Copper toxicosis in non-COMMD1 Bedlington terriers is associated with metal transport gene ABCA12.

Wilson's disease, caused by a mutation in the ATP-ase 7B gene, is the only genetically characterised human disease with inhibition of biliary copper excretion and toxic copper accumulation in liver and occasionally brain. A similar copper toxicosis occurs in Bedlington terriers (CT) with liver damage only. Although CT has been associated with a defect in the COMMD1 gene (COMMD1 (del/del)), Bedlington terriers with CT and lacking this mutation are also recognised (non-COMMD1 (del/del)). A study was designed to identify any other gene polymorphisms associated with copper toxicity in Bedlington terriers employing genome wide association studies (GWAS) followed by deep sequencing of the candidate region. Blood for DNA analysis and liver for confirmation of the diagnosis was obtained from 30 non-COMMD1 (del/del) Bedlington terriers comprising equal numbers of CT-affected dogs and controls. DNA was initially subjected to GWAS screening and then further sequencing to target the putative mutant gene. The study has identified a significant disease association with a region on chromosome 37 containing identified SNP's which are highly significantly associated with non-COMMD1 (del/del) Bedlington terrier CT. This region contains the ABCA12 gene which bears a close functional relationship to ATP-ase 7B responsible for Wilson's disease in man.

Elucidating the contribution of the elemental composition of fetal calf serum to antigenic expression of primary human umbilical-vein endothelial cells in vitro.

One of the major obstacles to obtaining human cells of a defined and reproducible standard suitable for use as medical therapies is the necessity for FCS (fetal calf serum) media augmentation in routine cell culture applications. FCS has become the supplement of choice for cell culture research, as it contains an array of proteins, growth factors and essential ions necessary for cellular viability and proliferation in vitro. It is, however, a potential route for the introduction of zoonotic pathogens and makes defining the cell culture milieu impossible in terms of reproducibility, as the precise composition of each batch of serum not only changes but is in fact extremely variable. The present study determined the magnitude of donor variations in terms of elemental composition of FCS and the effect these variations had on the expression of a group of proteins associated with the antigenicity of primary human umbilical-vein endothelial cells, using a combination of ICPMS (inductively coupled plasma MS) and flow cytometry. Statistically significant differences were demonstrated for a set of trace elements in FCS, with correlations made to variations in antigenic expression during culture. The findings question in detail the suitability of FCS for the in vitro supplementation of cultures of primary human cells due to the lack of reproducibility and modulations in protein expression when cultured in conjunction with sera from xenogeneic donors.

Kinetics of gold nanoparticles in the human placenta.

We studied the transfer of PEGylated gold nanoparticles through perfused human placenta. In 'once-through' perfusions using 15 and 30nm nanoparticles both maternal and fetal outflows were collected. Recirculating perfusions using 10 or 15nm nanoparticles lasted 6h. The gold concentration in samples was analysed on ICP-MS. The reference compound antipyrine crossed the placenta rapidly, as expected. In open perfusions nanoparticles were detected in maternal but not in fetal outflow, suggesting the lack of placental transfer. During 6h re-circulating perfusions, no particles were detected in fetal circulation. Using transmission electron microscopy (TEM) and silver enhancement, nanoparticles could be visualized in the placental tissue mainly in the trophoblastic cell layer. In in vitro experiments, nanoparticles were taken up by BeWo choriocarcinoma cells and retained inside the cells for an extended period of 48h. In conclusion, PEGylated gold nanoparticles of the size 10-30nm did not cross the perfused human placenta in detectable amounts into the fetal circulation within 6h. Whether PEGylated gold nanoparticles eventually are able to cross placenta and whether nanoparticles affect placental functions needs to be further studied.

Copper-associated liver disease: a proteomics study of copper challenge in a sheep model.

Sheep display a variant phenotype with respect to their susceptibility to copper and derivative pathology. The North Ronaldsay sheep are acutely sensitive to environmental copper while the Cambridge breed is much more copper-tolerant. A study of protein expression in the liver of the two different breeds of sheep as a result of copper challenge would aid in the understanding of their differing pathophysiologies and contribute to knowledge of copper toxicosis in man. In this initial study, Cambridge breed sheep were challenged with oral copper and liver proteins were analyzed by two-dimensional (2-D) gel electrophoresis. Proteins whose expression pattern was modified by copper exposure were then identified by peptide mass fingerprinting using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. In conclusion, the pattern of changes in protein expression were consistent with an early adaptive response to oxidative challenge. This was followed by evidence of an impaired ability of the liver to compensate as copper loading increased, accompanied by oxidative stress-induced injury.