Jonathan Tuke

Evaluation of the triple tibial osteotomy. A new technique for the management of the canine cruciate-deficient stifle.

The triple tibial osteotomy (TTO) is a technique which combines the features of tibial tuberosity advancement and wedge osteotomy for the treatment of complete and partial cruciate ligament injuries in dogs. In this paper, the technique is described and the results of a prospective study of 64 consecutive cases are presented. TTO provided a satisfactory clinical outcome in a very high percentage of cases. The technique is relatively easy to learn and has a low post-operative complication rate.

Low-dose endotoxin potentiates capsaicin-induced pain in man: evidence for a pain neuroimmune connection

Despite the wealth of evidence in animals that immune activation has a key role in the development and maintenance of chronic pain, evidence to support this in humans is scant. We have sought such evidence by examining the effect of a subtle immunological stimulus, low dose intravenous endotoxin, on the allodynia, hyperalgesia, flare and pain produced by intradermal capsaicin in healthy volunteers. Here we provide evidence of immune priming of this neuropathic-like pain response in humans. Specifically, in 12 healthy volunteers, activation of Toll-Like Receptor 4 by endotoxin (0.4ng/kg IV) caused significant 5.1-fold increase in the 90-min integral of areas of capsaicin-induced allodynia (95% CI 1.3-9.1), 2.2-fold increase in flare (95% CI 1.9-2.6) and 1.8-fold increase in hyperalgesia (95% CI 1.1-2.5) following 50μg intradermal capsaicin injected into the forearm 3.5h after endotoxin. These data demonstrate clinically a significant role for the neuroimmune pain connection in modifying pain, thus providing evidence that immune priming may produce pain enhancement in humans and hence offer a novel range of pharmacological targets for anti-allodynics and/or analgesics. Additionally, the simplicity of the model makes it suitable as a test-bed for novel immune-targeted pain therapeutics.

Aboriginal mitogenomes reveal 50,000 years of regionalism in Australia

Aboriginal Australians represent one of the longest continuous cultural complexes known. Archaeological evidence indicates that Australia and New Guinea were initially settled approximately 50 thousand years ago (ka); however, little is known about the processes underlying the enormous linguistic and phenotypic diversity within Australia. Here we report 111 mitochondrial genomes (mitogenomes) from historical Aboriginal Australian hair samples, whose origins enable us to reconstruct Australian phylogeographic history before European settlement. Marked geographic patterns and deep splits across the major mitochondrial haplogroups imply that the settlement of Australia comprised a single, rapid migration along the east and west coasts that reached southern Australia by 49–45 ka. After continent-wide colonization, strong regional patterns developed and these have survived despite substantial climatic and cultural change during the late Pleistocene and Holocene epochs. Remarkably, we find evidence for the continuous presence of populations in discrete geographic areas dating back to around 50 ka, in agreement with the notable Aboriginal Australian cultural attachment to their country.

Genetic, pathological and physiological determinants of transdermal fentanyl pharmacokinetics in 620 cancer patients of the EPOS study.

Objective This study aimed to investigate whether CYP3A4/5 genetic variants, together with clinical and patient factors, influence serum fentanyl and norfentanyl concentrations and their ratio in cancer pain patients receiving transdermal fentanyl. Methods CYP3A4*22 and CYP3A5*3 polymorphisms were analysed in 620 cancer pain patients receiving transdermal fentanyl (12.5–700 &mgr;g/h) from the European Pharmacogenetic Opioid Study. Using stepwise linear regression, CYP3A4/5 genetic variability was examined in combination with patient factors relating to organ drug elimination function and ABCB1 genetics for their association with serum fentanyl and norfentanyl concentrations and metabolic ratio (MR) (norfentanyl : fentanyl). Results Delivery rate-adjusted serum fentanyl concentrations (0.0012–1.1 nmol/l/&mgr;g.h) and MRs (0.08–499) varied widely. Only 43% of variability in serum fentanyl concentrations was accounted for by delivery rate and less than 50% by CYP3A4/5 genotypes and clinical variables (delivery rate, sex, comedications, kidney disease, BMI, serum albumin). CYP3A4*22 and CYP3A5*3 variants, CYP3A inhibitors and variables relating to liver and kidney function (serum albumin, glomerular filtration rate, kidney disease, BMI) were associated with MR, but accounted for only 14% of variability. Conclusion Serum fentanyl concentrations and MR vary considerably between cancer pain patients on transdermal fentanyl patches. CYP3A4*22 and CYP3A5*3 genotypes, and multiple clinical factors, combine to influence transdermal fentanyl pharmacokinetics, but accounted for only a small proportion of variability in this study. Identification of the remaining factors determining serum fentanyl concentrations, and their relationship to efficacy and adverse effects may aid in improving the safety and effectiveness of transdermal fentanyl.

Pleistocene Chinese cave hyenas and the recent Eurasian history of the spotted hyena, Crocuta crocuta

The living hyena species (spotted, brown, striped and aardwolf) are remnants of a formerly diverse group of more than 80 fossil species, which peaked in diversity in the Late Miocene (about 7–8 Ma). The fossil history indicates an African origin, and morphological and ancient DNA data have confirmed that living spotted hyenas (Crocuta crocuta) of Africa were closely related to extinct Late Pleistocene cave hyenas from Europe and Asia. The current model used to explain the origins of Eurasian cave hyena populations invokes multiple migrations out of Africa between 3.5–0.35 Ma. We used mitochondrial DNA sequences from radiocarbon‐dated Chinese Pleistocene hyena specimens to examine the origin of Asian populations, and temporally calibrate the evolutionary history of spotted hyenas. Our results support a far more recent evolutionary timescale (430–163 kya) and suggest that extinct and living spotted hyena populations originated from a widespread Eurasian population in the Late Pleistocene, which was only subsequently restricted to Africa. We developed statistical tests of the contrasting population models and their fit to the fossil record. Coalescent simulations and Bayes Factor analysis support the new radiocarbon‐calibrated timescale and Eurasian origins model. The new Eurasian biogeographic scenario proposed for the hyena emphasizes the role of the vast steppe grasslands of Eurasia in contrast to models only involving Africa. The new methodology for combining genetic and geological data to test contrasting models of population history will be useful for a wide range of taxa where ancient and historic genetic data are available.

Sex differences in mechanical allodynia: how can it be preclinically quantified and analyzed?

Translating promising preclinical drug discoveries to successful clinical trials remains a significant hurdle in pain research. Although animal models have significantly contributed to understanding chronic pain pathophysiology, the majority of research has focused on male rodents using testing procedures that produce sex difference data that do not align well with comparable clinical experiences. Additionally, the use of animal pain models presents ongoing ethical challenges demanding continuing refinement of preclinical methods. To this end, this study sought to test a quantitative allodynia assessment technique and associated statistical analysis in a modified graded nerve injury pain model with the aim to further examine sex differences in allodynia. Graded allodynia was established in male and female Sprague Dawley rats by altering the number of sutures placed around the sciatic nerve and quantified by the von Frey test. Linear mixed effects modeling regressed response on each fixed effect (sex, oestrus cycle, pain treatment). On comparison with other common von Frey assessment techniques, utilizing lower threshold filaments than those ordinarily tested, at 1 s intervals, appropriately and successfully investigated female mechanical allodynia, revealing significant sex and oestrus cycle difference across the graded allodynia that other common behavioral methods were unable to detect. Utilizing this different von Frey approach and graded allodynia model, a single suture inflicting less allodynia was sufficient to demonstrate exaggerated female mechanical allodynia throughout the phases of dioestrus and pro-oestrus. Refining the von Frey testing method, statistical analysis technique and the use of a graded model of chronic pain, allowed for examination of the influences on female mechanical nociception that other von Frey methods cannot provide.

The effects of air pollution on asthma hospital admissions in Adelaide, South Australia, 2003-2013: time-series and case-crossover analyses.

SummaryBackground Air pollution can have adverse health effects on asthma sufferers, but the effects vary with geographic, environmental and population characteristics. There has been no long time-series study in Australia to quantify the effects of environmental factors including pollen on asthma hospitalizations. Objectives This study aimed to assess the seasonal impact of air pollutants and aeroallergens on the risk of asthma hospital admissions for adults and children in Adelaide, South Australia. Methods Data on hospital admissions, meteorological conditions, air quality and pollen counts for the period 2003–2013 were sourced. Time-series analysis and case–crossover analysis were used to assess the short-term effects of air pollution on asthma hospitalizations. For the time-series analysis, generalized log-linear quasi-Poisson and negative binomial regressions were used to assess the relationships, controlling for seasonality and long-term trends using flexible spline functions. For the case–crossover analysis, conditional logistic regression was used to compute the effect estimates with time-stratified referent selection strategies. Results A total of 36,024 asthma admissions were considered. Findings indicated that the largest effects on asthma admissions related to PM2.5, NO2, PM10 and pollen were found in the cool season for children (0–17 years), with the 5-day cumulative effects of 30.2% (95% CI: 13.4–49.6%), 12.5% (95% CI: 6.6–18.7%), 8.3% (95% CI: 2.5–14.4%) and 4.2% (95% CI: 2.2–6.1%) increases in risk of asthma hospital admissions per 10 unit increments, respectively. The largest effect for ozone was found in the warm season for children with the 5-day cumulative effect of an 11.7% (95% CI: 5.8–17.9%) increase in risk of asthma hospital admissions per 10 ppb increment in ozone level. Conclusion Findings suggest that children are more vulnerable and the associations between exposure to air pollutants and asthma hospitalizations tended to be stronger in the cool season compared to the warm season, with the exception of ozone. This study has important public health implications and provides valuable evidence for the development of policies for asthma management.

Codeine-induced hyperalgesia and allodynia: investigating the role of glial activation

Chronic morphine therapy has been associated with paradoxically increased pain. Codeine is a widely used opioid, which is metabolized to morphine to elicit analgesia. Prolonged morphine exposure exacerbates pain by activating the innate immune toll-like receptor-4 (TLR4) in the central nervous system. In silico docking simulations indicate codeine also docks to MD2, an accessory protein for TLR4, suggesting potential to induce TLR4-dependent pain facilitation. We hypothesized codeine would cause TLR4-dependent hyperalgesia/allodynia that is disparate from its opioid receptor-dependent analgesic rank potency. Hyperalgesia and allodynia were assessed using hotplate and von Frey tests at days 0, 3 and 5 in mice receiving intraperitoneal equimolar codeine (21 mg kg−1), morphine (20 mg kg−1) or saline, twice daily. This experiment was repeated in animals with prior partial nerve injury and in TLR4 null mutant mice. Interventions with interleukin-1 receptor antagonist (IL-1RA) and glial-attenuating drug ibudilast were assessed. Analyses of glial activation markers (glial fibrillary acid protein and CD11b) in neuronal tissue were conducted at the completion of behavioural testing. Despite providing less acute analgesia (P=0.006), codeine induced similar hotplate hyperalgesia to equimolar morphine vs saline (−9.5 s, P<0.01 and −7.3 s, P<0.01, respectively), suggesting codeine does not rely upon conversion to morphine to increase pain sensitivity. This highlights the potential non-opioid receptor-dependent nature of codeine-enhanced pain sensitivity—although the involvement of other codeine metabolites cannot be ruled out. IL-1RA reversed codeine-induced hyperalgesia (P<0.001) and allodynia (P<0.001), and TLR4 knock-out protected against codeine-induced changes in pain sensitivity. Glial attenuation with ibudilast reversed codeine-induced allodynia (P<0.001), and thus could be investigated further as potential treatment for codeine-induced pain enhancement.

Gene profiling for determining pluripotent genes in a time course microarray experiment

In microarray experiments, it is often of interest to identify genes which have a prespecified gene expression profile with respect to time. Methods available in the literature are, however, typically not stringent enough in identifying such genes, particularly when the profile requires equivalence of gene expression levels at certain time points. In this paper, the authors introduce a new methodology, called gene profiling, that uses simultaneous differential and equivalent gene expression level testing to rank genes according to a prespecified gene expression profile. Gene profiling treats the vector of true gene expression levels as a linear combination of appropriate vectors, for example, vectors that give the required criteria for the profile. This gene profile model is fitted to the data, and the resulting parameter estimates are summarized in a single test statistic that is then used to rank the genes. The theoretical underpinnings of gene profiling (equivalence testing, intersection-union tests) are discussed in this paper, and the gene profiling methodology is applied to our motivating stem-cell experiment.

Synergistic influence of collagen I and BMP 2 drives osteogenic differentiation of mesenchymal stem cells: a cell microarray analysis

Cell microarrays are a novel platform for the high throughput discovery of new biomaterials. By re-creating a multitude of cell microenvironments on a single slide, this approach can identify the optimal surface composition to drive a desired cell response. To systematically study the effects of molecular microenvironments on stem cell fate, we designed a cell microarray based on parallel exposure of mesenchymal stem cells (MSCs) to surface-immobilised collagen I (Coll I) and bone morphogenetic protein 2 (BMP 2). This was achieved by means of a reactive coating on a slide surface, enabling the covalent anchoring of Coll I and BMP 2 as microscale spots printed by a robotic contact printer. The surface between the printed protein spots was passivated using poly (ethylene glycol) bisamine 10,000Da (A-PEG). MSCs were then captured and cultured on array spots composed of binary mixtures of Coll I and BMP 2, followed by automated image acquisition and quantitative, multi-parameter analysis of cellular responses. Surface compositions that gave the highest osteogenic differentiation were determined using Runx2 expression and calcium deposition. Quantitative single cell analysis revealed subtle concentration-dependent effects of surface-immobilised proteins on the extent of osteogenic differentiation obscured using conventional analysis. In particular, the synergistic interaction of Coll I and BMP 2 in supporting osteogenic differentiation was confirmed. Our studies demonstrate the value of cell microarray platforms to decipher the combinatorial interactions at play in stem cell niche microenvironments.