A validated regulatory network for Th17 cell specification.

Th17 cells have critical roles in mucosal defense and are major contributors to inflammatory disease. Their differentiation requires the nuclear hormone receptor RORγt working with multiple other essential transcription factors (TFs). We have used an iterative systems approach, combining genome-wide TF occupancy, expression profiling of TF mutants, and expression time series to delineate the Th17 global transcriptional regulatory network. We find that cooperatively bound BATF and IRF4 contribute to initial chromatin accessibility and, with STAT3, initiate a transcriptional program that is then globally tuned by the lineage-specifying TF RORγt, which plays a focal deterministic role at key loci. Integration of multiple data sets allowed inference of an accurate predictive model that we computationally and experimentally validated, identifying multiple new Th17 regulators, including Fosl2, a key determinant of cellular plasticity. This interconnected network can be used to investigate new therapeutic approaches to manipulate Th17 functions in the setting of inflammatory disease.

Development of a spontaneously active dorsal root ganglia assay using multiwell multielectrode arrays.

In vitro phenotypic assays of sensory neuron activity are important tools for identifying potential analgesic compounds. These assays are typically characterized by hyperexcitable and/or abnormally, spontaneously active cells. Whereas manual electrophysiology experiments provide high-resolution biophysical data to characterize both in vitro models and potential therapeutic modalities (e.g., action potential characteristics, the role of specific ion channels, and receptors), these techniques are hampered by their low throughput. We have established a spontaneously active dorsal root ganglia (DRG) platform using multiwell multielectrode arrays (MEAs) that greatly increase the ability to evaluate the effects of multiple compounds and conditions on DRG excitability within the context of a cellular network. We show that spontaneous DRG firing can be attenuated with selective Na(+) and Ca(2+) channel blockers, as well as enhanced with K(+) channel blockers. In addition, spontaneous activity can be augmented with both the transient receptor potential cation channel subfamily V member 1 agonist capsaicin and the peptide bradykinin and completely blocked with neurokinin receptor antagonists. Finally, we validated the use of this assay by demonstrating that commonly used neuropathic pain therapeutics suppress DRG spontaneous activity. Overall, we have optimized primary rat DRG cells on a multiwell MEA platform to generate and characterize spontaneously active cultures that have the potential to be used as an in vitro phenotypic assay to evaluate potential therapeutics in rodent models of pain.

Production diseases in smallholder pig systems in rural Lao PDR.

Pigs in Lao People's Democratic Republic are important for income and food security, particularly in rural households. The majority of pigs are reared in smallholder systems, which may challenge the implementation of any disease control strategies. To investigate risk factors for pig production diseases in such farming systems in the country a serological survey was conducted during 2011. A total of 647 pigs were sampled, accounting for 294 households in Luang Prabang and 353 in Savannakhet province representing upland and lowland, respectively. The results demonstrated that pigs in Lao PDR had antibodies against erysipelas (45.2%), CSF (11.2%), PRRS (8.6%), FMD O (17.2%) and FMD Asia 1, (3.5%). Differences in the housing systems influenced disease risk, for example, penned pigs had reduced odds of FMD and CSF, compared to those in scavenger systems. Pigs owned by farms using a sanaam (a communal area where pigs are kept for some time of the year) had 3.93 (95% confidence interval (CI): 1.09-14.7) times the odds of having pigs seropositive for FMD. Farms on which sudden piglet deaths had been experienced were more likely to have pigs seropositive for FMD O and erysipelas. These diseases constrain the development of village farming and the wider livestock industry due to their impact on productivity and trade. Vaccination coverage for FMD and CSF was low and there was a lack of national funding for livestock disease control at the time of the study. Further investigation into sustainable low-cost control strategies for these pathogens is warranted.

A network approach for provisional assay recognition of a Hendra virus antibody ELISA: test validation with low sample numbers from infected horses.

Obtaining statistically sound numbers of sera from Hendra virus (HeV)-infected horses is problematic because affected individuals usually die or are euthanized before developing a serum antibody response. As a consequence, test validation becomes a challenge. Our approach is an extension of OIE principles for provisional recognition and included 7 validation panels tested across multiple laboratories that provided estimates for test performance characteristics. At a 0.4 S/P cutoff, 16 of 19 sera from HeV-infected horses gave positive results in the HeV soluble G, indirect ELISA (HeVsG iELISA; DSe 84.2% [95% CI: 60.4-96.6%]); 463 of 477 non-infected horse sera tested negative (DSp 97.1% [95% CI: 95.1-98.4%]). The HeVsG iELISA eliminated almost all false-positive results from the previously used HeV iELISA, with marginally decreased relative sensitivity. Assay robustness was evaluated in inter-laboratory and proficiency testing panels. The HeVsG iELISA is considered to be fit for purpose for serosurveillance and international movement of horses when virus neutralization is used for follow-up testing of positive or inconclusive serum samples.

Susceptibility of domestic dogs and cats to Australian bat lyssavirus (ABLV).

The susceptibility of cats and dogs to Australian bat lyssavirus (ABLV; genotype VII) was investigated by intramuscular (IM) inoculation of 10(3.7)-10(5) 50% tissue culture infective doses (TCID(50)) of virus followed by observation of experimental animals for up to 3 months post-inoculation (pi). Each experiment also included positive and negative controls, animals inoculated with a bat variant of rabies virus (Eptesicus I, genotype I), or a 10% suspension of uninfected mouse brain, respectively. Each of the ABLV-inoculated cats showed occasional abnormal clinical signs, but none died. Necropsies performed at 3 months pi revealed no lesions, and no viral antigen, in the central nervous system of any cat. ABLV could not be recovered from any cats. However, rabies virus-neutralizing antibodies were detected between 4 and 14 weeks pi in the sera of all three ABLV-inoculated cats. At 2-3 weeks pi, three of the five ABLV-inoculated dogs showed very mild abnormal clinical signs that persisted for 1-2 days, after which the dogs recovered. At 3 months pi, when all dogs were necropsied, neither lesions nor ABLV antigen were detected in, and virus was not isolated from, any dog. No ABLV RNA was detected by polymerase chain reaction (PCR) in clinical or necropsy samples from the three ABLV-affected dogs. However, all ABLV-inoculated dogs seroconverted by 2 weeks pi, and serum antibody titres were higher than those observed in cats. CSF, collected at 3 months pi, was positive for rabies virus-neutralizing antibody in two ABLV-inoculated dogs.

Reprint of "Assessing the impact of a joint human-porcine intervention package for Taenia solium control: Results of a pilot study from northern Lao PDR".

Following confirmation that a remote village of approximately 300 inhabitants in northern Lao PDR was hyperendemic for the Neglected Tropical Disease Taenia solium, a pilot human-porcine therapeutic control intervention was implemented between October 2013 and November 2014. Mass drug administration with a three day albendazole 400mg protocol was offered to all eligible humans in October 2013 and March 2014. At these times, and again in October 2014, eligible village pigs received the anti-cysticercosis TSOL18 vaccination and an oral dose of oxfendazole anthelmintic at 30mg/kg, both repeated one month later. Community and individual human taeniasis prevalences were estimated via copro-antigen ELISA of volunteered human faecal samples prior to October 2013, and again in January 2015, in order to examine the short term impact of the intervention.

Assessing the impact of a joint human-porcine intervention package for Taenia solium control: Results of a pilot study from northern Lao PDR.

Following confirmation that a remote village of approximately 300 inhabitants in northern Lao PDR was hyperendemic for the Neglected Tropical Disease Taenia solium, a pilot human-porcine therapeutic control intervention was implemented between October 2013 and November 2014. Mass drug administration with a three day albendazole 400mg protocol was offered to all eligible humans in October 2013 and March 2014. At these times, and again in October 2014, eligible village pigs received the anti-cysticercosis TSOL18 vaccination and an oral dose of oxfendazole anthelmintic at 30mg/kg, both repeated one month later. Community and individual human taeniasis prevalences were estimated via copro-antigen ELISA of volunteered human faecal samples prior to October 2013, and again in January 2015, in order to examine the short term impact of the intervention.