A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABA(A) receptor chloride channels

There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an extemal I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Angiostrongrylus cantonensis as a cause of cerebrospinal disease in a yellow-tailed black cockatoo (Calyptorhynchus funereus) and two tawny frogmouths (Podargus strigoides)

A captive yellow-tailed black cockatoo (Calyptorhynchus funereus) and 2 free-living tawny frogmouths (Podargus strigoides), both native Australian species, were presented with neurologic signs including depression and pelvic limb paresis and paralysis. Despite supportive treatment, all 3 birds died or were euthanatized. On histologic examination, sections of metastrongyloid nematode larvae were found in the central nervous system of all 3 birds, whereas intact larvae, identified as Angiostrongylus cantonensis, were recovered from the brain and spinal cord of 2 birds. Angiostrongylus cantonensis, the rat lungworm. has an obligatory migratory phase through the host's central nervous system, which can cause severe pathologic lesions. Natural infections in accidental hosts have been documented only in mammals, and to our knowledge, angiostrongyliasis in avian hosts has not been previously reported.

Live Attenuated Chimeric Yellow Fever Dengue Type 2 (Chimeri Vax -DEN2) Vaccine: Phase 1 clinical trial for safety and immunogenicity

A randomized double-blind Phase I Trial was conducted to evaluate safety, tolerability, and immunogenicity of a yellow fever (YF)-dengue 2 (DEN2) chimera (ChimeriVax™-DEN2) in comparison to that of YF vaccine (YF-VAX®). Forty-two healthy YF naïve adults randomly received a single dose of either ChimeriVax™-DEN2 (high dose, 5 log plaque forming units [PFU] or low dose, 3 log PFU) or YF-VAXâ by the subcutaneous route (SC). To determine the effect of YF pre-immunity on the ChimeriVaxTM-DEN2 vaccine, 14 subjects previously vaccinated against YF received a high dose of ChimeriVax™-DEN2 as an open-label vaccine. Most adverse events were similar to YF-VAX® and of mild to moderate intensity, with no serious side-effects. One hundred percent and 92.3% of YF naïve subjects inoculated with 5.0 and 3.0 log10 PFU of ChimeriVaxTM-DEN2, respectively, seroconverted to wt DEN2 (strain 16681); 92% of subjects inoculated with YF-VAX® seroconverted to YF 17D virus but none of YF naïve subjects inoculated with ChimeriVax-DEN2 seroconverted to YF 17D virus. Low seroconversion rates to heterologous DEN serotypes 1, 3, and 4 were observed in YF naïve subjects inoculated with either ChimeriVax™-DEN2 or YF-VAX®. In contrast, 100% of YF immune subjects inoculated with ChimeriVax™-DEN2 seroconverted to all 4 DEN serotypes. Surprisingly, levels of neutralizing antibodies to DEN 1, 2, and 3 viruses in YF immune subjects persisted after 1 year. These data demonstrated that 1) the safety and immunogenicity profile of the ChimeriVax™-DEN2 vaccine is consistent with that of YF-VAX®, and 2) pre-immunity to YF virus does not interfere with ChimeriVaxTM-DEN2 immunization, but induces a long lasting and cross neutralizing antibody response to all 4 DEN serotypes. The latter observation can have practical implications toward development of a dengue vaccine.