Levamisole derivatives as immune modulators for the treatment of amyotrophic lateral sclerosis (ALS).

Aim: To discover derivatives of the anthelmintic drug levamisole, which has been reported to possess immune-modulatory properties, as treatments for amyotrophic lateral sclerosis (ALS), which has been suggested to be in part an autoimmune disease. Results: We have synthesized ten analogs of the racemic version of levamisole, tetramisole, as well as eleven analogs on a related system. All of the analogs have been tested for their ability to affect the response of five ALS-relevant cytokines. Conclusion: We have discovered a number of interesting derivatives that have encouraging cytokine response data and good metabolic stability, with the potential to have a positive impact on ALS either as single agents, or in combination.

Aim: To discover derivatives of the antiparasitic worm drug levamisole, which has been reported to be able to modulate the immune response, as treatments for amyotrophic lateral sclerosis (ALS), which has been suggested to be in part an autoimmune disease. Results: We have synthesized ten analogs of a variation of levamisole, tetramisole, as well as 11 analogs on a related system. All of the analogs have been tested for their ability to affect the response of five ALS-relevant immune-modulatory substances (cytokines). Conclusion: We have discovered a number of interesting derivatives that have encouraging cytokine response data and good metabolic stability, with the potential to have a positive impact on ALS either as single agents, or in combination.

Metals and Metalloids Increase in Cycas micronesica Seed Gametophyte Tissue in Shaded Growth Conditions.

Exposure to environmental toxins may be partly responsible for mammal neurodegenerative disorders. Consumption of seeds from Guam's cycad tree has been linked to the disorder known as amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC). The unambiguous identification of causal agents of ALS-PDC has been elusive. We have examined the levels of eight metals and metalloids in cycad seeds as a function of the ambient shade in which the plants were grown. Of these metals, the data strongly suggest that aluminum (Al) and selenium (Se) are present in washed flour prepared from southern Guam's cycad seed tissues at elevated levels, especially when the trees are grown in shade. Previous authors have speculated that Al and Se are involved in various ALS outcomes, and our results support this interpretation.

Computed tomography reveals hip dysplasia in the extinct Pleistocene saber-tooth cat Smilodon.

Reconstructing the behavior of extinct species is challenging, particularly for those with no living analogues. However, damage preserved as paleopathologies on bone can record how an animal moved in life, potentially reflecting behavioral patterns. Here, we assess hypothesized etiologies of pathology in a pelvis and associated right femur of a Smilodon fatalis saber-toothed cat, one of the best-studied species from the Pleistocene-age Rancho La Brea asphalt seeps, California, USA, using visualization by computed tomography (CT). The pelvis exhibits massive destruction of the right hip socket that was interpreted, for nearly a century, to have developed from trauma and infection. CT imaging reveals instead that the pathological distortions characterize chronic remodeling that began at birth and led to degeneration of the joint over the animal's life. These results suggest that this individual suffered from hip dysplasia, a congenital condition common in domestic dogs and cats. This individual reached adulthood but could not have hunted properly nor defended territory on its own, likely relying on a social group for feeding and protection. While extant social felids are rare, these fossils and others with similar pathologies are consistent with a spectrum of social strategies in Smilodon supported by a predominance of previous studies.

Early postnatal injections of whole vaccines compared to placebo controls: Differential behavioural outcomes in mice.

The present study was designed to evaluate the possible effects of the paediatric vaccination schedule in the United States on the central nervous system in a murine model. We compared the impact of treatment with the whole vaccines versus true placebo control. Seventy-six pups were divided into three groups: two vaccinated groups and unvaccinated control. The two vaccinated groups were treated between 7 and 21 post-natal days either with one or three times of the vaccine doses per body weight as used in children between newborn and eighteen months of age. The post-vaccination development, neuromotor behaviours and neurobehavioural abnormalities (NBAs) were evaluated in all mouse groups during the 67 post-natal weeks of mouse age. Mouse body weight was affected only in the vaccinated females compared to males and control. Some NBAs such as decreased sociability, increased anxiety-like behaviours, and alteration of visual-spatial learning and memory were observed in vaccinated male and female mice compared to controls. The present study also shows a slower acquisition of some neonatal reflexes in vaccinated female mice compared to vaccinated males and controls. The observed neurodevelopmental alterations did not show a linear relationship with vaccine dose, suggesting that the single dose gave a saturated response. The outcomes seemed to be sex-dependent and transient with age.

Investigating microglia during motor neuron degeneration using a zebrafish model.

Many different types of pathologies can arise in the central nervous system (CNS), such as neurodegeneration. The incidence of neurodegenerative diseases continues to increase, yet the pathogenesis underlying most neurodegenerative diseases, notably in amyotrophic lateral sclerosis (ALS), remains elusive. Neuronal support cells, or glia, are known to play a crucial role in ALS. Microglia are the resident immune cells of the CNS and also have neurotrophic support functions. These cells have a disease-modifying function in ALS, yet this role is not well understood. A likely reason for this is that the intact CNS is particularly challenging to access for investigation in patients and in most animal models, which has impeded research in this field. The zebrafish is emerging as a robust model system to investigate cells in vivo, and offer distinct advantages over other vertebrate models for investigating neurodegenerative diseases. Live imaging in vivo is a powerful technique to characterize the role of dynamic cells such as microglia during neurodegeneration, and zebrafish provide a convenient means for live imaging. Here, we discuss the zebrafish as a model for live imaging, provide a brief overview of available high resolution imaging platforms that accommodate zebrafish, and describe our own in vivo studies on the role of microglia during motor neuron degeneration. Live in vivo imaging is anticipated to provide invaluable advancements to defining the pathogenesis underlying neurodegenerative diseases, which may in turn allow for more specifically targeted therapeutics.

Neuroprotective effect of CuATSM on neurotoxin-induced motor neuron loss in an ALS mouse model.

CuATSM is a PET-imaging agent that has recently received attention for its success in extending the lifespan in animals in several neurodegenerative disease models. In the SOD1G93A model of ALS, CuATSM prolonged mouse longevity far longer than any previously tested therapeutic agents. The mechanism underlying this outcome has not been fully understood, but studies suggest that this copper complex contributes to maintaining copper homeostasis in mitochondria. More specifically for the SOD1 model, the molecule supplies copper back to the SOD1 protein. Additionally, CuATSM demonstrated similar protective effects in various in vivo Parkinson's disease mouse models. In the current pilot study, we utilized a neurodegenerative mouse model of motor neuron degeneration induced by the neurotoxin ß-sitosterol ß-D-glucoside. In this model, slow but distinct and progressive features of sporadic ALS occur. Treatment with CuATSM kept animal behavioural performance on par with the controls and prevented the extensive motor neuron degeneration and microglia activation seen in the untreated animals. These outcomes support a broader neuroprotective role for CuATSM beyond mutant SOD models of ALS.

Animal models of amyotrophic lateral sclerosis: A comparison of model validity.

Animal models are necessary to investigate the pathogenic features underlying motor neuron degeneration and for therapeutic development in amyotrophic lateral sclerosis (ALS). Measures of model validity allow for a critical interpretation of results from each model and caution from over-interpretation of experimental models. Face and construct validity refer to the similarity in phenotype and the proposed causal factor to the human disease, respectively. More recently developed models are restricted by limited phenotype characterization, yet new models hold promise for novel disease insights, thus highlighting their importance. In this article, we evaluate the features of face and construct validity of our new zebrafish model of environmentally-induced motor neuron degeneration and discuss this in the context of current environmental and genetic ALS models, including C9orf72, mutant Cu/Zn superoxide dismutase 1 and TAR DNA-binding protein 43 mouse and zebrafish models. In this mini-review, we discuss the pros and cons to validity criteria in each model. Our zebrafish model of environmentally-induced motor neuron degeneration displays convincing features of face validity with many hallmarks of ALS-like features, and weakness in construct validity. However, the value of this model may lie in its potential to be more representative of the pathogenic features underlying sporadic ALS cases, where environmental factors may be more likely to be involved in disease etiology than single dominant gene mutations. It may be necessary to compare findings between different strains and species modeling specific genes or environmental factors to confirm findings from ALS animal models and tease out arbitrary strain- and overexpression-specific effects.

Aluminum as a CNS and Immune System Toxin Across the Life Span.

In the following, I will consider the impact of aluminum on two major systems, the central nervous system (CNS) and the immune system, across the life span. The article will discuss the presence of aluminum in the biosphere, its history, and the sources of the element. These include food, water cosmetics, some vaccines, and a range of other sources. I will also consider aluminum's unique chemistry. Finally, in humans and animals, I will consider how aluminum may impact the CNS at various levels of organization and how it may be involved in various neurological disease states across the life span. These disorders include those of infancy and childhood, such as autism spectrum disorder (ASD), as well as those in adulthood, such as in Alzheimer's disease. The bidirectional nature of CNS-immune system interactions will be considered and put into the context of neurological disorders that have an autoimmune component. I will argue that the exposure to humans and animals to this element needs to be reduced if we are to diminish some CNS and immune system disorders.

Modeling Environmentally-Induced Motor Neuron Degeneration in Zebrafish.

Zebrafish have been used to investigate motor neuron degeneration, including as a model system to examine the pathogenesis of amyotrophic lateral sclerosis (ALS). The use of zebrafish for this purpose has some advantages over other in vivo model systems. In the current paper, we show that bisphenol A (BPA) exposure in zebrafish embryos results in motor neuron degeneration with affected motor function, reduced motor axon length and branching, reduced neuromuscular junction integrity, motor neuron cell death and the presence of activated microglia. In zebrafish, motor axon length is the conventional method for estimating motor neuron degeneration, yet this measurement has not been confirmed as a valid surrogate marker. We also show that reduced motor axon length as measured from the sagittal plane is correlated with increased motor neuron cell death. Our preliminary timeline studies suggest that axonopathy precedes motor cell death. This outcome may have implications for early phase treatments of motor neuron degeneration.

Is exposure to aluminium adjuvants associated with social impairments in mice? A pilot study.

BACKGROUND: Our group has shown that significant correlations exist between rates of Autism Spectrum Disorder (ASD) and total aluminum adjuvants given to children through vaccines in several Western countries. These correlations satisfied eight out of nine Hill criteria for causality. Experimental studies have demonstrated a range of behavioural abnormalities in young mice after postnatal exposure to aluminium. To build on our previous work, the current study will investigate the effect of aluminium adjuvants on social behaviour in mice. Anomalies in social interaction are a key characteristic of those with ASD. METHODS: Neonatal CD-1 mice pups were injected with either a total of 550µg of aluminum hydroxide gel (experimental group) or saline (control) spread out during the first two weeks of postnatal life. The mice were then subjected to behavioural tests for social interest and social novelty at postnatal week 8, 17 and 29. p-Values were calculated using the Mann-Whitney and Kruskal Wallis tests. RESULTS: Aluminum injected mice showed diminished social interest compared to controls at week 8 (p=0.016) and 17 (p=0.012). They also demonstrated abnormal social novelty from controls at week 8 (p=0.002) and week 29 (p=0.042). CONCLUSION: This is the first experimental study, to our knowledge, to demonstrate that aluminum adjuvants can impair social behaviour if applied in the early period of postnatal development. The study, however, is insufficient to make any assertive claims about the link between aluminium adjuvants and ASD in humans.

RETRACTED: Subcutaneous injections of aluminum at vaccine adjuvant levels activate innate immune genes in mouse brain that are homologous with biomarkers of autism.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief and Authors, due to evidence of incorrect data. The data of gel images in several figures (Fig. 2A and C and Fig. 4 A, B, C and D) are incorrectly presented. Given that the authors can no longer access the original gels and it would be necessary to redo the experiments, it is concluded that the data and results presented in this paper are clearly not reliable. In light of these concerns, the Editor-in-Chief and Authors have jointly decided to retract the article. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the preparation and submission process.

Skeletal trauma reflects hunting behaviour in extinct sabre-tooth cats and dire wolves.

Skeletal-injury frequency and distribution are likely to reflect hunting behaviour in predatory vertebrates and might therefore differ between species with distinct hunting modes. Two Pleistocene predators from the Rancho La Brea asphalt seeps, the sabre-tooth cat, Smilodon fatalis, and dire wolf, Canis dirus, represent ambush and pursuit predators, respectively. On the basis of a collection of over 1,900 pathological elements, the frequency of traumatic injury across skeletal elements in these two species was calculated. Here we show that the frequency of trauma in the sabre-tooth cat exceeds that of the dire wolf (4.3% compared to 2.8%), implying that the killing behaviour of S. fatalis entailed greater risk of injury. The distribution of traumatic injuries also differed between the two species. S. fatalis, an ambush predator, was injured more often than expected across the lumbar vertebrae and shoulders whereas C. dirus, a pursuit predator, had higher than expected levels of injury in the limbs and cervical vertebrae. Spatial analysis was used to quantify differences in the distribution of putative hunting injuries. Analysis of injury locations discriminated true hotspots from injury-dense areas and facilitated interpretation of predatory behaviour, demonstrating the use of spatial analyses in the study of vertebrate behaviour and evolution. These results suggest that differences in trauma distribution reflect distinct hazards of each species' hunting mode.

Behavioral abnormalities in female mice following administration of aluminum adjuvants and the human papillomavirus (HPV) vaccine Gardasil.

Vaccine adjuvants and vaccines may induce autoimmune and inflammatory manifestations in susceptible individuals. To date most human vaccine trials utilize aluminum (Al) adjuvants as placebos despite much evidence showing that Al in vaccine-relevant exposures can be toxic to humans and animals. We sought to evaluate the effects of Al adjuvant and the HPV vaccine Gardasil versus the true placebo on behavioral and inflammatory parameters in female mice. Six-week-old C57BL/6 female mice were injected with either, Gardasil, Gardasil + pertussis toxin (Pt), Al hydroxide, or, vehicle control in amounts equivalent to human exposure. At 7.5 months of age, Gardasil and Al-injected mice spent significantly more time floating in the forced swimming test (FST) in comparison with vehicle-injected mice (Al, p = 0.009; Gardasil, p = 0.025; Gardasil + Pt, p = 0.005). The increase in floating time was already highly significant at 4.5 months of age for the Gardasil and Gardasil + Pt group (p ≤ 0.0001). No significant differences were observed in the number of stairs climbed in the staircase test which measures locomotor activity. These results indicate that differences observed in the FST were unlikely due to locomotor dysfunction, but rather due to depression. Moreover, anti-HPV antibodies from the sera of Gardasil and Gardasil + Pt-injected mice showed cross-reactivity with the mouse brain protein extract. Immunohistochemistry analysis revealed microglial activation in the CA1 area of the hippocampus of Gardasil-injected mice. It appears that Gardasil via its Al adjuvant and HPV antigens has the ability to trigger neuroinflammation and autoimmune reactions, further leading to behavioral changes.

Necroptosis in amyotrophic lateral sclerosis and other neurological disorders.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive degeneration of upper and lower motor neurons. Cell death in ALS and in general was previously believed to exist as a dichotomy between apoptosis and necrosis. Most research investigating cell death mechanisms in ALS was conducted before the discovery of programmed necrosis thus did not use selective cell death pathway-specific markers. Recently, a new form of programmed cell death, termed "necroptosis", has been characterized and has been recently implicated in ALS as a primary mechanism driving motor neuron cell death in different forms of ALS. The present review is aimed at summarizing cell death pathways that are currently implicated in ALS and highlighting the emerging evidence on necroptosis as a major driver of motor neuron cell death.

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.

WITHDRAWN: Behavioral abnormalities in young female mice following administration of aluminum adjuvants and the human papillomavirus (HPV) vaccine Gardasil.

This article has been withdrawn at the request of the Editor-in-Chief due to serious concerns regarding the scientific soundness of the article. Review by the Editor-in-Chief and evaluation by outside experts, confirmed that the methodology is seriously flawed, and the claims that the article makes are unjustified. As an international peer-reviewed journal we believe it is our duty to withdraw the article from further circulation, and to notify the community of this issue. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.