No association between pyrite content and lung cell responses to coal particles.

There has been an increase in the identification of cases of coal workers' pneumoconiosis (CWP) in recent years around the world. While there are a range of possible explanations for this, studies have implicated the pyrite content of coal as a key determinant of CWP risk. However, experimental studies to support this link are limited. The aim of this study was to assess the association between the pyrite content, and subsequent release of bioavailable iron, in coal particles and the response of lung cells involved in the pathogenesis of CWP (epithelial cells, macrophages and fibroblasts). Using real-world Australian coal samples, we found no evidence of an association between the pyrite content of the coal and the magnitude of the detrimental cell response. We did find evidence of an increase in IL-8 production by epithelial cells with increasing bioavailable iron (p = 0.01), however, this was not linked to the pyrite content of the coal (p = 0.75) and we did not see any evidence of a positive association in the other cell types. Given the lack of association between the pyrite content of real-world coal particles and lung cell cytotoxicity (epithelial cells and macrophages), inflammatory cytokine production (epithelial cells, macrophages and fibroblasts), and cell proliferation (fibroblasts) our data do not support the use of coal pyrite content as a predictor of CWP risk.

The Contribution of Geogenic Particulate Matter to Lung Disease in Indigenous Children.

Indigenous children have much higher rates of ear and lung disease than non-Indigenous children, which may be related to exposure to high levels of geogenic (earth-derived) particulate matter (PM). The aim of this study was to assess the relationship between dust levels and health in Indigenous children in Western Australia (W.A.). Data were from a population-based sample of 1077 Indigenous children living in 66 remote communities of W.A. (>2,000,000 km2), with information on health outcomes derived from carer reports and hospitalisation records. Associations between dust levels and health outcomes were assessed by multivariate logistic regression in a multi-level framework. We assessed the effect of exposure to community sampled PM on epithelial cell (NuLi-1) responses to non-typeable Haemophilus influenzae (NTHi) in vitro. High dust levels were associated with increased odds of hospitalisation for upper (OR 1.77 95% CI [1.02-3.06]) and lower (OR 1.99 95% CI [1.08-3.68]) respiratory tract infections and ear disease (OR 3.06 95% CI [1.20-7.80]). Exposure to PM enhanced NTHi adhesion and invasion of epithelial cells and impaired IL-8 production. Exposure to geogenic PM may be contributing to the poor respiratory health of disadvantaged communities in arid environments where geogenic PM levels are high.

Pregnancy protects against the pro-inflammatory respiratory responses induced by particulate matter exposure.

BACKGROUND: Little is known about the effect of pregnancy on the response to particulate matter. The aim of this study was to determine if pregnancy increases the susceptibility to PM from different sources using a mouse model. METHODS: Pregnant, eight-week-old C57BL/6J mice were exposed intranasally to 50 µg of diesel exhaust particles (DEP), iron oxide (Fe2O3) or silica (SiO2) in 50 µL of saline, or saline alone, on gestational day (E)7.5, E12.5 and E17.5. Groups of non-pregnant mice were exposed on day (D)0, D5 and D10. Biological samples were collected 24 h after the last exposure. Serum IL-4 and IL-6 levels were quantified by ELISA. Bronchoalveolar lavage (BAL) fluid was collected for inflammatory cells counts and assessment of IFN-É£, IL-4, IL-5, IL-6, IL-8 and IL-10 levels by ELISA. The spleen and thymus were also collected and the percentage of B cells and CD4+, CD8+ and CD4+CD25 + T cells were determined by flow cytometry. RESULTS: Exposure to silica caused an influx of lymphocytes, eosinophils and neutrophils into the lung. The magnitude of this response was suppressed by pregnancy. Pregnancy also enhanced the production of CD4+CD25 + T cells in response to DEP and silica exposure. CONCLUSIONS: Collectively, our data suggest that pregnancy reduces the inflammatory response to silica and alters the immune response to DEP. These responses were accompanied by pregnancy related changes including increased IL-4 production, reduced IL-8 production and an increase in the proportion of CD4+CD25 + T cells in response to PM exposure.

Dual-energy X-ray absorptiometry scans accurately predict differing body fat content in live sheep.

BACKGROUND: There is considerable interest in implementing mobile scanning technology for on-farm body composition analysis on live animals. These experiments evaluated the use of dual energy X-ray absorptiometry (DXA) as an accurate method of total body fat measurement in live sheep. RESULTS: In Exp. 1, visceral and whole body fat analysis was undertaken in sheep with body condition scores (BCS) in the range 2 to 3.25 (scale 1: thin to 5: fat). The relationship of BCS was moderately correlated with visceral fat depot mass (r = 0.59, P < 0.01, n = 24) and whole body fat (r = 0.70, P < 0.001, n = 24). In Exp. 2, sheep with BCS in the range 2.25 to 3.75 were blood sampled to analyse circulating leptin concentrations, and were DXA scanned immediately post mortem for total body fat. Plasma leptin concentrations had low correlations with BCS (r = 0.50, P < 0.05, n = 17) and DXA body fat (r = 0.42, P < 0.05, n = 17), and no correlation with chemical body fat (r = 0.17, P > 0.05, n = 9). There was a moderate correlation between DXA body fat and BCS (r = 0.70, P < 0.01, n = 17), and DXA body fat was highly correlated with chemical body fat (r = 0.81, P < 0.001, n = 9). In Exp. 3, a series of five DXA scans, at 8-week intervals, was performed on growing sheep over a 32-week period. The average BCS ranged from 2.39 ± 0.07 (S.E.M.) to 3.05 ± 0.11 and the DXA body fat (%) ranged from 16.8 ± 0.8 to 24.2 ± 1.2. There was a moderate correlation between DXA body fat and BCS over the 32 weeks (r = 0.61, P < 0.001, n = 24). CONCLUSIONS: Overall, these experiments indicated that there was good agreement between BCS, DXA and chemical analysis for measuring total body fat in sheep, and that DXA scanning is a valid method for longitudinal measurement of total body fat in live sheep.

The Impact of Sex and 25(OH)D Deficiency on Metabolic Function in Mice.

Both dietary fat and vitamin D deficiency have been linked with increased incidence of non-alcoholic fatty liver disease and insulin resistance. While sex differences in disease prevalence and severity are well known, the impact on disease pathogenesis remains unclear. To further explore the effect of these exposures on metabolic function, C57BL/6 male and female mice were weaned onto one of four diets; low fat vitamin D replete, low fat vitamin D deficient, or two high fat diets, one vitamin D replete and one deficient. Visceral fat, hepatic adiposity, and insulin resistance were measured after five and a half weeks. Vitamin D deficiency, independent of dietary fat, increased hepatic fat accumulation in both sexes (p = 0.003), although did not increase hepatic expression of interleukin-6 (p = 0.92) or tumor necrosis factor-α (p = 0.78). Males were observed to have greater insulin resistance (glucose area under the curve: p < 0.001, homeostatic model assessment for insulin resistance: p = 0.046), and have greater visceral adiposity (p < 0.001), while female mice had greater hepatic fat accumulation (p < 0.001). This study is the first to demonstrate vitamin D deficiency alone can cause hepatic accumulation while also being the first to observe higher liver fat percentages in female mice.

PTEN depletion decreases disease severity and modestly prolongs survival in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is the second most common genetic cause of death in childhood. However, no effective treatment is available to halt disease progression. SMA is caused by mutations in the survival motor neuron 1 (SMN1) gene. We previously reported that PTEN depletion leads to an increase in survival of SMN-deficient motor neurons. Here, we aimed to establish the impact of PTEN modulation in an SMA mouse model in vivo. Initial experiments using intramuscular delivery of adeno-associated vector serotype 6 (AAV6) expressing shRNA against PTEN in an established mouse model of severe SMA (SMNΔ7) demonstrated the ability to ameliorate the severity of neuromuscular junction pathology. Subsequently, we developed self-complementary AAV9 expressing siPTEN (scAAV9-siPTEN) to allow evaluation of the effect of systemic suppression of PTEN on the disease course of SMA in vivo. Treatment with a single injection of scAAV9-siPTEN at postnatal day 1 resulted in a modest threefold extension of the lifespan of SMNΔ7 mice, increasing mean survival to 30 days, compared to 10 days in untreated mice. Our data revealed that systemic PTEN depletion is an important disease modifier in SMNΔ7 mice, and therapies aimed at lowering PTEN expression may therefore offer a potential therapeutic strategy for SMA.

Early detection of motor dysfunction in the SOD1G93A mouse model of Amyotrophic Lateral Sclerosis (ALS) using home cage running wheels.

The SOD1G93A mouse has been used since 1994 for preclinical testing in amyotrophic lateral sclerosis (ALS). Despite recent genetic advances in our understanding of ALS, transgenic mice expressing mutant SOD1 remain the best available, and most widely used, vertebrate model of the disease. We previously described an optimised and rapid approach for preclinical studies in the SOD1G93A mouse. Here we describe improvements to this approach using home cage running wheels to obtain daily measurements of motor function, with minimal intervention. We show that home cage running wheels detect reductions in motor function at a similar time to the rotarod test, and that the data obtained are less variable allowing the use of smaller groups of animals to obtain satisfactory results. This approach refines use of the SOD1G93A model, and reduces the number of animals undergoing procedures of substantial severity, two central principles of the 3Rs (replacement, reduction and refinement of animal use in research). The small group sizes and rapid timescales enable affordable large-scale therapeutic pre-screening in the SOD1G93A mouse, as well as rapid validation of published positive effects in a second laboratory, one of the major stumbling blocks in ALS preclinical therapy development.

Axonal Transport Defects in a Mitofusin 2 Loss of Function Model of Charcot-Marie-Tooth Disease in Zebrafish.

Charcot-Marie-Tooth disease (CMT) represents a group of neurodegenerative disorders typically characterised by demyelination (CMT1) or distal axon degeneration (CMT2) of motor and sensory neurons. The majority of CMT2 cases are caused by mutations in mitofusin 2 (MFN2); an essential gene encoding a protein responsible for fusion of the mitochondrial outer membrane. The mechanism of action of MFN2 mutations is still not fully resolved. To investigate a role for loss of Mfn2 function in disease we investigated an ENU-induced nonsense mutation in zebrafish MFN2 and characterised the phenotype of these fish at the whole organism, pathological, and subcellular level. We show that unlike mice, loss of MFN2 function in zebrafish leads to an adult onset, progressive phenotype with predominant symptoms of motor dysfunction similar to CMT2. Mutant zebrafish show progressive loss of swimming associated with alterations at the neuro-muscular junction. At the cellular level, we provide direct evidence that mitochondrial transport along axons is perturbed in Mfn2 mutant zebrafish, suggesting that this is a key mechanism of disease in CMT. The progressive phenotype and pathology suggest that zebrafish will be useful for further investigating the disease mechanism and potential treatment of axonal forms of CMT. Our findings support the idea that MFN2 mutation status should be investigated in patients presenting with early-onset recessively inherited axonal CMT.

Optimised and rapid pre-clinical screening in the SOD1(G93A) transgenic mouse model of amyotrophic lateral sclerosis (ALS).

The human SOD1(G93A) transgenic mouse has been used extensively since its development in 1994 as a model for amyotrophic lateral sclerosis (ALS). In that time, a great many insights into the toxicity of mutant SOD1 have been gained using this and other mutant SOD transgenic mouse models. They all demonstrate a selective toxicity towards motor neurons and in some cases features of the pathology seen in the human disease. These models have two major drawbacks. Firstly the generation of robust preclinical data in these models has been highlighted as an area for concern. Secondly, the amount of time required for a single preclinical experiment in these models (3-4 months) is a hurdle to the development of new therapies. We have developed an inbred C57BL/6 mouse line from the original mixed background (SJLxC57BL/6) SOD1(G93A) transgenic line and show here that the disease course is remarkably consistent and much less prone to background noise, enabling reduced numbers of mice for testing of therapeutics. Secondly we have identified very early readouts showing a large decline in motor function compared to normal mice. This loss of motor function has allowed us to develop an early, sensitive and rapid screening protocol for the initial phases of denervation of muscle fibers, observed in this model. We describe multiple, quantitative readouts of motor function that can be used to interrogate this early mechanism. Such an approach will increase throughput for reduced costs, whilst reducing the severity of the experimental procedures involved.

Direct evidence for axonal transport defects in a novel mouse model of mutant spastin-induced hereditary spastic paraplegia (HSP) and human HSP patients.

Mutations in spastin are the most common cause of hereditary spastic paraplegia (HSP) but the mechanisms by which mutant spastin induces disease are not clear. Spastin functions to regulate microtubule organisation, and because of the essential role of microtubules in axonal transport, this has led to the suggestion that defects in axonal transport may underlie at least part of the disease process in HSP. However, as yet there is no direct evidence to support this notion. Here we analysed axonal transport in a novel mouse model of spastin-induced HSP that involves a pathogenic splice site mutation, which leads to a loss of spastin protein. A mutation located within the same splice site has been previously described in HSP. Spastin mice develop gait abnormalities that correlate with phenotypes seen in HSP patients and also axonal swellings containing cytoskeletal proteins, mitochondria and the amyloid precursor protein (APP). Pathological analyses of human HSP cases caused by spastin mutations revealed the presence of similar axonal swellings. To determine whether mutant spastin influenced axonal transport we quantified transport of two cargoes, mitochondria and APP-containing membrane bound organelles, in neurons from mutant spastin and control mice, using time-lapse microscopy. We found that mutant spastin perturbs anterograde transport of both cargoes. In neurons with axonal swellings we found that the mitochondrial axonal transport defects were exacerbated; distal to axonal swellings both anterograde and retrograde transport were severely reduced. These results strongly support a direct role for defective axonal transport in the pathogenesis of HSP because of spastin mutation.

Nutritional influences on reproductive neuroendocrine output: insulin, leptin, and orexigenic neuropeptide signaling in the ovine hypothalamus.

This study investigated how changing nutritional status may alter reproductive neuroendocrine (LH) output via circulating leptin and insulin signaling through orexigenic hypothalamic pathways. Thin sheep were given an increasing nutritional plane (INP), sheep with intermediate adiposity a static nutritional plane (SNP), and fat sheep a decreasing nutritional plane (DNP) for 6 wk. Mean group adiposities converged by wk 6, LH output increased in INP, remained unchanged in SNP, and decreased in DNP sheep. Plasma and cerebrospinal fluid (CSF) insulin and plasma leptin concentrations increased in INP but did not change in the SNP and DNP groups. In INP sheep, LH output correlated positively with adiposity and plasma and CSF insulin concentrations and negatively with orexigenic neuropeptide Y gene expression in the hypothalamic arcuate nucleus (ARC). In DNP sheep, LH output correlated positively with adiposity, CSF leptin concentrations, and ARC proopiomelanocortin gene expression and negatively with leptin receptor (OB-Rb) and agouti-related peptide gene expression in the ARC. These data are consistent with the feedback response to an increasing nutritional plane being mediated by increasing circulating insulin entering the brain and stimulating LH via inhibition of hypothalamic neuropeptide Y and the response to a decreasing nutritional plane being mediated by altered hypothalamic leptin signaling brought about by increased OB-Rb expression and decreased melanocortin signaling. Because end point adiposity was similar yet LH output was different, the hypothalamus apparently retains a nutritional memory, based on changes in orexigenic neuropeptide expression, that influences contemporary neuroendocrine responses.

Interrelationships among plasma progesterone concentrations, luteal anatomy and function, and placental ontogeny during gestation in a viviparous lizard (Niveoscincus metallicus: Scincidae).

Plasma progesterone concentrations were measured at six stages of gestation in the viviparous lizard Niveoscincus metallicus. Anatomical and functional parameters of luteal activity were also investigated. The diameter of the corpus luteum (CL) decreased gradually though gestation, as did the diameter of the luteal cells. Major degenerative changes were observed in CLs post-partum. Plasma progesterone concentrations were basal both prior to, and just after, ovulation; a rapid increase occurred in early gestation. Plasma progesterone concentrations remained elevated until late gestation, but fell some 2 weeks before parturition. In vitro production of progesterone was greater in CLs in mid- than in late-gestation, and the addition of prostaglandin F(2alpha) to the incubation medium had no effect on progesterone production. Non-luteal ovarian tissue and adrenals produced progesterone, but at approximately one-tenth the rate of production by CLs. Temporal correlations between the plasma progesterone profile and stages of placental development were also assessed. The rise in plasma progesterone concentrations occurs before differentiation of the chorioallantoic placenta, but progesterone is still high when it degenerates. We conclude that the CLs are the major source of gestational progesterone in N. metallicus.