kg(-1)), Cu (264 mg kg(-1)), Pb (710 mg kg(-1)), Zn (522 mg kg(-1))]. Treatment impacts on soil microbial and biochemical features (i.e. microbial biomass-C, microbial counts, 16S rRNA PCR-TTGE of culturable bacteria, dehydrogenase, urease and ß-glucosidase activity, Biolog derived parameters-AWCD and richness) as well as bean (Phaseolus vulgaris) and wheat (Triticum vulgare) growth were also assessed. After 6 months equilibration, all the amendments (application rate 3% w/w) but RM reduced labile As while only Al-OH reduced the concentration of water-soluble heavy metals. Despite the highest bioavailability of contaminants, most of the soil microbial and biochemical features monitored (i.e. microbial biomass-C, total bacterial counts, dehydrogenase activity and AWCD) were significantly higher in the RM-soil. Bean germination was completely inhibited in RM-soil while wheat growth was similar to that of the control. The Al-OH treatment was best overall, promoting microbial abundance, diversity and activity while increasing bean and wheat growth and reducing As accumulated in plant shoots. Results suggest that Al-OH is a suitable candidate for field evaluations while the use of RM in the remediation of circumneutral or subalkaline contaminated soils should be reconsidered.

Toxicity and dose determination of quillaja saponin, aluminum hydroxide and squalene in olive flounder (Paralichthys olivaceus).

Adjuvants are substances added to vaccines to enhance the immune response of a given antigen. Most of the adjuvants are toxic at certain doses, and toxicity varies in different species. Moreover, there are no standard dosage limits set for adjuvant use in fish vaccines. We evaluated the acute toxicity, serum enzymes (AST/ALT) indicating hepatic injury and histopathological changes due to intra-peritoneal administration of different concentrations of a panel of adjuvants including quillaja saponin, aluminum hydroxide, squalene emulsion and Freund's incomplete adjuvant (FIA) with a dose ranging study of saponin (500, 160, 50, 16 and 5µgfish(-1)), aluminum hydroxide (5000, 1600, 500, 160 and 50µgfish(-1)), squalene emulsion (20, 10 and 5%), and FIA to determine the acceptable dosage for vaccination in olive flounder (Paralichthys olivaceus) fingerlings measuring 4.66±0.41g, 8.47±0.42cm. Saponin was highly toxic with a LD50 of approximately 105µgfish(-1) (22.4mgkg(-1)) causing severe histological damage and AST level was high at dose above 16µgfish(-1) and ALT, specific for liver damage was high only at 160µgfish(-1) (11U/L) and was safe at 5µgfish(-1). Aluminum hydroxide was toxic at 5000µgfish(-1) and was acceptable at dose below 1600µgfish(-1) with moderate histology and AST/ALT levels similar with control. Squalene emulsion showed increased inflammation at 20% and 10% emulsions and the inflammatory response was mild at a concentration of 5% oil emulsion and AST/ALT levels being similar to control in 10% and 5% emulsions and elevated in 20% on both sampling days. FIA was not lethal, but induced severe inflammation at injection site and around blood vessels. In comparison to FIA, saponin found to be safe at dose of 5µgfish(-1), aluminum hydroxide below 1600µgfish(-1), and squalene at 5% emulsion and could be accepted for vaccination studies. These results provide an insight for the selection of safer dose of adjuvants for intra-peritoneal vaccination of olive flounder.

New insight into artifactual phenomena during in vitro toxicity assessment of engineered nanoparticles: study of TNF-α adsorption on alumina oxide nanoparticle.

Biomolecules can be adsorbed on nanoparticles (NPs) and degraded during in vitro toxicity assays. These artifactual phenomena could lead to misinterpretation of biological activity, such as false-negative results. To avoid possible underestimation of cytokine release after contact between NP and cells, we propose a methodology to account for these artifactual phenomena and lead to accurate measurements. We focused on the pro-inflammatory cytokine tumor necrosis factor TNF-α. We studied well-characterized boehmite engineered NP [aluminum oxide hydroxide, AlO(OH)]. The rate of TNF-α degradation and its adsorption (on boehmite and on the walls of wells) were determined in cell-free conditions by adding a known TNF-α concentration (1500 pg/ml) under various experimental conditions. After a 24-h incubation, we quantified that 7 wt.% of the initial TNF-α was degraded over time, 6 wt.% adsorbed on the walls of 96-well plates, and 13 wt.% adsorbed on the boehmite surface. Finally, boehmite NP were incubated with murine macrophages (RAW 264.7 cell line). The release of TNF-α was assessed for boehmite NP and the experimental data were corrected considering the artifactual phenomena, which accounted for about 20-30% of the total.

Aluminium overload after 5 years in skin biopsy following post-vaccination with subcutaneous pseudolymphoma.

Aluminium hydroxide is used as an effective adjuvant in a wide range of vaccines for enhancing immune response to the antigen. The pathogenic role of aluminium hydroxide is now recognized by the presence of chronic fatigue syndrome, macrophagic myofasciitis and subcutaneous pseudolymphoma, linked to intramuscular injection of aluminium hydroxide-containing vaccines. The aim of this study is to verify if the subcutaneous pseudolymphoma observed in this patient in the site of vaccine injection is linked to an aluminium overload. Many years after vaccination, a subcutaneous nodule was discovered in a 45-year-old woman with subcutaneous pseudolymphoma. In skin biopsy at the injection site for vaccines, aluminium (Al) deposits are assessed by Morin stain and quantification of Al is performed by Zeeman Electrothermal Atomic Absorption Spectrophotometry. Morin stain shows Al deposits in the macrophages, and Al assays (in µg/g, dry weight) were 768.10±18 for the patient compared with the two control patients, 5.61±0.59 and 9.13±0.057. Given the pathology of this patient and the high Al concentration in skin biopsy, the authors wish to draw attention when using the Al salts known to be particularly effective as adjuvants in single or repeated vaccinations. The possible release of Al may induce other pathologies ascribed to the well-known toxicity of this metal.

Gene expression in primary cultured astrocytes affected by aluminum: alteration of chaperons involved in protein folding.

OBJECTIVES: Aluminum is notorious as a neurotoxic metal. The aim of our study was to determine whether endoplasmic reticulum (ER) stress is involved in aluminum-induced apoptosis in astrocytes. METHODS: Mitochondrial RNA (mRNA) was analyzed by reverse transcription (RT)-PCR following pulse exposure of aluminum glycinate to primary cultured astrocytes. Tunicamycin was used as a positive control. RESULTS: Gene expression analysis revealed that Ire1ß was up-regulated in astrocytes exposed to aluminum while Ire1α was up-regulated by tunicamycin. Exposure to aluminum glycinate, in contrast to tunicamycin, seemed to down-regulate mRNA expression of many genes, including the ER resident molecular chaperone BiP/Grp78 and Ca(2+)-binding chaperones (calnexin and calreticulin), as well as stanniocalcin 2 and OASIS. The down-regulation or non-activation of the molecular chaperons, whose expressions are known to be protective by increasing protein folding, may spell doom for the adaptive response. Exposure to aluminum did not have any significant effects on the expression of Bax and Bcl2 in astrocytes. CONCLUSIONS: The results of this study demonstrate that aluminum may induce apoptosis in astrocytes via ER stress by impairing the protein-folding machinery.

Evaluation of the intramuscular administration of Cervarix™ vaccine on fertility, pre- and post-natal development in rats.

Cervarix™ is a prophylactic human papillomavirus (HPV)-16/18 vaccine for the prevention of cervical cancer. It contains GSK Biologicals' proprietary Adjuvant System AS04. The objective of this study was to investigate the effects of Cervarix™ and of AS04 on female fertility and pre- and post-natal development in Sprague Dawley rats. Female rats were injected with vaccine, AS04, or saline 30 days before mating and on Gestation Days 6, 8, 11 and 15. Each dose of vaccine was one-fifth the human dose volume. Treatment of rats with vaccine or AS04 was not associated with any systemic toxicity and had no impact on female fertility. There were no adverse effects on pre- or post-natal development of litters from treated rats, as judged by fetal evaluation at Gestation Day 20, and growth and survival of pups to postnatal Day 25. These results support the use of the vaccine in the targeted human population.

Aluminum hydroxide injections lead to motor deficits and motor neuron degeneration.

Gulf War Syndrome is a multi-system disorder afflicting many veterans of Western armies in the 1990-1991 Gulf War. A number of those afflicted may show neurological deficits including various cognitive dysfunctions and motor neuron disease, the latter expression virtually indistinguishable from classical amyotrophic lateral sclerosis (ALS) except for the age of onset. This ALS "cluster" represents the second such ALS cluster described in the literature to date. Possible causes of GWS include several of the adjuvants in the anthrax vaccine and others. The most likely culprit appears to be aluminum hydroxide. In an initial series of experiments, we examined the potential toxicity of aluminum hydroxide in male, outbred CD-1 mice injected subcutaneously in two equivalent-to-human doses. After sacrifice, spinal cord and motor cortex samples were examined by immunohistochemistry. Aluminum-treated mice showed significantly increased apoptosis of motor neurons and increases in reactive astrocytes and microglial proliferation within the spinal cord and cortex. Morin stain detected the presence of aluminum in the cytoplasm of motor neurons with some neurons also testing positive for the presence of hyper-phosphorylated tau protein, a pathological hallmark of various neurological diseases, including Alzheimer's disease and frontotemporal dementia. A second series of experiments was conducted on mice injected with six doses of aluminum hydroxide. Behavioural analyses in these mice revealed significant impairments in a number of motor functions as well as diminished spatial memory capacity. The demonstrated neurotoxicity of aluminum hydroxide and its relative ubiquity as an adjuvant suggest that greater scrutiny by the scientific community is warranted.