Growth and differentiation of primary and passaged equine bronchial epithelial cells under conventional and air-liquid-interface culture conditions

<p>Abstract</p> <p>Background</p> <p>Horses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells <it>in vivo </it>would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic <it>in vivo </it>conditions.</p> <p>Results</p> <p>Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum. However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI. A pseudo-stratified muco-ciliary epithelium with basal cells was observed at differentiation. Further, transepithelial resistance (TEER) was more consistent and higher in P₁cultures compared to P₀cultures while ciliation was delayed in P₁cultures.</p> <p>Conclusions</p> <p>This study provides an efficient method for obtaining a high-yield of EBECs and for generating highly differentiated cultures. These EBEC cultures can be used to study the formation of tight junction or to identify epithelial-derived inflammatory factors that contribute to lung diseases such as asthma.</p

Dynamic Locomotor Capabilities Revealed by Early Dinosaur Trackmakers from Southern Africa

BACKGROUND: A new investigation of the sedimentology and ichnology of the Early Jurassic Moyeni tracksite in Lesotho, southern Africa has yielded new insights into the behavior and locomotor dynamics of early dinosaurs. METHODOLOGY/PRINCIPAL FINDINGS: The tracksite is an ancient point bar preserving a heterogeneous substrate of varied consistency and inclination that includes a ripple-marked riverbed, a bar slope, and a stable algal-matted bar top surface. Several basal ornithischian dinosaurs and a single theropod dinosaur crossed its surface within days or perhaps weeks of one another, but responded to substrate heterogeneity differently. Whereas the theropod trackmaker accommodated sloping and slippery surfaces by gripping the substrate with its pedal claws, the basal ornithischian trackmakers adjusted to the terrain by changing between quadrupedal and bipedal stance, wide and narrow gauge limb support (abduction range = 31 degrees ), and plantigrade and digitigrade foot posture. CONCLUSIONS/SIGNIFICANCE: The locomotor adjustments coincide with changes in substrate consistency along the trackway and appear to reflect 'real time' responses to a complex terrain. It is proposed that these responses foreshadow important locomotor transformations characterizing the later evolution of the two main dinosaur lineages. Ornithischians, which shifted from bipedal to quadrupedal posture at least three times in their evolutionary history, are shown to have been capable of adopting both postures early in their evolutionary history. The substrate-gripping behavior demonstrated by the early theropod, in turn, is consistent with the hypothesized function of pedal claws in bird ancestors

Phase 1 Safety and Immunogenicity Evaluation of ADVAX, a Multigenic, DNA-Based Clade C/B' HIV-1 Candidate Vaccine

BACKGROUND: We conducted a Phase I dose escalation trial of ADVAX, a DNA-based candidate HIV-1 vaccine expressing Clade C/B' env, gag, pol, nef, and tat genes. Sequences were derived from a prevalent circulating recombinant form in Yunnan, China, an area of high HIV-1 incidence. The objective was to evaluate the safety and immunogenicity of ADVAX in human volunteers. METHODOLOGY/PRINCIPAL FINDINGS: ADVAX or placebo was administered intramuscularly at months 0, 1 and 3 to 45 healthy volunteers not at high risk for HIV-1. Three dosage levels [0.2 mg (low), 1.0 mg (mid), and 4.0 mg (high)] were tested. Twelve volunteers in each dosage group were assigned to receive ADVAX and three to receive placebo in a double-blind design. Subjects were followed for local and systemic reactogenicity, adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA. Cellular immunogenicity was assessed by a validated IFNgamma ELISpot assay and intracellular cytokine staining. ADVAX was safe and well-tolerated, with no vaccine-related serious adverse events. Local and systemic reactogenicity events were reported by 64% and 42% of vaccine recipients, respectively. The majority of events were mild. The IFNgamma ELISpot response rates to any HIV antigen were 0/9 (0%) in the placebo group, 3/12 (25%) in the low-dosage group, 4/12 (33%) in the mid-dosage group, and 2/12 (17%) in the high-dosage group. Overall, responses were generally transient and occurred to each gene product, although volunteers responded to single antigens only. Binding antibodies to gp120 were not detected in any volunteers, and HIV seroconversion did not occur. CONCLUSIONS/SIGNIFICANCE: ADVAX delivered intramuscularly is safe, well-tolerated, and elicits modest but transient cellular immune responses. TRIAL REGISTRATION: Clinicaltrials.gov NCT00249106.published_or_final_versio

Immunomic investigation of Holocyclotoxins to produce the first protective Anti-Venom vaccine against the Australian paralysis tick, Ixodes holocyclus

Venom producing animals are ubiquitously disseminated among vertebrates and invertebrates such as fish, snakes, scorpions, spiders, and ticks. Of the ~890 tick species worldwide, 27 have been confirmed to cause paralysis in mammalian hosts. The Australian paralysis tick (Ixodes holocyclus) is the most potent paralyzing tick species known. It is an indigenous three host tick species that secretes potent neurotoxins known as holocyclotoxins (HTs). Holocyclotoxins cause a severe and harmful toxicosis leading to a rapid flaccid paralysis which can result in death of susceptible hosts such as dogs. Antivenins are generally polyclonal antibody treatments developed in sheep, horses or camels to administer following bites from venomous creatures. Currently, the methods to prevent or treat tick paralysis relies upon chemical acaricide preventative treatments or prompt removal of all ticks attached to the host followed by the administration of a commercial tick-antiserum (TAS) respectively. However, these methods have several drawbacks such as poor efficacies, non-standardized dosages, adverse effects and are expensive to administer. Recently the I. holocyclus tick transcriptome from salivary glands and viscera reported a large family of 19 holocyclotoxins at 38-99% peptide sequence identities. A pilot trial demonstrated that correct folding of holocyclotoxins is needed to induce protection from paralysis. The immunogenicity of the holocyclotoxins were measured using commercial tick antiserum selecting HT2, HT4, HT8 and HT11 for inclusion into the novel cocktail vaccine. A further 4 HTs (HT1, HT12, HT14 and HT17) were added to the cocktail vaccine to ensure that the sequence variation among the HT protein family was encompassed in the formulation. A second trial comparing the cocktail of 8 HTs to a placebo group demonstrated complete protection from tick challenge. Here we report the first successful anti-venom vaccine protecting dogs from tick paralysis

Laboratory evaluations of the 3-month efficacy of oral lotilaner (Credelio™) against experimental infestations of dogs with the Australian paralysis tick, Ixodes holocyclus

Abstract Background From three days following host attachment, the Australian paralysis tick, Ixodes holocyclus, secretes a neurotoxin that annually causes paralysis in approximately 10,000 domestic pets. Lotilaner, a novel isoxazoline formulated in a chewable flavoured tablet (CredelioTM), produces rapid onset of acaricidal activity in dogs, with an efficacy duration of at least one month. Two studies were performed to determine the efficacy of lotilaner against I. holocyclus infestations over 3 months. Methods Both studies included 16 dogs, ranked according to I. holocyclus counts on Day -5 (from infestations on Day -8) and blocked into pairs. One dog in each pair was randomized to be a sham-treated control, the other to receive lotilaner at a minimum dose rate of 20 mg/kg on Day 0. Dogs were dosed in a fed state. Infestations were performed in both studies on Days -8 (to determine the tick carrying capacity of each dog) -1, 28, 56, 70, 77 and 84, and additionally in Study 1 on Day 91, in Study 2 on Days 14 and 42. In Study 1, ticks were counted and assessed as alive or dead at 24, 48 and 72 h post-initial infestation and post-subsequent re-infestations. In study 2, ticks were counted at 24, 48 and 72 h post-dosing or post-re-infestation. Efficacy was determined by the percent reduction in live attached tick counts in the lotilaner group compared to control. Results Within 48 h post-treatment in Study 1 and within 72 h post-treatment in Study 2 all lotilaner-group dogs were free of live ticks. By 72 h post-infestation, efficacy in Study 1 remained at 100% through Day 87, except on Day 31 when a single tick was found on one dog, and through Day 59 in Study 2. Efficacy exceeded 95% through the final assessment in each study (Days 94 and 87 in Studies 1 and 2, respectively). Conclusion These results demonstrate that lotilaner quickly kills existing I. holocyclus infestations. By providing 95.3–100.0% protection through at least 87 days post-treatment, lotilaner can be a valuable tool in reducing the risk of tick paralysis in dogs

Immunomic Investigation of Holocyclotoxins to Produce the First Protective Anti-Venom Vaccine Against the Australian Paralysis Tick, Ixodes holocyclus

Venom producing animals are ubiquitously disseminated among vertebrates and invertebrates such as fish, snakes, scorpions, spiders, and ticks. Of the ~890 tick species worldwide, 27 have been confirmed to cause paralysis in mammalian hosts. The Australian paralysis tick (Ixodes holocyclus) is the most potent paralyzing tick species known. It is an indigenous three host tick species that secretes potent neurotoxins known as holocyclotoxins (HTs). Holocyclotoxins cause a severe and harmful toxicosis leading to a rapid flaccid paralysis which can result in death of susceptible hosts such as dogs. Antivenins are generally polyclonal antibody treatments developed in sheep, horses or camels to administer following bites from venomous creatures. Currently, the methods to prevent or treat tick paralysis relies upon chemical acaricide preventative treatments or prompt removal of all ticks attached to the host followed by the administration of a commercial tick-antiserum (TAS) respectively. However, these methods have several drawbacks such as poor efficacies, non-standardized dosages, adverse effects and are expensive to administer. Recently the I. holocyclus tick transcriptome from salivary glands and viscera reported a large family of 19 holocyclotoxins at 38-99% peptide sequence identities. A pilot trial demonstrated that correct folding of holocyclotoxins is needed to induce protection from paralysis. The immunogenicity of the holocyclotoxins were measured using commercial tick antiserum selecting HT2, HT4, HT8 and HT11 for inclusion into the novel cocktail vaccine. A further 4 HTs (HT1, HT12, HT14 and HT17) were added to the cocktail vaccine to ensure that the sequence variation among the HT protein family was encompassed in the formulation. A second trial comparing the cocktail of 8 HTs to a placebo group demonstrated complete protection from tick challenge. Here we report the first successful anti-venom vaccine protecting dogs from tick paralysis.</p

Assess, compare and enhance the <i>status</i> of persons with Multiple Sclerosis (MS) in Europe: a European Register for MS

Objectives – Persons with multiple sclerosis (PwMS) experience health-related quality of life (HRQoL) problems greatly differing across Europe, and the European Union (EU) faces deep inequalities in MS management from country to country. Through the establishment of a European MS Register (EUReMS), an effective action is proposed to improve the overall knowledge on MS and support effective intervention programmes at EU and national political level. EUReMS aims to achieve consensus on its mission and vision, to define existing data providers, to develop models driving future MS health policies and research, to develop an information technology (IT) infrastructure for a data set, to develop a European shared governance and to secure providers’ data provision into EUReMS. Materials and methods – EUReMS is meant to build on a minimum set of core data from existing national and regional population-based MS registries and from PwMS’ perspectives. EUReMS’ main partner is the European MS Platform (EMSP) acting in collaboration with associated and collaborating European partners. Results – EUReMS was launched in July 2011. A Consensus Statement on purposes, vision, mission and strategies was produced in December 2011, and a comprehensive survey on existing MS data collections in Europe has been performed, and the EUReMS data mask is currently being discussed. Conclusions – EUReMS will represent a tool to provide up to date, comparable and sustainable MS data through an effective and credible register, which will encourage extensive knowledge building of MS, more equitable policies and higher standards in MS treatment and services.</br