A Sting In The Tail (Trust Investigations Ltd Book 1)
When Martin Cumberworth and his wife, Ann, travelled to Peru earlier this year on an organised trip, they followed medical advice to the letter. Before they left their home in Hertfordshire, they had vaccines for yellow fever, typhoid and tetanus and started taking malaria tablets. But despite their precautions, they were both bitten on the head by an insect and ended up with larvae the size of fishing maggots living in their scalps. But it could have been a lot worse if we'd been bitten in the eye socket or the groin, or they had hatched into flies and laid more eggs in our scalps.
The Cumberworths had fallen foul of the South American botfly while on a jungle trip but, undaunted, they are preparing for another intrepid break in Chile and Patagonia next year. Yet despite the threat of serious illness, many travellers routinely jet off across the world with no protection whatsoever.
The availability of cheap long haul flights and last-minute deals have led people to bypass crucial medical advice on vaccinations, medications and even simple hygiene practices, laying them open to the threat of malaria, intestinal worms, blood-sucking parasites and burrowing insects.
Green Motion’s response
The most acute - and intractable cases are treated at London's Hospital for Tropical Diseases. Earlier this year film-makers were allowed inside the hospital for three months, and the Cumberworths feature in the programme, which, despite its jocular title, Help! I Caught i t Abroad, makes for harrowing viewing. Patients include a prison officer who went to Gambia at the last minute and didn't take anti-malarial drugs. He was bitten by mosquitoes just four times but contracted the disease, leading to the amputation of his fingers and lower legs.
Also in the programme is a woman with a cyst in her brain caused by tapeworms contracted in Ethiopia and a man who was bitten by a sandfly in Greece and has been infected by a parasite that is attacking his major organs and destroying his eyesight. There's been a worrying rise in malignant tertian malaria, which, if untreated, will kill you.
Measurement was made of distance traveled in terms of grid squares crossed and total number of rears. Weekly measurements of open field activity were recorded for 3 min from when animals were placed facing any corner of the arena. The open end was raised by 3 cm above the floor by a wooden support to prevent nonpurposeful displacement of the contents. Mice were placed individually in the cages for 2 hr, at which point the food remaining in the cages was weighed and the amount displaced burrowed was calculated.
Weekly measurements were taken during prion disease progression.
The horizontal bar was designed to assess forelimb muscular strength and coordination. Each mouse was held by the tail and allowed to grip the central point of the bar with its front paws only. The inverted screen assessed muscular strength for all four limbs.
The mouse was placed on the screen, which was then slowly 2 s inverted. Padding was provided to cushion mice falling from both bar and screen apparatus. The skin overlying the sternum was incised, and the chest cavity opened to expose the heart. A butterfly needle connected to a peristaltic pump Gilson, Villiers le Bel, France was inserted into the left ventricle.
The right atrium was cut, and the animal was perfused transcardially with heparinized saline 0. Those tissues used for cell isolation were prepared as below. Sorted cells were collected in 1.
Purity of sorted astrocytes and microglia was determined by quantitative polymerase chain reaction qPCR Figure 1 d,e. Sorted microglia and astrocytes samples were centrifuged at 10, g for 10 min. Where possible, probes were designed to cross an intron such that they were cDNA specific. Primer and probe sequences, along with accession numbers for mRNA sequence of interest may be found in Table 1. Table 1 lists the sequences for primers and probes for those assays that have not been published in our prior studies Cox et al. A standard curve was plotted of C t value versus the log of the concentration assigned an arbitrary value since the absolute concentration of cytokine transcripts is not known.
Table 2 lists the antibodies and dilution and incubation times. All blots except those for PrP used the above protocol. Proteinase K treatment was used to determine protease resistance of PrP Sc. The blots were quantified using Image J software. Slides were cooled to room temperature by gently rinsing with cold tap water. The reaction was completed using the ABC method with peroxidase as enzyme, diaminobenzidine as chromagen, and 0.
Slides were counterstained with hematoxylin, dehydrated, and coverslips mounted using DPX Sigma, Poole, UK , except those for NeuN and Sy38 to preserve high contrast for quantification. Thereafter, the reaction continued as above. After rehydration, sections were treated with 0. The DAB reaction had the additional component of 0. Synaptophysin density was assessed using transmittance of synaptic layers assessed in ImageJ after image capture using a Leica DM microscope and CellA software Olympus. A mean transmittance was calculated for corpus callosum cc , overlying cortex, stratum oriens, CA1, stratum radiatum, and the stratum lacunosum.
Synaptophysin density was measured in two thalamic areas in the same sections: the ventral posteromedial nucleus of the thalamus VPM and the posterior nucleus of the thalamus Po. Once again, the cc served as an internal control.
An average of this measure was taken from each animal and collated to form a group average, which represented the extent of neuronal loss in the posterior nucleus. Thereafter, the reaction was continued as per the general protocol.
All statistical analyses were performed using GraphPad Prism 5 for Windows. Bonferroni post hoc tests were performed following significant main effects or interactions in ANOVA analysis. Survival time days of animals postinoculation with ME7 was assessed by Kaplan—Meier log rank survival analysis. Statistical analyses are shown in Table 4.
Arg1 was neither altered by disease nor by genotype 4f. These data are strongly suggestive of an altered microglial phenotype; but, because some of these transcripts may also be astrocytic in origin, we then isolated microglia and astrocytes from additional animals and assessed key markers Figure 5. Cathepsin S and the NADPH oxidase subunits also showed increases in astrocytes, but the overall level of expression of these genes in astrocytes was much lower than in microglia.
Therefore, at the transcriptional level, it is clear that the lack of IFNAR1 signaling results in significant phenotypic changes in microglia and astrocytes. Cathepsin D labeling was most obvious surrounding the large rounded nuclei of neuronal cells in the thalamus of NBH animals, with small microglial cells white arrows sparsely distributed. Conversely, in the thalamus of WT ME7 animals, the predominant pattern of labeling was a dense labeling of the soma of small cells with small nuclei and these strongly positive cells were clearly increased in number.
These cells are microglia, based on their size and nuclear morphology. These data indicate that IFNAR1 deficiency limits the extent of lysosomal cathepsin D expression in microglia in the key area of neurodegeneration in the ME7 prion disease model Reis et al. Based on these white matter CD68 changes, we assessed both IBA1 and cathepsin D labeling in the cc, directly dorsal to the degenerating hippocampus.
Soothing a Sting to the Integrity of Science - Aquatic Informatics
We show Figure 8 m,q that in both cases, there is a clear increase in labeling in this white matter tract in ME7 that contains axons exiting the degenerating hippocampus. Both the hippocampus and the thalamus showed severe synaptic loss in ME7 animals as previously shown Reis et al. Similarly, we have previously described significant neuronal loss in the posterior thalamic nucleus Reis et al.
This is also manifest when examining 6D11 labeling of extracellular PrP in histological sections in the hippocampus and thalamus Figure 11 e. The absence of IFNAR1 led to a slowing of disease progression and reduced synaptic and neuronal loss. These possibilities require further study. We have previously shown major increases in phagocytic and lysosomal transcripts and increased phagocytic activity in ME7 animals Hughes et al. Initial experiments used RNA isolated from homogenates of the hippocampus and thalamus, the major regions of pathology in the ME7 model, and the patterns observed in those crude preparations were almost entirely replicated in microglial cells isolated, by FACS sorting, from these same regions.
Green Motion has low car hire rates – but users say there’s a sting in the tail
The major pattern observed in the current transcriptional analysis was a suppression of genes associated with phagocytosis and lysosomal activity including scavenger receptors, components of the complement pathway, and NADPH oxidase and lysosomal cathepsins. There is also direct evidence that inhibition of microglial phagocytosis limits inflammatory neuronal death in other model systems Neher et al.
Therefore, there is evidence to support the idea that mitigating these pathways, in this case via deletion of IFNAR1, will slow degenerative processes. Cathepsin D is a relatively ubiquitously expressed lysosomal protein. Although cathepsin D is also important for neuronal integrity Shacka et al. However, unlike in the acute models described above, it also appears to contribute to chronic neurological decline.
Importantly, the early signs of disease, including burrowing and open field hyperactivity Cunningham et al. This is an important observation because it indicates that neither early clearance of the surgically inoculated infectious material Beringue et al. Such longitudinal analysis has typically not been performed in other model systems in which IFNAR1 has been shown to be influential.
Cunningham has received speaker's fees from Eli Lilly but has no other financial relationships to disclose. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries other than missing content should be directed to the corresponding author for the article.