Development and use of an Arctic charr cell line to study antiviral responses at extremely low temperatures.

Arctic charr (Salvelinus alpinus) are the northernmost distributed freshwater fish and can grow at water temperatures as low as 0.2 °C. Other teleost species have impaired immune function at temperatures that Arctic charr thrive in, and thus, charr may maintain immune function at these temperatures. In this study, a fibroblastic cell line, named ACBA, derived from the bulbus arteriosus (BA) of Arctic charr was developed for use in immune studies at various temperatures. ACBA has undergone more than forty passages at 18 °C over 3 years, while showing no signs of senescence-associated ß-galactosidase activity and producing nitric oxide. Remarkably, ACBA cells survived and maintained some mitotic activity even at 1 °C for over 3 months. At these low temperatures, ACBA also continued to produce MH class I proteins. After challenge with poly I:C, only antiviral Mx proteins were induced while MH proteins remained constant. When exposed to live viruses, ACBA was shown to permit viral infection and replication of IPNV, VHSV IVa and CSV at 14 °C. Yet at the preferred temperature of 4 °C, only VHSV IVa was shown to replicate within ACBA. This study provides evidence that Arctic charr cells can maintain immune function while also resisting infection with intracellular pathogens at low temperatures.

Mutational spectrum in Usher syndrome type II.

Usher syndrome type II is an autosomal recessive disorder characterized by moderate to severe hearing impairment and progressive visual loss due to retinitis pigmentosa (RP). We carried out a mutation screening of the USH2A gene in 88 probands with Usher syndrome type II to determine the frequency of USH2A mutations as a cause for USH2. Six mutations, including 2299delG, 921-922insCAGC, R334W, N346H, R626X, and N357T were identified, with 2299delG mutation being the most frequent (16.5% of alleles), accounting for 77.5% of the pathologic alleles. Thirty-five percent (31/88) of the probands had a USH2A mutation. Nine of them carried two pathogenic mutations: six cases were homozygotes and three were compound heterozygotes. Twenty-two probands (25%) were found to carry only single USH2A mutations. One new missense mutation (N357T) occuring within the laminin N-terminal (type VI) domain of usherin was identified. Eight polymorphisms were found, five of which are novel. Our data support the view that the 2299delG is the most common mutation in USH2A.

[18F]-EF5, a marker for PET detection of hypoxia: synthesis of precursor and a new fluorination procedure.

There is a great deal of clinical and experimental interest in determining tissue hypoxia using non-invasive imaging methods. We have developed EF5, 2-(2-nitro-1[H]-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide, with both invasive and non-invasive hypoxia detection in mind. EF5 and other 2-nitroimidazoles are used to detect hypoxia, because the rate of their bioreductive metabolism is inversely dependent on oxygen partial pressure. Such metabolism leads to the formation of covalent adducts within the metabolizing cells. Previously, we have described the invasive detection of these adducts by highly specific monoclonal antibodies after tissue biopsy. In this report, we demonstrate the synthesis of 18F-labeled EF5, [18F]-2-(2-nitro-1[H]-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide, in greater than 10% yield by direct fluorination of the newly synthesized precursor 2-(2-nitro-1[H]-imidazol-1-yl)-N-(2,3,3-trifluoroallyl)-acetamide by [18F]-F2 in trifluoroacetic acid. Our objective was to optimize the electrophilic fluorination of the fluorinated alkene bond with fluorine gas, a new method of 18F-labeling of polyfluorinated molecules. Previous biodistribution studies in mice have demonstrated uniform access of EF5 to all tissues with bioelimination dominated by renal excretion. When [18F]-EF5 was injected into a rat followed by urine collection and analysis, we found no detectable metabolism to other radioactive compounds. Thus, [18F]-EF5 should be well suited for use as a non-invasive hypoxia marker with detection using positron emission tomography (PET).

Synthesis of new hypoxia markers EF1 and [18F]-EF1.

We report on the preparation of a hypoxia marker 2-(2-nitroimidazol-1[H]-yl)-N-(3-fluoropropyl)acetamide (EF1) and its 18F analog, 2-(2-nitroimidazol-1[H]-yl)-N- (3-[18F]fluoropropyl)acetamide ([18F]-EF1). Two methods for the preparation of 3-fluoropropylamine, the EF1 side chain, are described. [18F]-EF1 was prepared with a radiochemical yield of 2% by nucleophilic substitution of bromine in 2-(2-nitroimidazol-1[H]-yl)-N-(3-bromopropyl)acetamide (EBr1) by carrier-added 18F in DMSO at 120 degrees C. Our results demonstrate the preparation of clinically relevant amounts of [18F]-EF1 for use as a non-invasive hypoxia marker with detection using positron emission tomography (PET).

[An experimental study on the treatment of asthmatics with diethylcarbamazine].

This study was designed to assess the effectiveness of oral administration of diethylcarbamazine (DEC) on the treatment of asthmatics. When compared to baseline and placebo control DEC failed to improve significantly lung ventilation function within 3 hours in 28 asthmatics. In 11 patients with stable asthma a significant increase in MCH-PC20-FEV1 was noticed at 3 days after the use of DEC. In 24 atopic asthmatics DEC given 90 minutes prior to exercise led to significantly less post-exercise percentage fall in FEV1 when compared to placebo. These findings indicate: 1. DEC has no bronchodilation effect. 2. DEC inhibits MCH-induced bronchospasm and reduces the nonspecific airway hyperreactivity. 3. DEC given 90 minutes before exercise protects from exercise-induced asthma.